Patent application title: METHOD FOR ASSESSMENT OF POTENTIAL FOR DEVELOPMENT OF DRAVET SYNDROME AND USE THEREOF
Inventors:
Iori Ohmori (Okayama-Shi, JP)
Mamoru Ouchida (Okayama-Shi, JP)
Assignees:
NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
IPC8 Class: AA01K67027FI
USPC Class:
800 3
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of using a transgenic nonhuman animal in an in vivo test method (e.g., drug efficacy tests, etc.)
Publication date: 2013-02-07
Patent application number: 20130036482
Abstract:
Provided is a method of assessing a potential for development of Dravet
syndrome with high accuracy, and use thereof. The method according to the
present invention of assessing a potential for development of Dravet
syndrome includes, with use of a sample taken from a subject, detecting
whether or not a mutation is on α-subunit type 1 of voltage-gated
sodium ion channel NaV1.1, and detecting whether or not a mutation
is on α-subunit type 1 of voltage-gated calcium ion channel
CaV2.1.Claims:
1. A method of obtaining data for assessing potential for development of
Dravet syndrome, the method comprising: with use of a sample taken from a
subject, detecting whether or not a mutation exists on α-subunit
type 1 of voltage-gated sodium ion channel NaV1.1; and detecting
whether or not a mutation is on α-subunit type 1 of voltage-gated
calcium ion channel CaV2.1.
2. The method according to claim 1, wherein the mutation on the α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 is at least one of mutations recited in Table 1, and the mutation on the α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 is at least one of mutations recited in Table 2.
3. The method according to claim 1, further comprising: detecting a change in activity of the voltage-gated sodium ion channel NaV1.1; and detecting a change in activity of the voltage-gated calcium ion channel CaV2.1.
4. A kit for assessing a potential for development of Dravet syndrome, the kit comprising: a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
5. A model animal of Dravet syndrome, having a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
6. A method of producing a model animal of Dravet syndrome as set forth in claim 5, the method comprising: introducing a mutation on a α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1; and introducing a mutation on a α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
7. A cell, having a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
8. A method of producing a cell as set forth in claim 7, the method comprising: introducing a mutation on a α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1; and introducing a mutation on a α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
9. A screening method of a drug for treating Dravet syndrome, the method comprising: administering a candidate agent to the model animal of Dravet syndrome as set forth in claim 5; and assessing whether or not the administering of the candidate agent has made Dravet syndrome improve or cure in the model animal of Dravet syndrome.
10. A screening method of a drug for treating Dravet syndrome, the method comprising: administering a candidate agent to the cell as set forth in claim 7; and assessing whether or not the administering of the candidate agent has made activity of the voltage-gated sodium ion channel NaV1.1 and/or activity of the voltage-gated calcium ion channel CaV2.1 change in the cell.
11. The method according to claim 2, further comprising: detecting a change in activity of the voltage-gated sodium ion channel NaV1.1; and detecting a change in activity of the voltage-gated calcium ion channel CaV2.1.
Description:
TECHNICAL FIELD
[0001] The present invention relates to a method for assessing a potential for development of Dravet syndrome, and use thereof.
BACKGROUND ART
[0002] Febrile seizure is a disease that has a high incidence rate of approximately 8% in infants. A main symptom of febrile seizure is known as a continuation of generalized convulsions for 1 to 5 minutes while suffering a fever at or over 38° C. caused by a viral or bacterial infection such as a cold, or microbism. Most cases of febrile seizure that have an onset of between 6 months after birth and around 5 years old cure by the time when the patient turns 6 years old. In many cases, febrile seizure does not require active treatment. Therefore, febrile seizure is considered, in principle, as a benign disease.
[0003] However, among patients whose onset of febrile seizure was under the age of one, other than the patients of the benign disease which cease as a regular febrile seizure, there are some patients who suffer from convulsions continuously even after turning 6 years old, and there are some patients who are patients of Dravet syndrome (previously called "Severe Myoclonic Epilepsy in Infancy; SMEI"), which are patients of an intractable epilepsy disease.
[0004] The patients of Dravet syndrome are triggered in the onset of convulsions under the age of one. An average age of the onset of convulsions for patients of Dravet syndrome is 4 months to 6 months after birth. An incipient seizure of convulsion for a patient of Dravet syndrome is generally a systemic or a unilateral tonic-clonic or clonic convulsion, and during infancy, may lead to status epilepticus. Moreover, this convulsion seizure is easily induced by fever or bathing.
[0005] Conventionally, febrile seizure was diagnosed and treated by a general pediatrician or a family doctor, and Dravet syndrome is also diagnosed based on clinical symptoms characteristic of Dravet syndrome such as convulsion seizure or the like. However, by the time the patients of Dravet syndrome turn two to three years old, that is around when the clinical symptoms of Dravet syndrome have all appeared, these patients would have suffered repetitive convulsions many times and would often have had experienced critical conditions such as status epilepticus or the like. Hence, it is necessary to develop a diagnosis method that enables detection of Dravet syndrome in its possible earliest stage by a general pediatrician or family doctor, who is engaged in primary medical care. Detection of Dravet syndrome at an earlier stage would allow for the patent to see an epilepsy specialist in advance, which would allow for preventing the patient from reaching a critical condition.
[0006] Recently, it has been reported that 30% to 80% of Dravet syndrome patients find missense mutation (mutation causing a substitution of an amino acid) and nonsense mutation (mutation causing protein synthesis to stop in an incomplete state) on a SCN1A gene that encodes a voltage-gated sodium ion channel NaV1.1 α-subunit type 1 (see Non Patent Literature 1 and 2). From such a point in view, attempts have been made to examine abnormalities in the SCN1A gene to diagnose Dravet syndrome on the basis of genes.
[0007] For example, Patent Literatures 1 to 4 disclose that mutation of the SCN1A gene is related to SMEI. Moreover, Patent Literatures 1 to 4 disclose that SMEI can be diagnosed by use of the mutation of the SCN1A gene as an indicator.
[0008] More specifically, Patent Literature 1 discloses the diagnosis of SMEI by assessing a plurality of mutations on the SCN1A gene that relate to SMEI, as a whole.
[0009] Patent Literature 2 discloses the diagnosis of SMEI performed by detecting a presence of a mutation that frequently occurs on the SCN1A gene of a nerve that is affected by SMEI.
[0010] Patent Literatures 3 and 4 disclose a method of diagnosing epilepsy syndromes including SMEI and syndromes associated with SMEI, by detecting a change in the SCN1A gene and confirming whether that change is known as being related to SMEI or a syndrome associated with SMEI or is known as not being related to SMEI or a syndrome associated with SMEI.
CITATION LIST
Patent Literature
Patent Literature 1
[0011] Japanese Patent Application Publication, Tokukai, No. 2004-329153 A (Publication Date: Nov. 25, 2004)
Patent Literature 2
[0011] [0012] Japanese Patent Application Publication, Tokukai, No. 2004-73058 A (Publication Date: Mar. 11, 2004)
Patent Literature 3
[0012] [0013] Published Japanese Translations of PCT International Publication, Tokuhyo, No. 2008-546376 A (Publication Date: Dec. 25, 2008)
Patent Literature 4
[0013] [0014] Published Japanese Translations of PCT International Publication, Tokuhyo, No. 2006-524490 A (Publication Date: Nov. 2, 2006)
Non Patent Literature
Non Patent Literature 1
[0014] [0015] Sugawara T, Mazaki-Miyazaki E, Fukushima K, Shimomura J, Fujiwara T, Hamano S, Inoue Y, Yamakawa K. 2002. Frequent mutations of SCN1A in severe myoclonic epilepsy in infancy. Neurology 58: 1122-1124.
Non Patent Literature 2
[0015] [0016] Ohmori I, Ouchida M, Ohtsuka Y, Oka E, Shimizu K. 2002. Significant correlation of the SCN1A mutations and severe myoclonic epilepsy in infancy. Biochem Biophys Res Commun 295: 17-23.
Non Patent Literature 3
[0016] [0017] Escayg A, Heils A, MacDonald B T, Haug K, Sander T, and Meisler M H. 2001. A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus--and prevalence of variants in patients with epilepsy. Am J Hum Genet. 68: 866-873.
SUMMARY OF INVENTION
Technical Problem
[0018] As described above, the mutation on the SCN1A gene is found in an extremely large number of Dravet syndrome patients (30% to 80%). However, it is becoming revealed that the presence of a mutation on the SCN1A gene does not necessarily mean that the symptoms of Dravet syndrome would appear.
[0019] For example, Non Patent Literature 3 reports that not just the patients of the intractable Dravet syndrome, but also patients of febrile seizure and patients with a certain kind of benign epilepsy (e.g. GEFS+ (Generalized epilepsy with febrile seizure plus)) have a mutation on the SCN1A gene.
[0020] As such, the mutation on the SCN1A gene is not a phenomenon specific to Dravet syndrome. Hence, the conventional methods of examining just the abnormalities on the SCN1A gene as described in Patent Literatures 1 to 4 can be said as insufficient for specifically diagnosing Dravet syndrome.
[0021] Therefore, in order to distinguish between the patients with benign febrile seizure and the patients with Dravet syndrome and to allow for the patients with Dravet syndrome to receive appropriate treatment by a specialist, further development is required in techniques for more accurately diagnosing Dravet syndrome.
[0022] The present invention is accomplished in view of the foregoing problems, and an object thereof is to provide a method of (specifically) assessing with high accuracy a potential for development of Dravet syndrome.
Solution to Problem
[0023] Patients of GEFS+ and the patients of Dravet syndrome are common in a point that the SCN1A gene has a mutation. Meanwhile, the inventors performed diligent study based on their unique point of view of focusing on the difference in malignancy between the diseases; they considered that the development of Dravet syndrome is related to not just the mutation on the SCN1A gene but also another factor, and that another cause is related to the worsening and intractableness of Dravet syndrome. As a result, the inventors uniquely found out that many Dravet syndrome patients have a mutation on the SCN1A gene and further a mutation on the CACNA1A gene that encodes a P/Q type voltage-gated calcium ion channel CaV2.1 α1 subunit.
[0024] Furthermore, based on this finding, the inventors produced a rat having both the mutations on the SCN1A gene and the CACNA1A gene, and demonstrated that the rat having both the mutations on the SCN1A gene and the CACNA1A gene experienced more serious convulsion seizures as compared to rats having just the mutation on the SCN1A gene.
[0025] Based on these results of analyzing genes and animal testing results, it was found that the potential for development of Dravet syndrome can be assessed with high accuracy by detecting mutations for both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1, and accomplished the present invention.
[0026] Namely, the present invention includes the following inventions.
[0027] An assessment method according to the present invention is a method of assessing a potential for development of Dravet syndrome, the method including:
[0028] with use of a sample taken from a subject,
[0029] detecting whether or not a mutation is on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and
[0030] detecting whether or not a mutation is on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. It is preferable that the assessment method according to the present invention is a method of obtaining data for assessing potential for development of Dravet syndrome.
[0031] A kit according to the present invention is a kit for assessing a potential for development of Dravet syndrome, the kit comprising:
[0032] a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and
[0033] a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. The kit according to the present invention may be a kit for obtaining data for assessing a potential for development of Dravet syndrome.
[0034] A model animal of Dravet syndrome according to the present invention has a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
[0035] A production method according to the present invention of a model animal of Dravet syndrome is a method of producing the model animal of Dravet syndrome described above, which method includes:
[0036] introducing a mutation on a α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1; and
[0037] introducing a mutation on a α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
[0038] A cell according to the present invention has a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
[0039] A method of producing a cell according to the present invention is a method of producing the cell described above, which method includes:
[0040] introducing a mutation on a α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1; and
[0041] introducing a mutation on a α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
[0042] A screening method according to the present invention of a drug for treating Dravet syndrome includes:
[0043] administering a candidate agent to the model animal of Dravet syndrome according to the present invention; and
[0044] assessing whether or not the administering of the candidate agent has made Dravet syndrome improve or cure in the model animal of Dravet syndrome.
[0045] A screening method according to the present invention of a drug for treating Dravet syndrome includes:
[0046] administering a candidate agent to the cell according to the present invention; and
[0047] assessing whether or not the administering of the candidate agent has made activity of the voltage-gated sodium ion channel NaV1.1 and/or activity of the voltage-gated calcium ion channel CaV2.1 change in the cell.
[0048] For a fuller understanding of the nature and advantages of the invention, reference should be made to the ensuing detailed description taken in conjunction with the accompanying drawings.
Advantageous Effects of Invention
[0049] The method according to the present invention of assessing a potential for development of Dravet syndrome allows for obtaining data for assessing the potential for development of Dravet syndrome, by detecting mutations for both α-subunit type 1 of voltage-gated sodium ion channel
[0050] NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
[0051] Patients of GEFS+, being a benign epilepsy, inherit the mutation of the SCN1A gene within the family. In comparison, in patients of Dravet syndrome, approximately 90% of the mutations on SCN1A gene are de novo mutation, i.e. are anew mutations in which a mutation arises even though their parents have no mutation. As such, although the GEFS+ patients and the Dravet syndrome patients are common in that a mutation is on the SCN1A gene, the cause for the difference in malignancy of the disease was unknown. However, it was clarified by the present inventors for the first time, that the presence of mutations on both the SCN1A gene and the CACNA1A gene is related to the worsening and intractableness of Dravet syndrome.
[0052] As described above, reports have already been made that a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 (hereinafter, referred to as "sodium ion channel α1 subunit") is related to the development of Dravet syndrome. However, no reports have been made whatsoever that Dravet syndrome is related to a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1 (hereinafter, referred to as "calcium ion channel α1 subunit").
[0053] Reports have been made that a mutation on a subunit other than the α 1 subunit of voltage-gated calcium ion channel CaV2.1 is associated with Dravet syndrome (see Iori Ohmori et. Al., Neurobiology of Disease 32 (2008) 349-354). More specifically, this literature (Iori Ohmori et. Al.) discloses that a mutation on β4 subunit of voltage-gated calcium ion channel CaV2.1 (hereinafter, simply referred to as "calcium ion channel (34 subunit") is associated with Dravet syndrome.
[0054] However, the foregoing literature strongly teaches regarding Dravet syndrome that a mutation on the "calcium ion channel β4 subunit" is important together with the mutation on the "α-subunit of sodium ion channel NaV1.1". This description in the literature hinders a motivation to arrive at a point that a mutation suitable for detecting Dravet syndrome is present in the calcium ion channel α 1 subunit.
[0055] In the first place, a skilled person would not arrive at considering, just because a relationship of a mutation on a specific subunit with a disease is known for a specific channel, that other subunits would also have a mutation related to that disease. At least, the finding that the voltage-gated sodium ion channel NaV1.1 is related to Dravet syndrome is only known regarding the mutation on the "α 1 subunit"; this does not give motivation for analyzing mutations on other subunits.
[0056] As to a mutation on the calcium ion channel α 1 subunit, reports have been made stating a relationship with (1) epixodic ataxia type 2 (characterized in paroxysmal cerebellar ataxia), (2) familial hemiplegic migraine type 1 (e.g. hemiplegia, hemianopsia, dysphagia, throbbing headache), and (3) spinocerebellar ataxia type 6 (e.g. ataxic gait, limb ataxia, cerebellar dysarthria, nystagmus) (see Keiji IMOTO et al., "Igaku no Ayumi" (Development in Medical Science), Vol. 201, No. 13 (Issued Jun. 29, 2002); Taiji TSUNEMI et al., "Igaku no Ayumi" (Development in Medical Science), Vol. 201, No. 13 (Issued Jun. 29, 2002)). However, the diseases of (1) to (3) all show no symptoms of epilepsy, and neither are diseases related to Dravet syndrome. At least, although the finding regarding the mutation on the calcium ion channel α 1 subunit is known as related to the diseases of (1) to (3), it is not one that gives motivation for analyzing a mutation on the calcium ion channel α 1 subunit in Dravet syndrome, which disease is completely unrelated to the diseases of (1) to (3).
[0057] The assessment method according to the present invention detects a mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1. Hence, it is possible to detect Dravet syndrome with high accuracy. Consequently, the assessment method of the present invention brings about an effect that it is possible to improve reliability of a potential for detecting Dravet syndrome as compared to the conventional method by detecting a mutation on the SCN1A gene. Furthermore, detection of a mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and a mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 is possible even with an infant under the age of one. Hence, according to the assessment method of the present invention, an effect is brought about that data for assessing the potential for development in Dravet syndrome can be obtained from a patient in an early stage of development or in a stage prior to the onset of the intractable disease, in particular of an infant under the age of one.
[0058] Moreover, as shown in Examples later described, an effect is brought about that by detecting a mutation on both α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1, the detection sensitivity of Dravet syndrome patients dramatically improve.
[0059] Furthermore, with use of the kit according to the present invention, it is possible to easily detect the mutation on both α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1. Hence, the kit according to the present invention is useful for a general pediatrician to screen, at an early stage of disease of under the age of one, a patient of Dravet syndrome that requires treatment by a specialist, among benign febrile epilepsy.
[0060] By using the assessment method and kit according to the present invention, it is possible to detect the patients of Dravet syndrome with high accuracy at the point in time of an age under one, which is an age difficult to detect until now. Moreover, by sending a blood sample to an examination center and examining its abnormal genes, it is possible to detect a Dravet syndrome patient with high accuracy even in a private hospital at a remote location or the like.
[0061] Moreover, the Dravet syndrome model animal and cell according to the present invention can be usefully used for resolving a development mechanism of the intractable Dravet syndrome, and for development and the like of medicament for Dravet syndrome.
BRIEF DESCRIPTION OF DRAWINGS
[0062] FIG. 1 is a view illustrating an amino acid sequence of a protein encoded by a human SCN1A gene and an amino acid sequence of a protein encoded by a rat Scn1a gene.
[0063] FIG. 2 is a view illustrating a result of performing function analysis of sodium ion channel, by use of patch clamping. Illustrated in (a) is a typical example of a sodium current effected by a change in potential of a normal sodium ion channel and a mutant sodium ion channel. Illustrated in (b) is a result of examining a time constant (τ) at inactivation.
[0064] FIG. 3 is a view illustrating a result of performing function analysis of a sodium ion channel, by use of patch clamping. Illustrated in (a) is a current-voltage relationship, illustrated in (b) is an activation curve of the sodium ion channel, illustrated in (c) is an inactivation curve of the sodium ion channel, and illustrated in (d) is a recovery curve from the inactivation of the sodium ion channel.
[0065] FIG. 4 is a view illustrating a result of performing function analysis of a sodium ion channel, by use of patch clamping. Illustrated in (a) is a sodium current flowing in the sodium ion channel, and illustrated in (b) is a relative value (%) of a persistent sodium current amount flowing into the sodium ion channel.
[0066] FIG. 5 is a view illustrating genotypes of parent rats (P), first filial generation (F1) rats, and second filial generation (F2) rats. Illustrated in (a) is a view showing genotypes of the parent rats (P) and the F1 rats. Illustrated in (b) are genotypes of the F1 rats and the F2 rats.
[0067] FIG. 6 is a view illustrating a method of identifying genotypes of the Scn1a gene and the Cacna1a gene of the F2 rat, by sequencing.
[0068] FIG. 7 is a view illustrating a method of identifying a genotype of the Scn1a gene of the F2 rat, by restriction enzyme digestion. Illustrated in (a) is a nucleotide sequence of where mutation is on a mutant Scn1a gene (N1417H), and a nucleotide sequence of a wild-type Scn1a gene corresponding to that nucleotide sequence of the mutant Scn1a gene. Illustrated in (b) is a size of a DNA fragment expected by the restriction enzyme digestion. Illustrated in (c) is a result of electrophoresis.
[0069] FIG. 8 is a view illustrating a method of identifying a genotype of the Cacna1a gene in a F2 rat, by restriction enzyme digestion. Illustrated in (a) is a nucleotide sequence of where a mutation is on a mutant Cacna1a gene (M251K), and a nucleotide sequence of a wild-type Cacna1a gene corresponding to that nucleotide sequence of the mutant Cacna1a gene. Illustrated in (b) is a size of a DNA fragment expected by the restriction enzyme digestion. Illustrated in (c) is a result of electrophoresis.
[0070] FIG. 9 is a view illustrating a result of examining an effect of a mutation on the Cacna1a gene, in a rat having a mutation on Scn1a gene. Illustrated in (a) is a body temperature at a time of convulsion onset (convulsion threshold), illustrated in (b) is a severity score, and illustrated in (c) is duration of the convulsion.
[0071] FIG. 10 is a view illustrating a part of an electroencephalogram at a time of seizure of a rat in group (3) (Scn1a mutant (homo)+Cacna1a mutant (hetero)).
[0072] FIG. 11 is a view illustrating an amino acid sequence of a protein encoded by a human CACNA1A gene and an amino acid sequence of a protein encoded by a rat Cacna1a gene.
[0073] FIG. 12 is a view illustrating a result of detecting a mutation on voltage-gated calcium ion channel CaV2.1 a 1 subunit. Illustrated in (a) is a result of a mutation analysis of the CACNA1A gene, and schematically illustrated in (b) is a part where a mutation was detected in the calcium ion channel α1 subunit.
[0074] FIG. 13 is a view illustrating a result of performing function analysis of the calcium ion channel, by use of patch clamping. Illustrated in (a) is a barium current record effected by a change in potential of a normal calcium ion channel and a mutant calcium ion channel. Illustrated in (b) is a current-voltage relationship, and illustrated in (c) is peak current value (pA), a total charge (pF) and a peak current density (pA/pF).
[0075] FIG. 14 is a view illustrating a result of performing function analysis of a calcium ion channel, by use of patch clamping. Illustrated in (a) is an activation curve of the calcium ion channel. Illustrated in (b) is a time constant of voltage-gated activation of the calcium ion channel. Illustrated in (c) is a time constant of voltage-gated activation at 20 mV. Illustrated in (d) is a voltage-gated inactivation curve of the calcium ion channel. Illustrated in (e) is a result of examining fast and slow inactivation time constants (τ).
DESCRIPTION OF EMBODIMENTS
[0076] Described below is an embodiment of the present invention in detail. The present invention is not limited to this embodiment however, and may be carried out in modes of various modifications that are made within the described scope. Moreover, all academic literature and patent literature disclosed in the present specification are incorporated as reference. Unless mentioned otherwise, numerical ranges expressed as "A to B" denote "not less than A but not more than B".
[0077] 1. Assessment method according to the present invention
[0078] A method of assessing a potential for development of Dravet syndrome according to the present invention (also referred to as "assessment method according to the present invention") is a method of assessing a potential for development of Dravet syndrome in a subject, by use of a sample taken from the subject. In the present specification, the "potential for development of Dravet syndrome" includes a potential that the Dravet syndrome is already developed and a potential that the Dravet syndrome may develop in the future.
[0079] The subject is not particularly limited, and may be an individual in which Dravet syndrome has developed (individual having potential for development) or may be an individual in which the Dravet syndrome is not developed (individual having no potential for development). Out of such individuals, it is preferable that the subject is of either infants or children.
[0080] The assessment method according to the present invention, more specifically, may be of any method as long as it includes, with use of a sample taken from the subject: detecting whether or not a mutation is on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and detecting whether or not a mutation is on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. Any other specific configurations are not limited in particular.
[0081] In the embodiment, the voltage-gated sodium ion channel NaV1.1 is made up of α-subunit type 1, β1 subunit, and β2 subunit. The β1 subunit and the β2 subunit are auxiliary subunits.
[0082] The α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 (hereinafter, referred to as "sodium ion channel α1 subunit") is for example a polypeptide that is registered as GenBank accession No. AB093548 (i.e. amino acid sequence represented by SEQ ID NO. 1). Moreover, an example of a gene that encodes the α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 (hereinafter, called "sodium ion channel α1 subunit gene") is, as a SCN1A gene, a polynucleotide made up of a nucleotide sequence registered as GenBank accession No. AB093548 (i.e. nucleotide sequence represented by SEQ ID NO. 2).
[0083] The voltage-gated calcium ion channel CaV2.1 is made up of α-subunit type 1, β subunit, γ subunit, and α2δ subunit.
[0084] The voltage-gated calcium ion channel CaV2.1 α-subunit type 1 (hereinafter, referred to as "calcium ion channel al subunit") is for example a polypeptide registered as GenBank accession No. NM 023035 (i.e. amino acid sequence represented by SEQ ID NO. 3). Moreover, an example of a gene that codes the α-subunit type 1 of voltage-gated calcium ion channel CaV2.1 (hereinafter, referred to as "calcium ion channel α1 subunit gene") is, as a CACNA1A gene, a polynucleotide made up of a nucleotide sequence registered as GenBank accession No. NM 023035 (i.e. nucleotide sequence represented by SEQ ID NO. 4).
[0085] In the present specification, for example, the term "α-subunit type 1 of voltage-gated sodium ion channel NaV1.1" denotes "α-subunit type 1 protein of voltage-gated sodium ion channel NaV1.1". Namely, in the present specification, unless it is clearly described as indicating a gene as like "gene encoding α-subunit type 1 of voltage-gated sodium ion channel NaV1.1" or "α-subunit type 1 gene of voltage-gated sodium ion channel NaV1.1", a protein is denoted. This way of description is not limited to the "α-subunit type 1 of voltage-gated sodium ion channel NaV1.1", and "α-subunit type 1 of voltage-gated calcium ion channel CaV2.1" is denoted similarly thereto.
[0086] It is preferable that the assessment method according to the present invention further includes, in addition to the detecting the presence of a mutation: detecting a change in activity of the voltage-gated sodium ion channel NaV1.1; and detecting a change in activity of the voltage-gated calcium ion channel CaV2.1.
[0087] The assessment method according to the present invention may include, for detecting the mutation, a step such as preprocessing of a sample that is taken from the living organism. The "preprocessing" indicates, for example, a process of extracting DNA from the sample taken from the living organism, a process of extracting RNA from the sample taken from the living organism, a process of extracting protein from the sample taken from the living organism, or like process. These preprocessing can be carried out by use of conventionally known methods.
[0088] The assessment method according to the present invention may be a method of obtaining data for assessing a potential for development of Dravet syndrome. In this case, the present invention does not include the step of determining by a doctor.
[0089] (1-1. Detecting Presence of Mutation)
[0090] In the present specification, the "detecting presence of a mutation" denotes detecting a presence of a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and detecting a presence of a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
[0091] In the assessment method according to the present invention, the detecting of the presence of a mutation on the α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 may be performed prior to the detecting of the presence of a mutation on the α-subunit type 1 of voltage-gated calcium ion channel CaV2.1 or vice versa, or may be performed simultaneously.
[0092] By detecting the presence of a mutation in both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit, it is possible to obtain the data that enables accurate assessment of the potential for development of Dravet syndrome.
[0093] The mutation detected by the assessment method according to the present invention may be a mutation on a nucleotide sequence of a gene, or may be a mutation on an amino acid of a protein. The "mutation on a nucleotide sequence of a gene" is not limited in particular by a specific kind of mutation as long as it is a mutation that causes a change in an amino acid sequence of a protein encoded by a gene having a mutation on its nucleotide sequence as compared to an amino acid sequence of a protein encoded by a wild-type gene. Mutations on the nucleotide sequence as described above are, for example, missense mutation (substitution of an amino acid), nonsense mutation (synthesis of an amino acid stops in an incomplete state), frameshift (a frame of an amino acid codon shifts caused by insertion or deletion of a nucleotide, which causes an amino acid sequence downstream of the mutation position to change, thereby losing its original function), splicing defect (e.g. deletion of its exon region), minority nucleotide insertion or deletion (a part of amino acids is newly added or lost however its downstream is synthesized as normal amino acid), and minor deletion of an exon region (loss of one or a plurality of exon). Variations on the nucleotide sequence as such are not limited to mutations, and may also include gene polymorphism.
[0094] Moreover, in the assessment method according to the present invention, the detection of mutation may be performed to mRNA, cDNA, and proteins obtained from these genes.
[0095] In the present specification, "gene" can be replaced by "polynucleotide", "nucleic acid" or "nucleic acid molecule".
[0096] The "polynucleotide" means a polymer of a nucleotide. Hence, the term "gene" in the present specification includes not only the double stranded DNA but also a single stranded DNA and RNA (mRNA, etc.) such as a sense strand and an antisense strand that construct the double stranded DNA.
[0097] The term "DNA" encompasses cDNA, genomic DNA and the like that can be obtained by cloning, a chemically synthesized technique or a combination of these. Namely, DNA may be a "genome" type DNA, which includes a noncoding sequence such as intron or the like that is a form included in an animal genome, or may be a cDNA obtained from mRNA with use of reverse transcriptase or polymerase, i.e. "transcription" type DNA that does not include a noncoding sequence such as intron.
[0098] Examples of the mutation on sodium ion channel a 1 subunit is, more specifically, a mutation of asparagine (N) at position 1417 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, and is preferably a mutation of asparagine (N) at position 1417 to histidine (H) ("N1417H" in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 4249 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 4249 with cytosine (C) (A4249C).
[0099] Moreover, another embodiment is a mutation of lysine (K) at position 1027 of the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of lysine (K) at position 1027 to a stop codon ("K1027X" in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 3079 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 3079 with thymine (T) (A3079T).
[0100] Yet another embodiment is a mutation of glutamine (Q) at position 1450 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of glutamine (Q) at position 1450 to arginine (R) ("Q1450R" in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 4349 of a nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 4349 with guanine (G) (A4349G).
[0101] Yet another embodiment is a mutation of threonine (T) at position 1082 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1086 by frameshift ("T1082fsX1086" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 3245 of a nucleotide sequence of sodium ion channel a 1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 3245 (C3245de1).
[0102] Yet another embodiment is a mutation of lysine (K) at position 547 of the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 570 by frameshift ("K547fsX570" in Table 1). This mutation is caused by, for example, a mutation at position 1641 of the nucleotide sequence of the sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably an insertion of adenine (A) into position 1641 (1641insA).
[0103] Yet another embodiment is a mutation of proline (P) at position 707 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 714 by frameshift ("P707fsX714" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2120 in the nucleotide sequence of sodium ion channel al subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 2120 (C2120de1).
[0104] Yet another embodiment is a mutation of arginine (R) at position 712 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of arginine (R) at position 712 to a stop codon ("R712X" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2134 of the nucleotide sequence of the sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 2134 with thymine (T) (C2134T).
[0105] Yet another embodiment is a mutation of leucine (L) at position 1265 of the amino acid sequence of the sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a mutation of leucine (L) at position 1265 to proline (P) ("L1265P" in Table 1). This mutation is caused by, for example, a mutation of thymine (T) at position 3794 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of thymine (T) at position 3794 with cytosine (C) (T3794C).
[0106] Yet another embodiment is a deletion of amino acid of positions 460 to 554 of the amino acid sequence of the sodium ion channel α 1 subunit represented by SEQ ID NO. 1 ("Exon10" in Table 1). This mutation is caused by, for example, a deletion of nucleotide at positions 1378 to 1662 (exon 10) of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2.
[0107] Yet another embodiment is a mutation of arginine (R) at position 865 of the amino acid sequence of the sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a mutation of arginine (R) at position 865 to a stop codon ("R865X" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2593 of the nucleotide sequence of the sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 2593 with thymine (T) (C2593T).
[0108] Yet another embodiment is a mutation of arginine (R) at position 1648 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a substitution of arginine (R) at position 1648 with cysteine (C) ("R1648C" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4942 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 4942 with thymine (T) (C4942T).
[0109] Yet another embodiment is a mutation of arginine (R) at position 931 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of arginine (R) at position 931 with cysteine (C) ("R931C" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2791 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 2791 with thymine (T) (C2791T).
[0110] Yet another embodiment is a mutation of arginine (R) at position 501 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 543 by frameshift ("R501fsX543" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 1502 of the nucleotide sequence of sodium ion channel al subunit gene represented by SEQ ID NO. 2, preferably a deletion of guanine (G) at position 1502 (G1502de1).
[0111] Yet another embodiment is a mutation of alanine (A) at position 1002 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1009 by frameshift ("A1002fsX1009" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 3006 of the nucleotide sequence of sodium ion channel al subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 3006.
[0112] Yet another embodiment is a mutation of phenylalanine (F) at position 902 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of phenylalanine (F) at position 902 to cysteine (C) ("F902C" in Table 1). This mutation is caused by, for example, a mutation of thymine (T) at position 2705 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of thymine (T) at position 2705 with guanine (G) (T2705G).
[0113] Yet another embodiment is a mutation of glycine (G) at position 1674 of the amino acid sequence of aodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of glycine (G) at position 1674 with arginine (R) ("G1674R" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 5020 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 5020 with cytosine (C) (G5020C).
[0114] Yet another embodiment is a mutation of valine (V) at position 1390 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of valine (V) at position 1390 to methionine (M) ("V1390M" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 4168 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 4168 with adenine (A) (G4168A).
[0115] Yet another embodiment is a mutation of serine (S) at position 607 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 622 by frameshift ("S607fsX622" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 1820 of the nucleotide sequence of sodium ion channel al subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 1820 (C1820de1).
[0116] Yet another embodiment is a mutation of tryptophan (W) at position 1434 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a substitution of tryptophan (W) at position 1434 with arginine (R) ("W1434R" in Table 1). This mutation is caused by a mutation of thymine (T) at position 4300 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of thymine (T) at position 4300 with cytosine (C) (T4300C).
[0117] Yet another embodiment is a mutation of threonine (T) at position 1909 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a substitution of threonine (T) at position 1909 with isoleucine (I) ("T1909I" in Table 1). This mutation is caused by, for example, the mutation of cytosine (C) at position 5726 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 5726 with thymine (T) (C5726T).
[0118] Yet another embodiment is a mutation of phenylalanine (F) at position 1289 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a deletion of phenylalanine (F) at position 1289 ("F1289de1" in Table 1). This mutation is caused by, for example, mutations of cytosine (C) at position 3867, thymine (T) at position 3868, and thymine (T) at position 3869, each in the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 3867, thymine (T) at position 3868, and thymine (T) at position 3869.
[0119] Yet another embodiment is a mutation of tryptophan (W) at position 1271 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of tryptophan (W) at position 1271 to a stop codon ("W1271X" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 3812 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of guanine (G) at position 3812 with adenine (A) (G3812A).
[0120] Yet another embodiment is a mutation of alanine (A) at position 1429 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1443 by frameshift ("A1429fsX1443" in Table 1). This mutation is caused by, for example, a mutation of five-nucleotide CCACA between positions 4286 to 4290 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of CCACA at positions 4286 to 4290, with ATGTCC.
[0121] Moreover, another embodiment is a mutation of glycine (G) at position 1880 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1881 by frameshift ("G1880fsX1881" in Table 1). This mutation is caused by mutation of six-nucleotide AGAGAT between positions 5640 to 5645 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of six-nucleotide AGAGAT between positions 5640 to 5645 with CTAGAGTA.
[0122] Yet another embodiment is a mutation of alanine (A) at position 1685 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of alanine (A) at position 1685 with aspartic acid (D) ("A1685D" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 5054 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 5054 with adenine (A) (C5054A).
[0123] Yet another embodiment is a mutation of arginine (R) at position 377 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of arginine (R) at position 377 with leucine (L) ("R377L" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 1130 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by substitution of guanine (G) at position 1130 with thymine (T) (G1130T).
[0124] Yet another embodiment is a mutation of serine (S) at position 1574 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a mutation of serine (S) at position 1574 to a stop codon ("51574X" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4721 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 4721 with guanine (G) (C4721G).
[0125] Yet another embodiment is a mutation of glutamine (Q) at position 1277 in the amino acid sequence of the sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a mutation of glutamine (Q) at position 1277 to a stop codon ("Q1277X" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 3829 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 3829 with thymine (T) (C3829T).
[0126] Yet another embodiment is a mutation of glycine (G) at position 177 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of glycine (G) at position 177 to arginine (R) ("G 177R" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 529 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of guanine (G) at position 529 with adenine (A) (G529A).
[0127] Yet another embodiment is a mutation of glutamic acid (E) at position 788 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a substitution of glutamic acid (E) at position 788 with lysine (K) ("E788K" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 2362 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of guanine (G) at position 2362 with adenine (A) (G2362A).
[0128] Yet another embodiment is splicing defects at positions 1429 and subsequent positions of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a deletion of positions at and subsequent to 1429 ("intron 21" in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at a second last position (position -2), preferably a mutation in which adenine (A) at a second last position (position -2) of the intron 21 is substituted with guanine (G) (intron 21 ag(-2)gg), out of the intron 21 present in a genomic DNA between positions 4284 and 4285 of the nucleotide sequence of sodium ion channel a 1 subunit gene represented by SEQ ID NO. 2. Namely, the second last nucleotide sequence of the intron 21 present in the genomic DNA between positions 4284 (exon 21) and 4285 (exon 22) of the nucleotide sequence of sodium ion channel a 1 subunit gene represented by SEQ ID NO. 2 is ag, and is connected to the beginning of the exon 22. Generally, since the ag of the intron 21 is a recognition sequence that is spliced, in a case in which an abnormality exists at that position, the intron is determined as still continuing, which thus causes the exon immediately after (or in its downstream) to be abnormally spliced. This makes it impossible to generate a full-length protein.
[0129] Yet another embodiment is a mutation of serine (S) at position 1574 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a mutation of serine (S) at position 1574 to a stop codon ("51574X" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4721 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 4721 with guanine (G).
[0130] Yet another embodiment is a mutation of valine (V) at position 212 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of valine (V) at position 212 with alanine (A) ("V212A" in Table 1). This mutation is caused by, for example, a mutation of thymine (T) at position 635 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of thymine (T) at position 635 with cytosine (C) (T635C).
[0131] Yet another embodiment is a mutation of threonine (T) at position 1539 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a mutation of threonine (T) at position 1539 to proline (P) ("T1539P" in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 4615 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 4615 with cytosine (C) (A4615C).
[0132] Yet another embodiment is a mutation of tryptophan (W) at position 738 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably by mutation causing generation of a stop codon at position 746 by frameshift ("W738fsX746" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 2213 in the nucleotide sequence of the sodium ion channel a 1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of guanine (G) at position 2213 (G2213de1).
[0133] Yet another embodiment is a mutation of leucine (L) at position 990 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably by a mutation of leucine (L) at position 990 to phenylalanine (F) ("L990F" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 2970 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 2970 with thymine (T) (G2970T).
[0134] Yet another embodiment is a mutation of glycine (G) at position 163 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of glycine (G) at position 163 to glutamic acid (E) ("G163E" in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 488 of the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 488 with adenine (A) (G488A).
[0135] Yet another embodiment is a mutation of alanine (A) at position 1662 of the amino acid sequence of sodium ion channel α 1 subunit represented by SEQ ID NO. 1, preferably a mutation of alanine (A) at position 1662 to valine (V) ("A1662V" in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4985 in the nucleotide sequence of sodium ion channel α 1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 4985 with thymine (T) (C4985T).
[0136] Yet another embodiment is a mutation of lysine (K) at position 1057 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1073 by frameshift ("K1057fsX1073" in Table 1). This mutation is caused by, for example, a mutation of 14 nucleotides (AGAAAGACAGTTGT) between positions 3170 to 3183 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of the 14 nucleotides between the positions 3170 to 3183 with TCATTCTGTATG.
[0137] It is needless to say that the mutation on the α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 is not limited to the mutations exemplified above.
[0138] Examples of mutations on a calcium ion channel al subunit encompass, more specifically, a mutation on methionine (M) at position 249 of an amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on methionine (M) at position 249 to lysine (K) ("M249K" in Table 2). This mutation is caused by, for example, a mutation on thymidine (T) at position 746 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a mutation on thymidine (T) at position 746 substituted with adenine (A) (T746A).
[0139] Moreover, another embodiment is a mutation on glutamic acid (E) at position 921 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation on glutamic acid (E) at position 921 to aspartic acid (D) ("E921D" in Table 2). This mutation is, for example, caused by a mutation on adenine (A) at position 2762 of the nucleotide sequence of calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of adenine (A) at position 2762 with cytosine (C) (A2762C).
[0140] Yet another embodiment is a mutation on glutamic acid (E) at position 996 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation on glutamic acid (E) at position 996 to valine (V) ("E996V" in Table 2). This mutation is, for example, caused by a mutation on adenine (A) at position 2987 of the nucleotide sequence of the calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of adenine (A) at position 2987 with thymine (T) (A2987T).
[0141] Yet another embodiment is a mutation on arginine (R) at position 1126 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation on arginine (R) at position 1126 to histidine (H) ("R1126H" in Table 2). This mutation is, for example, caused by a mutation on guanine (G) at position 3377 of the nucleotide sequence of calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of guanine (G) at position 3377 with adenine (A) (G3377A).
[0142] Yet another embodiment is a mutation on arginine (R) at position 2201 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation on arginine (R) at position 2201 to glutamine (Q) ("R2201Q" in Table 2). This mutation is, for example, caused by mutation on guanine (G) at position 6602 of the nucleotide sequence of calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably by a substitution of guanine (G) at position 6602 with adenine (A) (G6602A).
[0143] Yet another embodiment is a mutation on glycine (G) at position 1108 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation on glycine (G) at position 1108 to serine (S) ("G1108S" in Table 2). This mutation is, for example, caused by a mutation on guanine (G) at position 3322 of the nucleotide sequence of calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of guanine (G) at position 3322 with adenine (A) (G3322A).
[0144] Yet another embodiment is a mutation on alanine (A) at position 924 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation of alanine (A) at position 924 to glycine (G) ("A924G" in Table 2). This mutation is, for example, caused by a mutation on cytosine (C) at position 2771 of the nucleotide sequence of calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of cytosine (C) at position 2771 with guanine (G) (C2771G).
[0145] Yet another embodiment is a mutation on glycine (G) at position 266 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation on glycine (G) at position 266 to serine (S) ("G2665" in Table 2). This mutation is, for example, caused by a mutation on guanine (G) at position 796 of the nucleotide sequence of calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably by a substitution of guanine (G) at position 796 with adenine (A) (G796A).
[0146] Yet another embodiment is a mutation on lysine (K) at position 472 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3, preferably a mutation on lysine (K) at position 472 to arginine (R) ("K472R" in Table 2). This mutation is, for example, caused by a mutation on adenine (A) at position 1415 of the nucleotide sequence of calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4, preferably by a substitution of adenine (A) at position 1415 with guanine (G) (A1415G).
[0147] Yet another embodiment is a deletion of an amino acid at positions 2202 to 2205 of the amino acid sequence of calcium ion channel α 1 subunit represented by SEQ ID NO. 3 ("de12202-2205" in Table 2). This mutation is, for example, caused by a mutation on ACCAGGAGCGGG of positions 6605 to 6616 of the nucleotide sequence of calcium ion channel a 1 subunit gene represented by SEQ ID NO. 4, preferably a deletion of ACCAGGAGCGGG at positions 6605 to 6616 (de16605-6616).
[0148] It is needless to say that the mutations related to the function abnormality of voltage-gated calcium ion channel CaV2.1 is not limited to the mutations exemplified above.
[0149] The mutations on the foregoing sodium ion channel a 1 subunit and the mutations on the foregoing calcium ion channel α 1 subunit are organized into Table 1 and Table 2.
TABLE-US-00001 TABLE 1 Mutations on sodium ion channel α1 subunit 1289de1F, G177R, Q1450R, T1539P, A1002fsX1009, G1880fsX1881, R1648C, T1909I, A1429fsX1443, intron 21, R377L, V1390M, A1662V, K1027X, R501fsX543, V212A, A1685D, K1057fsX1073, R712X, W1271X, E788K, K547fsX570, R865X, W1434R, Exon10*, L1265P, R931C, W738fsX746, F902C, L990F, S1574X, N1417H, G163E, P707fsX714, S607fsX622, G1674R, Q1277X, T1082fsX1086, Exon10* exon deletion detected by MLPA
TABLE-US-00002 TABLE 2 Mutations on calcium ion channel α1 subunit A924G, E996V, K472R, del 2202-2205, G1108S, R1126H, E921D, G266S, R2201Q, M249K
[0150] In the assessment method according to the present invention, it is preferable that the mutation on sodium ion channel α 1 subunit is, more specifically, at least one mutation shown in Table 1, and the mutation on calcium ion channel al subunit is, more specifically, at least one mutation shown in Table 2.
[0151] The assessment method according to the present invention is not limited in particular of how the presence of a mutation is detected for both the sodium ion channel a 1 subunit and the calcium ion channel α 1 subunit, and any method conventionally known may be used.
[0152] Examples of methods for detecting the presence of the mutation for both the sodium ion channel α 1 subunit gene and the calcium ion channel α 1 subunit gene encompass mutation detecting methods such as DNA sequencing method using PCR, SSCP method (Single strand conformation polymorphism), DHPLC method (denaturing high performance liquid chromatography); polymorphism detecting methods using real-time PCR or DNA chip; method of detecting micro-deletion of exons of a gene; and Northern blotting, RT-PCR, Real-time PCR, and cDNA array, each of which detect an increase and decrease of mRNA. Moreover, when the presence of mutation is to be detected for both of sodium ion channel α 1 subunit protein and calcium ion channel α 1 subunit protein, a method such as Western blotting, immunostaining, protein array or the like may be used.
[0153] The following provides more specific descriptions, by separating into the following embodiments: (A) an embodiment detecting a gene mutation with use of a genomic DNA included in a sample taken from a subject, (B) an embodiment detecting a gene mutation with use of mRNA (cDNA) included in a sample taken from a subject, and (C) an embodiment detecting a protein mutation with use of a protein included in a sample taken from a subject.
[0154] (A) Embodiment Using Genomic DNA
[0155] In the embodiment detecting a gene mutation with use of a genomic DNA included in a sample taken from a subject, first, a genomic DNA is extracted from the sample taken from the subject, by a conventionally known method.
[0156] The "sample taken from the subject" is not limited in particular, and any sample from which a genomic DNA is extractable can be used. More specifically, a sample of blood, oral mucosa cells, bone marrow fluid, hair, various organs, peripheral lymphocytes, synovial cells or the like can be used. Moreover, cells taken from the subject may be cultured and a genomic DNA may be extracted from its proliferated cells.
[0157] Moreover, the extracted genomic DNA may be used upon amplification by a gene amplification method generally performed, for example, PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequence based amplification), TMA (Transcription-mediated amplification), SDA (Strand Displacement Amplification), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids).
[0158] The method of detecting the presence of mutation for both the sodium ion channel α 1 subunit gene and the calcium ion channel α 1 subunit gene with use of a sample including a genomic DNA prepared as such is not limited in particular, and examples encompass allele-specific oligonucleotide probe method, Oligonucleotide Ligation Assay, PCR-SSCP, PCR-CFLP, PCR-PHFA, invader method, RCA (Rolling Circle Amplification), Primer Oligo Base Extension, and like methods.
[0159] More specifically, a polynucleotide for detecting a mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and a polynucleotide for detecting a mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 are used to detect, from the genomic DNA, the presence of a mutation for both the sodium ion channel α 1 subunit gene and the calcium ion channel α1 subunit gene.
[0160] The "polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1" is indicative of a polynucleotide having a nucleotide sequence complementary to a set region in a sodium ion channel al subunit gene (e.g. a region including an exon, or boundary region between an exon and an intron). The "polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1" is indicative of a polynucleotide having a nucleotide sequence complementary to a set region in the calcium ion channel α1 subunit gene (e.g. a region including an exon, or a boundary region between an exon and an intron).
[0161] The "polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1" is, more specifically, a polynucleotide having a nucleotide sequence represented by any one of SEQ ID NOs.: 5, 6, and 9 to 62, for example. Moreover, the "polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1" is, more specifically, a polynucleotide having a nucleotide sequence represented by any one of SEQ ID NOs.: 7, 8, and 63 to 143.
[0162] Two kinds of the polynucleotides may be used in combination as a primer pair, or one kind may be used as a probe. When the two kinds are used in combination as a primer pair, the polynucleotides may be used in combinations as exemplified in Examples described later.
[0163] When two kinds of the polynucleotides are used in combination as a primer pair, it is possible, for example, to amplify a set region in the gene by PCR with use of a corresponding primer pair, and thereafter, directly sequence the obtained PCR product, to detect the presence of the mutation in the gene.
[0164] Moreover, two kinds of fluorescence-labeled polynucleotides may be used as a primer pair, to amplify a set region of the gene by PCR, perform gel electrophoresis or capillary electrophoresis with the obtained PCR product, and study a strength of the signals, so as to detect the presence of a mutation in the gene.
[0165] Moreover, when one kind of the polynucleotides is to be solely used as a probe, the presence of the mutation on the gene can be detected by, for example, digesting the genomic DNA with an appropriate restriction enzyme and detecting a difference in size of the digested genomic DNA fragment by Southern blotting or the like.
[0166] As such, by detecting the presence of mutations for both the sodium ion channel α 1 subunit gene and calcium ion channel α 1 subunit gene with use of the genomic DNA included in the sample taken from the subject, it is possible to obtain data for assessing a potential for development of Dravet syndrome in the subject. More specifically, when a mutation is found on both the sodium ion channel α 1 subunit gene and the calcium ion channel α 1 subunit gene in the obtained data, it can be assessed that the subject has a high potential for development of Dravet syndrome.
[0167] The primer pair and probe used in the method of detecting the mutation may be prepared by a DNA synthesizer or the like, as in law of the art.
[0168] (B) Embodiment Using mRNA (cDNA)
[0169] In the embodiment of detecting a mutation with use of mRNA included in a sample taken from the subject, first, mRNA is extracted from a sample taken from the subject, with use of a conventionally known method.
[0170] The "sample taken from the subject" is not limited in particular, and any sample can be used as long as mRNA can be extracted therefrom and a gene that can be subjected to the detection of a mutation is expressed or is possibly expressed. The "sample taken from the subject" is preferably, for example, a peripheral blood leukemic cell, dermal fibroblast, oral mucosa cell, neuron, or muscle cell, each of a patient.
[0171] Subsequently, cDNA is prepared from the extracted mRNA by reverse transcription reaction. Furthermore, if necessary, the obtained cDNA may be amplified by a gene amplification method generally performed, for example PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequence based amplification), TMA (Transcription-mediated amplification), SDA (Strand Displacement Amplification), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids).
[0172] The method of detecting the presence of the mutation for both the sodium ion channel α 1 subunit gene and calcium ion channel α 1 subunit gene with use of a sample including cDNA prepared as such is not limited in particular; whether or not a gene mutation is present in a subject that is subjected to mutation detection may be detected with use of a similar method as with a case in which a gene mutation is detected with use of a genomic DNA, as described in the foregoing "(A) Embodiment using genomic DNA".
[0173] By detecting the presence of the mutation for both the sodium ion channel α 1 subunit gene and calcium ion channel α1 subunit gene with use of mRNA included in the sample that is taken from the subject, it is possible to obtain data for assessing a potential for development of Dravet syndrome in the subject. More specifically, when a mutation is found in both the sodium ion channel α 1 subunit gene and the calcium ion channel α 1 subunit gene in the obtained data, it can be assessed that the subject has a high potential for the development of Dravet syndrome.
[0174] (C) Embodiment Using Protein
[0175] In the embodiment of detecting a mutation using protein included in the sample taken from a subject, first, protein is extracted from the sample taken from the subject with use of a conventionally known method.
[0176] The sample taken from the subject is not limited in particular, and may be any sample from which protein is extractable and in which both of sodium ion channel a 1 subunit protein and calcium ion channel α 1 subunit protein are expressed or is possibly expressed.
[0177] The method of detecting the presence of mutation for both the sodium ion channel α 1 subunit protein and the calcium ion channel α 1 subunit protein with use of the sample including the protein prepared as described above is not limited in particular, and for example an antibody which specifically recognizes just a protein having a set mutation may be prepared, to detect the mutation by ELISA or Western blotting using that antibody. In the present specification, the term "protein" may be used replaceable with "polypeptide" or "peptide".
[0178] Moreover, mutation may be detected by isolating a protein to be subjected to the mutation detection from the sample including the foregoing protein, and digesting the isolated protein with an enzyme or the like directly or if necessary, with use of a protein sequencer or a mass spectrometer. Alternatively, the mutation may be detected on the basis of an isoelectric point of the isolated protein.
[0179] As such, by detecting the presence of a mutation for both of the sodium ion channel α1 subunit protein and the calcium ion channel α1 subunit protein with use of a protein included in the sample taken from the subject, it is possible to obtain data for assessing potential for development of Dravet syndrome in the subject. More specifically, when a mutation is found on both the sodium ion channel α1 subunit protein and the calcium ion channel α 1 subunit protein in the obtained data, it is possible to assess that the subject has a high potential for development of Dravet syndrome.
[0180] (1-2. Step of Detecting Change in Activity)
[0181] In the present specification, the "step of detecting change in activity" is indicative of a step of detecting whether activity of the voltage-gated sodium ion channel NaV1.1 has changed and a step of detecting whether activity of the voltage-gated calcium ion channel CaV2.1 has changed.
[0182] As described in Examples later described, it is considered that the change in activity in both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1, caused by the mutations on the sodium ion channel α1 subunit and on the calcium ion channel α1 subunit, is related to the development of Dravet syndrome. Hence, although the mutation on the sodium ion channel α1 subunit is not particularly limited in its position, it is preferable that the mutation is on a position that causes a change in the activity of the voltage-gated sodium ion channel NaV1.1. Moreover, although the mutation on the calcium ion channel α1 subunit is not particularly limited in its position, it is preferable that the mutation is on a position that causes a change in the activity of the voltage-gated calcium ion channel CaV2.1.
[0183] Here, the activity of the voltage-gated sodium ion channel NaV1.1 is, more specifically, an activity to allow transmission of sodium ion (Na+) into the cell by depending on membrane potential. The change in activity of the voltage-gated sodium ion channel NaV1.1 is not limited in particular, and may be an increase of activity or may be a decrease in activity. Namely, the change is sufficiently one that shows an abnormality in the activity of the voltage-gated sodium ion channel NaV1.1.
[0184] In the present specification, "the activity of the voltage-gated sodium ion channel NaV1.1 is changed" indicates that an activity of a mutant voltage-gated sodium ion channel NaV1.1 including the sodium ion channel α1 subunit on which the mutation is present is of a value having a statistically significant difference based on a significant test as compared to an activity of a wild-type voltage-gated sodium ion channel NaV1.1, and preferably indicates that p is equal to or smaller than 0.05 by Student's t-test.
[0185] Moreover, the activity of the voltage-gated calcium ion channel CaV2.1 is, more specifically, an activity that causes transmission of calcium ion (Ca2+) into the cell to be membrane voltage-gated. The change in function of the voltage-gated calcium ion channel CaV2.1 is not particularly limited, and may be the increase of activity or the decrease in activity. Namely, the change is sufficiently one that shows abnormality of the activity of the voltage-gated calcium ion channel CaV2.1.
[0186] In the present specification, "the activity of the voltage-gated calcium ion channel CaV2.1 is changed" indicates that the activity of a mutant voltage-gated calcium ion channel CaV2.1 including the calcium ion channel al subunit on which a mutation is present is of a value having a statistically significant difference based on a significant test as compared to an activity of a wild-type voltage-gated calcium ion channel CaV2.1, and preferably indicates that p is equal to or smaller than 0.05 by Student's t-test.
[0187] An example of a method of detecting that the activity of the voltage-gated sodium ion channel NaV1.1 is changed by the mutation is, for example, (i) coexpressing, in a culture cell with use of a expression vector or the like, a sodium ion channel α1 subunit gene on which a mutation is present with a wild-type gene (β1 subunit gene and β2 subunit gene) that encodes a subunit (β1 subunit and β2 subunit) other than the α1 subunit, which wild-type gene makes up the voltage-gated sodium ion channel NaV1.1, (ii) measuring an activity of the voltage-gated sodium ion channel NaV1.1 on which a mutation is present with use of the obtained cultured cell, and (iii) comparing the activity with an activity of the wild-type voltage-gated sodium ion channel NaV1.1, to confirm whether the activity of the voltage-gated sodium ion channel NaV1.1 is changed. The method of measuring the activity of the voltage-gated sodium ion channel NaV1.1 is not particularly limited, however it is possible to use the conventionally known patch clamping, imaging with use of a fluorescence probe, or like method.
[0188] An example of a method of detecting that the activity of the voltage-gated calcium ion channel CaV2.1 is changed by mutation is by (i) coexpressing, in a culture cell with use of an expression vector or the like, a calcium ion channel al subunit gene on which a mutation is present with a wild-type gene (β subunit gene, γ subunit gene, and α2δ subunit gene) that encodes a subunit (β subunit, γ subunit, and α2δ subunit) other than the α1 subunit, which wild-type gene makes up the voltage-gated calcium ion channel CaV2.1, (ii) measuring, with the obtained cultured cell, an activity of the voltage-gated calcium ion channel CaV2.1 on which the mutation is present, and (iii) comparing the activity with an activity of the wild-type voltage-gated calcium ion channel CaV2.1, to confirm whether the activity of the voltage-gated calcium ion channel CaV2.1 is changed. The method of measuring the activity of the voltage-gated calcium ion channel CaV2.1 is not limited in particular, however it is possible to use the conventionally known patch clamping, imaging using an optical probe, a calcium indicator, or a caged compound, for example.
[0189] The assessment method according to the present invention, since it includes the foregoing configuration, it is possible to obtain data for assessing a potential for development of Dravet syndrome in the subject. Hence, with the assessment method according to the present invention, it is possible to find out, with high accuracy and at an early stage, Dravet syndrome having the unfavorable prognosis, which thus allows for preparing a treatment management system by an epilepsy specialist from an earlier stage for a Dravet syndrome patient. As a result, it is possible to improve treatment intervention of the patient, reduce the mental burden on their families, and reduce the economical burden. Furthermore, it is possible to provide appropriate treatment for the patient of Dravet syndrome; this hence reduces medical fees.
[0190] 2. Kit According to the Present Invention
[0191] The present invention also encompasses a kit for assessing the potential for development of Dravet syndrome, with use of the assessment method according to the present invention (hereinafter, also referred simply as "kit according to the present invention").
[0192] The kit according to the present invention is not limited in its specific configuration in particular as long as it includes at least a reagent for detecting the presence of mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and a reagent for detecting the presence of mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
[0193] As described in "1. Assessment method according to the present invention", ways considered to detect the presence of mutation for both of α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 are (A) detecting a gene mutation with use of a genomic DNA included in a sample taken from a subject, or (B) detecting a gene mutation with use of mRNA (cDNA) included in a sample taken from the subject.
[0194] Hence, in order to detect a mutation using a genomic DNA included in the sample taken from the subject or mRNA (cDNA) included in the sample taken from the subject, the kit according to the present invention includes a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. Such polynucleotides can be used as, for example, a primer pair or a probe. These polynucleotides may be included solely or may be included as a combination of a plurality thereof.
[0195] The kit according to the present invention encompasses (A) a kit for detecting a mutation with use of a genomic DNA included in a sample taken from a subject and (B) a kit for detecting a mutation with use of a mRNA (cDNA) included in a sample taken from a subject. The following specifically describes the reagents included in the embodiments of the kits in (A) or (B).
[0196] (A) Kit for detecting mutation with use of genomic DNA included in sample taken from subject
[0197] For example, a configuration of the sodium ion channel α1 subunit and the calcium ion channel α 1 subunit may include a primer pair designed so as to allow amplification of the genomic DNA of each of the genes or a part of its region, or may include a probe designed so that one of genomic DNA of its mutant type or wild-type can be specifically detected. These polynucleotides are as described in the foregoing (A) Embodiment using genomic DNA in "1. Assessment method according to the present invention", so hence its description has been omitted here.
[0198] Furthermore, such a kit may be configured to include, in addition to the primer pair or probe, a combination of one or more reagent necessary for detecting the presence of the mutation on the gene, such as a reagent used in PCR, Southern blotting, and nucleic acid sequencing.
[0199] The reagent is selected and employed as appropriate in accordance with the detection method of the present invention, and examples thereof are dATP, dCTP, dTTP, dGTP, DNA polymerase and the like. Furthermore, the kit according to the present invention may include a suitable buffer solution and a washing solution that can be used in the PCR, Southern blotting, and nucleic acid sequencing.
[0200] (B) Kit detecting mutation with use of mRNA (cDNA) included in sample taken from subject For example, a configuration of the sodium ion channel α1 subunit and the calcium ion channel α 1 subunit may include a primer pair designed so as to allow amplification of the cDNA of each of the genes or a part of its region, or include a probe designed so that one of mRNA of its mutant type or wild-type can be specifically detected. These polynucleotides are as described in (B) Embodiment using mRNA (cDNA) in "1. Assessment method according to the present invention", so hence its description has been omitted here.
[0201] Furthermore, such a kit may be configured to include, in addition to the primer pair or probe, a combination of one or more reagent necessary for detecting the presence of a mutation on the gene, such as a reagent used in RT-PCR, Northern blotting, nucleic acid sequencing or the like.
[0202] The reagent is selected and employed as appropriate in accordance with the detection method of the present invention, and examples thereof are dATP, dCTP, dTTP, dGTP, DNA polymerase and the like. Furthermore, the kit according to the present invention may include a suitable buffer solution and a washing solution that can be used in RT-PCR, Northern blotting, and nucleic acid sequencing.
[0203] The kit according to the present invention may include the exemplified configuration in any combination. Furthermore, the kit may include other reagents other than the reagents exemplified above.
[0204] As described in the item "1. Assessment method according to the present invention", in order to detect the presence of mutation for both the sodium ion channel a 1 subunit and the calcium ion channel α 1 subunit, it is further considerable to (C) detect the mutation with use of a protein included in the sample taken from a subject.
[0205] Therefore, the kit according to the present invention may include, for example, an antibody that specifically bonds to just the wild-type or mutant protein among the proteins of the sodium ion channel α 1 subunit and the calcium ion channel α 1 subunit. Furthermore, the configuration may be one which, in addition to the antibody, includes one or more reagent in combination, which reagent is used for ELISA or Western blotting.
[0206] Furthermore, the kit according to the present invention may include a reagent used for measuring activity of the voltage-gated sodium ion channel NaV1.1, a reagent used for measuring activity of the voltage-gated calcium ion channel CaV2.1, or the like.
[0207] With use of the kit according to the present invention as described above, it is possible to easily obtain data for assessing the potential for development of Dravet syndrome in the subject. A subject to which the kit may be applied is not particularly limited, however is preferably applied to infants or children.
[0208] 3. Model Animal of Dravet Syndrome According to the Present Invention and its Production Method
[0209] The present invention encompasses a model animal of Dravet syndrome, and its production method.
[0210] (3-1. Model Animal of Dravet Syndrome According to the Present Invention)
[0211] The model animal of Dravet syndrome according to the present invention has a mutation on both the sodium ion channel α 1 subunit and the calcium ion channel α 1 subunit. The mutation on the sodium ion channel α 1 subunit and the mutation on the calcium ion channel α 1 subunit are as described in the item "1. Assessment method according to the present invention" described above, so therefore specific descriptions thereof are omitted here.
[0212] It is preferable in the model animal of the Dravet syndrome that both the activity of the voltage-gated sodium ion channel NaV1.1 and the activity of the voltage-gated calcium ion channel CaV2.1 are changed as compared to a wild-type animal. This change in activity is not particularly limited, and may be an increase of activity or may be a decrease in activity. The method of confirming whether or not an activity of the voltage-gated sodium ion channel NaV1.1 of the model animal of Dravet syndrome according to the present invention is changed from that of a wild-type, and the method of confirming whether or not an activity of the voltage-gated calcium ion channel CaV2.1 of the model animal of Dravet syndrome according to the present invention is changed from that of a wild-type, are both not particularly limited. For example, with an individual of a model animal of Dravet syndrome according to the present invention or cells collected from the model animal of Dravet syndrome according to the present invention, confirmation may be made by measuring the activity by use of the conventionally known patch clamping, slice patching, imaging with use of fluorescence probe and like method.
[0213] The model animal of Dravet syndrome according to the present invention has the mutation on both the sodium ion channel α 1 subunit and the calcium ion channel α 1 subunit, so therefore develops Dravet syndrome. Such a model animal of Dravet syndrome can be used advantageously for clarification of the development mechanism of the intractable Dravet syndrome, and for development of medicament for Dravet syndrome.
[0214] In the present specification, "model animal" denotes an experiment animal used for developing a prevention method or treatment against human diseases, and more specifically is a non-human mammal such as a mouse, rat, rabbit, monkey, goat, pig, sheep, cow, or dog, and other vertebrates.
[0215] (3-2. Production Method of Model Animal of Dravet Syndrome According to the Present Invention)
[0216] A method of producing a model animal of Dravet syndrome, according to the present invention, includes: introducing a mutation on sodium ion channel α 1 subunit and introducing a mutation on calcium ion channel α 1 subunit.
[0217] More specifically, a mutation can be introduced on each of the genes by manipulating the gene of the model animal. Here, the "manipulating the gene of the model animal" intends to mean manipulation of a gene of a model animal by use of a conventionally known gene manipulation technique. More specifically, this encompasses all of destruction of a gene of the model animal, an introduction of a mutation to that gene, a substitution of that gene with a mutant gene, and furthermore, introduction of a foreign gene into the model animal, and crossing of model animals.
[0218] The production method according to the present invention of the model animal of Dravet syndrome may include steps other than those described above. Specific steps, materials, conditions, used devices, used equipment and the like are not limited in particular.
[0219] With the production method according to the present invention of a model animal of Dravet syndrome, it is possible to produce a model animal developed in Dravet syndrome by manipulating genes of a model animal so that a mutation is introduced into the genes of the sodium ion channel a 1 subunit and the calcium ion channel α 1 subunit.
[0220] 4. Cells According to the Present Invention and its Production Method
[0221] The present invention also encompasses cells having a mutation on both the sodium ion channel α 1 subunit and the calcium ion channel α 1 subunit, and its production method.
[0222] (4-1. Cell According to the Present Invention)
[0223] The cell according to the present invention is a cell having a mutation on both the sodium ion channel α 1 subunit and the calcium ion channel α 1 subunit. The mutation on the sodium ion channel α 1 subunit and the mutation on the calcium ion channel α 1 subunit are as described in the item "1. Assessment method according to the present invention" described above, so therefore specific description thereof have been omitted here.
[0224] The cell according to the present invention intends to mean experimental culture cells having a mutation on both the sodium ion channel α 1 subunit and the calcium ion channel α1 subunit. More specifically, the cell is an experimental culture cell derived from a mammal such as a human, mouse, rat, hamster, rabbit, monkey and the like, and other vertebrates.
[0225] It is preferable that with such a cell, both of activity of the voltage-gated sodium ion channel NaV1.1 and activity of the voltage-gated calcium ion channel CaV2.1 are changed. This change in activity is not particularly limited, and may be an increase of activity or a decrease in activity. The method of confirming whether or not the activity of the voltage-gated sodium ion channel NaV1.1 of the cell according to the present invention is changed from that of a wild-type, and a method of confirming whether or not the activity of both of the voltage-gated calcium ion channel CaV2.1 of the cell according to the present invention is changed from that of the wild-type are as described in "1. Assessment method according to the present invention" described above, so hence specific description thereof have been omitted here.
[0226] Such a cell can be used for clarification of a development mechanism of the intractable Dravet syndrome, and for the development in medicament for Dravet syndrome. For example, it is possible to suitably use this for screening of a drug for treating Dravet syndrome. Namely, this cell can also be said as a screening cell for a drug for treating Dravet syndrome. Accordingly, the present invention also encompasses a screening cell of a drug for treating Dravet syndrome (hereinafter, simply called "screening cell"), and its production method.
[0227] (4-2. Production Method of Cell According to Present Invention)
[0228] A method of producing a cell according to the present invention is a method of producing a cell that has the foregoing properties, and includes: introducing a mutation on a sodium ion channel α 1 subunit; and introducing a mutation on a calcium ion channel α 1 subunit. More specifically, the following three embodiments can be raised. The following three embodiments are described specifically below, however the present invention is not limited to these.
[0229] (1) Method of Using Expression Vector Etc.
[0230] This method produces a cell that expresses a mutant voltage-gated sodium ion channel NaV1.1 and mutant voltage-gated calcium ion channel CaV2.1, with use of an expression vector or the like. More specifically described, in order to make a cell express the mutant voltage-gated sodium ion channel NaV1.1, for example, a sodium ion channel a 1 subunit gene having a mutation that causes a change in an amino acid is coexpressed, in a culture cell that serves as a host, with a wild-type gene (β1 subunit gene and β2 subunit gene) making up the voltage-gated sodium ion channel NaV1.1, which wild-type gene encodes a subunit other than the α1 subunit (β1 subunit and β2 subunit), with use of an expression vector or the like. This enables the cell to express the mutant voltage-gated sodium ion channel NaV1.1 that includes the mutant sodium ion channel α 1 subunit.
[0231] Similarly, in order to make the cell express the mutant voltage-gated calcium ion channel CaV2.1, for example, a calcium ion channel α 1 subunit gene having a mutation that causes a change in an amino acid is coexpressed, in a culture cell that serves as a host, with a wild-type gene (β subunit gene, γ subunit gene, and α2δ subunit gene) making up a voltage-gated calcium ion channel CaV2.1, which wild-type gene encodes a subunit other than the α 1 subunit (β subunit, γ subunit, and α2δ subunit), with the expression vector or the like. This hence enables the cell to express a mutant voltage-gated calcium ion channel CaV2.1 that includes the mutant calcium ion channel α 1 subunit.
[0232] At this time, it is preferable that the culture cell serving as a host is a cell from which no voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1 is expressed. With use of such a cell, no effect is caused by the residing voltage-gated sodium ion channel NaV1.1 and residing voltage-gated calcium ion channel CaV2.1.
[0233] (2) Method of Using Artificial Mutation Introduction
[0234] This method introduces mutation for both of the sodium ion channel α 1 subunit and the calcium ion channel a 1 in a culture cell expressing both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1.
[0235] The method of introducing the mutation on the culture cell is not particularly limited, and a conventionally known gene manipulation technique is used in combination as appropriate.
[0236] (3) Method of Using Model Animal of Dravet Syndrome According to the Present Invention
[0237] This method extracts a tissue from the model animal of Dravet syndrome according to the present invention as described above, and prepares a culture cell from that tissue. The model animal of Dravet syndrome according to the present invention is as described in "3. Model animal of Dravet syndrome according to the present invention and its production method", and so therefore specific description thereof has been omitted here. Of course, the "tissue" that is extracted is intended to mean a tissue in which both the sodium ion channel α 1 subunit on which a mutation is introduced and the calcium ion channel α 1 subunit on which a mutation is introduced are expressed.
[0238] This hence allows for easy production of a cell that has a mutation on both the sodium ion channel α 1 subunit and the calcium ion channel α 1 subunit. The kinds of tissues extracted from the model animal of Dravet syndrome is not limited in particular, and may be selected as appropriate depending on its purpose.
[0239] The method according to the present invention of producing a cell may include steps other than the steps described above. Specific steps, materials, conditions, used devices, used equipment and the like are not limited in particular.
[0240] 5. Screening Method of Drug for Treating Dravet Syndrome
[0241] The model animal of Dravet syndrome according to the present invention and the cell according to the present invention can be used in development of a new treatment method and drug for treating Dravet syndrome. Hence, the present invention encompasses a screening method of a drug for treating Dravet syndrome, which screens a drug for treating Dravet syndrome (hereinafter, also called "screening method according to the present invention").
[0242] In the specification, an embodiment using a model animal of Dravet syndrome according to the present invention and an embodiment using a screening cell have been explained as embodiments of the screening method according to the present application. However, the present invention is not limited to these embodiments.
[0243] Namely, for example, the embodiment may use another model animal of Dravet syndrome instead of the model animal of Dravet syndrome according to the present invention.
[0244] (1) Case of using model animal of Dravet syndrome according to the present invention
[0245] The method is sufficient as long as it includes administering a candidate agent to the model animal of Dravet syndrome according to the present invention, and assessing whether or not Dravet syndrome shows improvement or is cured in the model animal of Dravet syndrome to which the candidate agent is administered.
[0246] Namely, according to the screening method of the drug for treating Dravet syndrome according to the present invention, a candidate agent is administered to the model animal of Dravet syndrome, to assess whether or not that candidate agent can serve as a drug for treating Dravet syndrome in the model animal of Dravet syndrome to which the candidate agent is administered, by having the improvement or curing of Dravet syndrome serve as an indicator.
[0247] The method of assessing whether or not Dravet syndrome is improved or cured in the model animal of Dravet syndrome to which the candidate agent is administered is not limited in particular, and is sufficiently assessed by use of characteristic symptoms of Dravet syndrome as indicators. For example, it is possible to determine whether Dravet syndrome is improved or cured by comparing a control animal not having a mutation that causes an amino acid change on the sodium ion channel α1 subunit gene and the calcium ion channel α 1 subunit gene (i.e. an animal not having a mutation on both of α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1) with the model animal of Dravet syndrome according to the present invention, in terms of "body temperature at convulsion onset (convulsion threshold)", "severity score", "duration of convulsion", and the like each shown in the Examples later described.
[0248] The candidate agent is not limited in particular, however it is preferable that it is a compound expectable of giving effect on the expression of voltage-gated sodium ion channel NaV1.1 and/or expression of voltage-gated calcium ion channel CaV2.1, or a compound expectable of giving effect on the activity of the voltage-gated sodium ion channel NaV1.1 and/or the activity of voltage-gated calcium ion channel CaV2.1 (e.g. an inhibitor or candidate substance of an inhibitor, or an agonist or a candidate substance of an agonist, each of which has effect on both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1).
[0249] Moreover, the candidate agent may be an expression plasmid vector or a virus vector that includes a polynucleotide made of a sodium ion channel α 1 subunit gene or a part of its nucleotide sequence. Moreover, the candidate agent may be an expression plasmid vector or a virus vector that includes a polynucleotide made of the calcium ion channel α 1 subunit gene or a part of its nucleotide sequence.
[0250] The method of administering such a candidate agent to the Dravet syndrome model animal according to the present invention is not limited in particular, and a suitable method is sufficiently selected from conventionally known methods in accordance with physical properties of that candidate agent.
[0251] (2) Case of Using Screening Cell According to the Present Invention
[0252] The method at least includes administering a candidate agent to a screening cell according to the present invention, and assessing whether or not activity of voltage-gated sodium ion channel NaV1.1 and/or activity of voltage-gated calcium ion channel CaV2.1 in the screening cell of a drug for treating Dravet syndrome to which the candidate agent was administered, is changed.
[0253] Namely, with the screening method according to the present embodiment, it is possible to assess whether a candidate agent can serve as a drug for treating Dravet syndrome, by administering the candidate agent to the screening cell according to the present invention, based on an indicator of whether the activity of the voltage-gated sodium ion channel NaV1.1 and/or the activity of the voltage-gated calcium ion channel CaV2.1 in the screening cell to which the candidate agent is administered, is changed.
[0254] Moreover, the method of assessing, in the screening cell to which the candidate agent is administered, whether or not the activity of the voltage-gated sodium ion channel NaV1.1 is changed and whether or not the activity of the voltage-gated calcium ion channel CaV2.1 is changed are not limited in particular, and the assessments are sufficiently carried out by use of an electrophysiologic measurement device, fluorescence observation device, or the like.
[0255] The candidate agent is not limited in particular, and similar substances as those described in the foregoing "(1) Case of using model animal of Dravet syndrome according to the present invention" may be used.
[0256] The method of administering such a candidate agent to a cell according to the present invention is not limited in particular, and a suitable method based on the physical properties and the like of that candidate agent is selected and used from conventionally known methods.
[0257] It is preferable in the assessment method according to the present invention that the mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 is at least one of a mutation shown in Table 1, and
[0258] the mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 is at least one of a mutation shown in Table 2.
[0259] It is preferable in the assessment method according to the present invention to further include:
[0260] detecting a change in activity of the voltage-gated sodium ion channel NaV1.1; and
[0261] detecting a change in activity of the voltage-gated calcium ion channel CaV2.1.
[0262] The present invention is not limited to the description of the embodiments above, but may be altered by a skilled person within the scope of the claims. An embodiment based on a proper combination of technical means disclosed in different embodiments is encompassed in the technical scope of the present invention.
EXAMPLES
[0263] The following describes more specifically of the present invention with use of Examples, however the present invention is not limited to the Examples.
Example 1
Identification of Risk Factors for Predicting Development of Dravet Syndrome
[0264] DNA were extracted from peripheral blood of 47 Dravet syndrome patients who visited Okayama University Hospital and/or its related hospitals, and mutations on various genes were analyzed. This study was performed upon receiving approval from Okayama University, Institutional Review Board of Human Genome and Gene Analysis Research.
[0265] More specifically, a genomic DNA was extracted from peripheral blood of a patient with use of a DNA extraction kit (WB kit; Nippon gene, Tokyo, Japan), and all exons were amplified by PCR. In PCR, a reaction solution of 25 μl was used, which includes 50 ng of human genomic DNA, 20 μmol of various primers, 0.8 mM of dNTPs, 1 reaction buffer, 1.5 mM of MgCl2, and 0.7 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, Calif., USA). As to the nucleotide sequence (SEQ ID NOs.: 9-62) of the primer pair used, see "Sequence of primers" described later.
[0266] An obtained PCR product was purified with use of PCR products pre-sequencing kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England). Subsequently, with use of Big Dye Terminator FS ready-reaction kit (Applied Biosystems), a sequence reaction was performed, and with use of a fluorescence sequencer (ABI PRISM3100 sequencer; Applied Biosystems), a nucleotide sequence of the obtained PCR product was determined.
[0267] First, mutation analysis was performed of SCN1A gene that encodes α-subunit type 1 (also called "α1 subunit") making up the voltage-gated sodium ion channel NaV1.1, for the 47 Dravet syndrome patients. As a result, a mutation in the SCN1A gene was found in 38 patients out of the 47 Dravet syndrome patients. For the 9 patients in which no mutation was detected, a further analysis was performed on the number of gene copies of the SCN1A gene, with use of Multiplex Ligation-dependent Probe Amplification (MLPA; MRC-Holland; SALSA MLPA kit P137). As a result, a deletion of exon 10 was detected in 1 patient. The number of patients in which no mutation of the SCN1A gene was found was 8. The mutation detected in the SCN1A gene is as shown in Table 1.
[0268] Next, with use of the DNA of the 47 patients, gene analysis was performed for GABRG2 gene, CACNA1A gene, CACNB4 gene, SCN1B gene, and SCN3A gene. These genes encode proteins as follows:
[0269] GABRG2: GABAA receptor γ2 subunit gene
[0270] CACNA1A: α1 subunit of voltage-gated calcium ion channel CaV2.1
[0271] CACNB4: β4 subunit of voltage-gated calcium ion channel
[0272] SCN1B: β1 subunit of voltage-gated sodium ion channel
[0273] SCN3A: α3 subunit of voltage-gated sodium ion channel NaV1.3
[0274] The nucleotide sequence (SEQ ID NOs.: 63-143) of the primer pair used for the gene analysis of the CACNA1A gene is shown in "Sequence of primers" described later.
[0275] As a result, various kinds of gene mutations were found in the CACNA1A gene that encodes α-subunit type 1 (also called "α1 subunit") making up the voltage-gated calcium ion channel CaV2.1 (see Table 2 and FIG. 12).
[0276] Table 3 shows the gene mutations of SCN1A and CACNA1A that were detected in the Dravet syndrome patients.
TABLE-US-00003 TABLE 3 SCN1A and CACNA1A gene mutations detected in Dravet syndrome patients P. No. SCN1A gene CACNA1A gene 1 G177R G266S 2 W738fsX746 K472R 3 V1390M A924G 4 V212A E921D E996V 5 R377L E921D E996V 6 Deletion of exon 10 E921D E996V (Exon10*) 7 P707fsX714 E921D E996V 8 R865X E921D E996V 9 F902C E921D E996V 10 T1082fsX1086 E921D E996V 11 Q1277X E921D E996V 12 Q1450R E921D E996V 13 A1685D E921D E996V 14 T1909I E921D E996V R1126H R2201Q 15 G163E R1126H R2201Q 16 K547fsX570 R1126H R2201Q 17 S1574X R1126H R2201Q 18 R712X G1108S 19 R1648C G1108S 20 negative G1108S 21 negative Del2202-2205 22 R501fsX543 negative 23 S607fsX622 negative 24 E788K negative 25 R931C negative 26 R931C negative 27 L990F negative 28 A1002fsX1009 negative 29 K1027X negative 30 K1057fsX1073 negative 31 L1265P negative 32 W1271X negative 33 1289delF negative 34 Intron 21 splicing negative error 35 A1429fsX1443 negative 36 W1434R negative 37 T1539R negative 38 S1574X negative 39 G1674R negative 40 A1662V negative 41 G1880fsX1881 negative 42 negative negative 43 negative negative 44 negative negative 45 negative negative 46 negative negative 47 negative negative P. No. Patient Number Exon10* exon deletion detected by MPLA
[0277] The following mutations are mutations of the CACNA1A gene detected this time. These mutations were mutations that cause an amino acid substitution, mutations that cause no amino acid substitution, and intron mutations.
(1) Missense Mutations
TABLE-US-00004 [0278] G266S 1 case K472R 1 case E921D 11 cases A924G 1 case E996V 11 cases G1108S 3 cases R1126H 4 cases R2201Q 4 cases
(2) Deletion of Amino Acids
[0279] 4 amino acid deletions (deletion 2202-2205) 1 case
(3) Gene Mutation Causing No Amino Acid Change in Exon E292E (rs16006), E394E (rs2248069), 15251 (rs16010), T698T (rs16016), R1023R (rs16025), F1291F (rs16030), T1458T (new SNP or mutation), S1472S (new SNP or mutation), V1890V (rs17846921), H2225H (rs16051)
(4) Gene Mutation in Intron
[0280] exon 1 upstream (rs16000), intron 1 (rs16003), intron 3 (rs17846942), intron 8 (rs2306348), intron 11 (rs10407951), intron 17 (rs16018), intron 39 (rs3816027), intron 40 (rs17846925), intron 42 (new SNP or mutation).
[0281] The missense mutations and deletion mutations detected in coding regions of the CACNA1A gene shown in the foregoing (1), and (2) are shown in Table 4.
TABLE-US-00005 TABLE 4 Summary of mutations detected in coding region of CACNA1A gene Coding Region Mutation SNP Reg. Exon No. Amino acid type No. 1 Exon 6 G266S Missense -- 2 Exon 11 K472R Missense -- 3 Exon 19 E921D Missense rs16022 4 Exon 19 A924G Missense -- 5 Exon 19 E996V Missense rs16023 6 Exon 20 G1108S Missense rs16027 7 Exon 20 R1126H Missense -- 8 Exon 47 R2201Q Missense -- 9 Exon 47 Del 2202-2205 Deletion -- SNP Reg. No.: Single Nucleotide Polymorphism Registration Number
[0282] These mutations were compared and studied with a gene polymorphism (Single Nucleotide Polymorphism; SNP) database of NCBI (National Center for Biotechnology Information). As a result, it was found that 3 kinds of the mutations out of the 9 kinds of mutations were registered in the SNP database as gene polymorphism (Single Nucleotide Polymorphism; SNP).
[0283] The gene mutation shown in (3) and (4) were either a gene polymorphism registered in the SNP database, or a new gene polymorphism or mutation. The registered number in the SNP database is shown in the brackets.
[0284] Out of the SNP already reported, the mutations which caused a change in the amino acid were considered probably that although no seizure occurs just by that individual case having the CACNA1A gene SNP, but when an abnormality of SCN1A gene is simultaneously present, this is somewhat involved in the worsening of the symptom.
[0285] A comparison of patients having a mutation in either of the SCN1A gene and the CACNA1A gene or both of the SCN1A gene and CACNA1A gene, out of the 47 Dravet syndrome patients, resulted as follows.
[0286] Patients having a mutation on both SCN1A and CACNA1A: 19 cases
[0287] Patients having a mutation on just SCN1A: 20 cases
[0288] Patients having a mutation on just CACNA1A: 2 cases
[0289] Patients having no mutation on either of SCN1A or CACNA1A: 6 cases.
[0290] No reports whatsoever have been made regarding abnormalities in the CACNA1A gene of the patients of Dravet syndrome, until now. The result of the present study shows that Dravet syndrome patients highly frequently has a mutation in SCN1A, i.e. a α1 subunit gene of the voltage-gated sodium ion channel NaV1.1, and in CACNA1A, i.e. a α1 subunit gene of the voltage-gated calcium ion channel CaV2.1.
[0291] A literature disclosing that a mutation on a β4 subunit of the voltage-gated calcium ion channel CaV2.1 (hereinafter, simply referred to as "calcium ion channel β4 subunit") is involved with Dravet syndrome (Iori Ohmori et al., Neurobiology of Disease 32 (2008) 349-354) describes that out of 38 patients in which a mutation was detected in the sodium ion channel α 1 subunit, 1 Dravet syndrome patient had a mutation on both the sodium ion channel α 1 subunit and the calcium ion channel β4 subunit.
[0292] In comparison, out of 39 patients in which a mutation was detected on the sodium ion channel α 1 subunit, the patients of Dravet syndrome having a mutation on both the sodium ion channel α 1 subunit and the calcium ion channel α1 subunit were 19 patients (6 patients when excluding patients having registered SNP that cause a change in an amino acid in an exon). This result shows that by detecting the mutation for both the sodium ion channel α 1 subunit and the calcium ion channel α 1 subunit, the detection sensitivity of Dravet syndrome patients dramatically increase as compared to detecting the mutation for both the sodium ion channel a 1 subunit and the calcium ion channel β4 subunit.
[0293] In the present specification, a nucleotide number in mRNA of the SCN1A gene and an amino acid number in a protein of SCN1A were made to be in line with GenBank accession No. AB093548; methionine, encoded by the initiation codon (ATG), was numbered as the first amino acid, and the initial A of the initiation codon was numbered as the first nucleotide.
[0294] Moreover, a genome sequence of the CACNA1A gene was in line with the GenBank accession number NC--000019. The number of the nucleotide in mRNA of CACNA1A gene and the number of the amino acid in CACNA1A protein was made to be in line with the GenBank accession number NM 023035; methionine, encoded by the initiation codon (ATG), was numbered as the primacy amino acid, and the initial A of the initiation codon was numbered as the primacy nucleotide.
Example 2
Study of Gene Mutation in Benign Febrile Seizure Patient
[0295] A study was performed of a SCN1A gene and CACNA1A gene abnormality in a benign febrile seizure patient. DNA was extracted from peripheral blood of 50 patients of benign generalized epilepsy with febrile seizure plus (GEFS+), who visited Okayama University Hospital and/or its related hospitals, and mutations on various genes were analyzed. The DNA extraction, PCR amplification of the gene, and sequencing reactions were performed by the methods described above.
[0296] First, mutation analysis of voltage-gated sodium ion channel SCN1A gene was performed, which resulted in detecting gene mutation that caused amino acid changes in 6 patients. Next, mutation analysis was performed for 9 kinds of mutations of missense mutations and deletion mutations that were detected in the coding region of the CACNA1A gene, which resulted in detecting a mutation in 16 patients. Each of the mutations are shown in Table 5.
TABLE-US-00006 TABLE 5 SCN1A and CACNA1A gene mutations detected in benign febrile seizure Patient No. SCN1A CACNA1A 1 2 M1856T 3 del 2202-2205 4 5 del 2202-2205 6 R1575C 7 E921D E996V 8 E921D E996V 9 E921D E996V 10 11 12 I1616T 13 14 15 16 17 18 E921D E996V 19 20 21 22 E921D E996V 23 E921D E996V 24 25 26 E921D E996V 27 28 E921D E996V 29 A924G 30 E921D E996V 31 32 33 E921D E996V 34 G1108S 35 36 I1616T 37 I1616T 38 39 Y1769H 40 E921D E996V 41 42 43 44 45 46 47 48 E921D E996V 49 50
[0297] Out of the 50 benign epilepsy patients, it was confirmed that no patient had mutations simultaneously on both SCN1A gene and CACNA1A gene.
[0298] The following shows a result of gene mutation analysis of a total of 97 patients, of 47 malignant Dravet syndrome cases and 50 benign febrile seizure patient cases.
[0299] (1) As a result of screening patients having a mutation on the SCN1A gene among the 97 patients, 39 Dravet syndrome patients (39 cases out of 47 cases) and 6 benign epilepsy patients (6 cases out of 50 cases) were detected.
[0300] (2) As a result of screening patients having a mutation on both the SCN1A gene and CACNA1A gene out of the 97 patients, 19 Dravet syndrome patients (19 cases out of 47) were detected, and no (0) benign epilepsy patients were detected.
[0301] These results suggest that by examining both the SCN1A gene mutation and the CACNA1A gene mutation, it is possible to eliminate the false positive (benign febrile seizure patients) better than examining just the SCN1A gene mutation, and suggest a possibility of detecting the Dravet syndrome patients with higher accuracy.
Example 3
Study of Gene Mutation in a Healthy Person
[0302] To investigate whether the remaining 6 kinds of gene mutations excluding the registered 3 kinds out of the 9 kinds of missense mutations and deletion mutations detected in the coding region of the CACNA1A gene are of the gene polymorphism (SNP), gene mutation of the CACNA1A gene was similarly analyzed for DNA extracted from blood of 190 healthy persons. Results of the 9 kinds of the missense mutations and deletion mutations detected in the coding region of the CACNA1A gene are shown in Table 6. As a result, one kind of the CACNA1A gene mutation (G266S) was not detected from the healthy persons. From this result, it was found that the CACNA1A gene mutation of G266S is not an SNP, and is a novel gene mutation (gene abnormality) not found in the 190 healthy persons, which neither is in the NCBI SNP database.
TABLE-US-00007 TABLE 6 CACNA1A gene mutation detected in healthy persons and Dravet syndrome Nucleotide Amino Acid Control Exon Substitution Substitution Dravet (n = 47) (n = 188-190) p-value Frequency of variants 6 A876G G266S 1/47 2.1% 0/188 0% 0.20 11 A1415G K472R 1/47 2.1% 1/188 0.53% 0.36 19 A2762C E921D 11/47 23.4% 49/188 26.06% 0.71 19 C2771G A924G 1/47 2.1% 7/190 3.68% 1.00 19 A2987T E996V 11/47 23.4% 49/188 26.06% 0.71 20 G3322A G1108S 3/47 6.4% 16/189 8.46% 0.77 20 G3377A R1126H 4/47* 8.5% 1/188 0.53% 0.0061 47 G6602A R2201Q 4/47 8.5% 4/189 2.12% 0.052 47 6605-6616del DQER2202- 1/47 2.1% 3/190 1.58% 1.00 2205del Frequency of combined mutations 19 E921D + E996V 11/47 23.4% 49/188 26.06% 0.71 20 + 47 R1126H + R2201Q 4/47* 8.50% 0/188 0% 0.0014
[0303] As a result of studying the comparison of frequencies in which mutations occur in healthy persons and Dravet syndrome patients, it was shown that the CACNA1A gene mutation R1126H was of a larger number with Dravet syndrome in terms of statistical significance (p=0.0061), and it was found that the CACNA1A gene mutation R2201Q also had a trend having a larger number with Dravet syndrome patients (p=0.052). The patients simultaneously having both mutations of R1126H and R2201Q on the CACNA1A gene were detected significantly in just the Dravet syndrome patients (4 cases out of 47 cases), and no healthy persons were detected (p=0.0014). Examination of DNA of the parents of these four patients revealed that the two mutations of R1126H and R2201Q were simultaneously present on one chromosome, i.e. within the same CACNA1A protein molecule, and that this double mutation was inherited from the parents.
Example 4
Study of Relation Between Genotype and Symptoms
[0304] A study was performed on how the 9 kinds of missense mutations and deletion mutations detected in the coding region of CACNA1A gene give effect on the worsening of symptoms of the disease. Out of Dravet syndrome patients whose seizure symptom data is managed in detail, the seizure symptoms under the age of 1 were compared between 20 patients who have just the SCN1A gene mutation and 19 patients who have a mutation on both the SCN1A gene and the CACNA1A gene. A result thereof is shown in Table 7. Note that "GTC" in Table 7 is an abbreviation of a generalized tonic-clonic seizure, and "CPS" is an abbreviation of a complex partial seizure.
TABLE-US-00008 TABLE 7 Relation of symptoms under the age of 1 with genotype Total no. of Type of Seizures Seizure Total no. prolonged Hemi- Myoclonic onset of (>10 min) GTC CPS convulsion seizure Genotype N (months) seizures seizures (%) (%) (%) (%) SCN1A 20 5.6 ± 0.3 10.2 ± 1.2 2.4 ± 0.4 95 45 50 15 mutation + No CACNA1A variants SCN1A 19 4.6 ± 0.4* 10.7 ± 1.3 4.4 ± 0.7* 95 26 84* 11 mutation + CACNA1A variants GTC: generalized tonic-clonc seizure. CPS: complex partial seizure *p < 0.05
[0305] It was found that the patients having a CACNA1A variant, as compared to the patients having no CACNA1A variant, are (i) significantly quicker in seizure onset (p=0.049), (ii) significantly greater in the number of times prolonged seizures occur, which prolonged seizure is a convulsion seizure that continues for 10 or more minutes (p=0.019), and (iii) significantly higher in the frequency that a hemiconvulsion occurs (p=0.041). This indicates that when there is a variation of the CACNA1A gene including the polymorphism in addition to a SCN1A gene abnormality, there is a possibility that the symptom may worsen.
Example 5
Analysis on Functions of Mutant Voltage-Gated Calcium Ion Channel
[0306] An analysis was performed on functions of a mutant calcium ion channel and a normal (wild-type) calcium ion channel, with use of culture cells. First, cDNA of a human CACNA1A gene (SEQ ID NO.: 4) was used to prepare an expression vector having a mutant CACNA1A (double mutation of G266S; R1126H; R2201Q; deletion 2202-2205; double mutation of R1126H and R2201Q) gene. After obtaining DNA fragments including the mutated parts by PCR, regions of a normal cDNA corresponding to those fragments were substituted with those fragments, to prepare the mutant cDNA. As a control, an expression vector (pMO14×2-CACNA1A) having a normal (wild-type) CACNA1A gene was used.
[0307] Analysis was performed on functions of the mutant calcium ion channel and the normal calcium ion channel, with use of the culture cells. A α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1, which is a CACNA1A gene product, had been subjected to function adjustment by the α2δ subunit and β4 subunit that similarly configure the voltage-gated calcium ion channel CaV2.1. Hence, an expression vector having a CACNA1A gene that encodes a α-subunit type 1, and an expression vector having a human CACNB4 gene (GenBank accession No. U95020) (SEQ ID NO.: 151) encoding a P4 subunit and a rabbit α2δ gene (GenBank accession No. NM--001082276) (SEQ ID NO.: 152) encoding a α2δ subunit. were coexpressed on a human renal cell HEK293 with use of a transfection reagent. Electrophysiologic properties were studied by patch clamping of a whole cell record.
[0308] More specifically, recording of a calcium ion channel current was carried out at room temperature of 22° C. to 24° C., 72 hours after transfection. With use of a multistage P-97 Flaming-Brown micropipette puller, a patch electrode was prepared from borosilicate glass.
[0309] The composition of intracellular fluid was 110 mM CsOH, 20 mM CsCl, 5 mM MgCl2, 10 mM EGTA, 5 mM MgATP, 5 mM creatine-phosphate, and 10 mM HEPES. On the other hand, the composition of the used extracellular fluid was 5 mM BaCl, 150 mM TEA-C1, 10 mM glucose, and 10 mM HEPES. The amplifier used was Axopatch200B (Axon Instruments).
[0310] Electrophysiologic properties of the mutation channel were compared with those of a normal channel, by studying voltage-gated channel activation, inactivation, recovery from inactivation, and duration current. The activation curve and the inactivation curve were analyzed by Boltzmann function, to find a half-maximal activation/inactivation (V1/2) and a slope factor (k). The recovery curve from the inactivation was analyzed by a two exponential function. Statistics used the unpaired Student's t test. Clampfit 8.2 software and OriginPro 7.0 (OriginLab) were used for data analysis.
[0311] FIG. 13 and FIG. 14 are views illustrating results of performing function analysis of the calcium ion channel, by patch clamping. In the graphs in FIG. 13 and FIG. 14, the normal calcium ion channel is shown as "WT", and the mutant calcium ion channels are shown as "R266S", "R1126H", "R2201Q", "De12202", and "RH+RQ". The mutation "De12202" means the mutation "Deletion 2202-2205", and the mutation "RH+RQ" means the mutation "R1126H+R2201Q".
[0312] Illustrated in (a) of FIG. 13 is a barium current record in accordance with a change in potential of the normal calcium ion channel and the mutant calcium ion channel. Illustrated in (b) is a current-voltage relationship, and illustrated in (c) are a peak current value (pA), a total charge (pF), and a peak current density (pA/pF).
[0313] More specifically, (a) of FIG. 13 illustrates a current record of measuring barium current that is depolarized by changing a depolarizing stimulus by 10 mV each from -40 mV to +60 mV and is flowed therein. The current-voltage relationship illustrated in (b) of FIG. 13 is a graph obtained by (i) measuring a flowing barium current for every membrane potential while having a holding potential, being deeper than a resting membrane potential, as -100 mV, and a depolarizing stimulus being changed by 10 mV each from -40 mV to +60 mV, and (ii) plotting the membrane potential on a horizontal axis and a current value on a vertical axis. The view illustrated on the lower right of the graph in (b) of FIG. 13 shows that in this experiment, "the depolarizing stimulus was changed by 10 mV each from -40 mV to +60 mV for 30 ms (milliseconds), with the holding potential being -100 mV, which holding potential is deeper than the resting membrane potential".
[0314] As a result, it was found that the mutant calcium ion channel "Deletion2202-2205" and "R1126H+R2201Q" significantly increased in its flowed current amount, peak current value, and peak current density, as compared to the normal calcium ion channel.
[0315] Next, in order to specifically study the electrophysiologic properties of the calcium ion channel, a voltage-gated activity of the calcium ion channel ((a) of FIG. 14), a time constant (τ) at activation ((b) and (c) of FIG. 14), inactivation of the calcium ion channel ((d) of FIG. 14), and a time constant (τ) at inactivation ((e) FIG. 14) were measured.
[0316] The activation curve illustrated in (a) of FIG. 14 shows a barium current value flowing per membrane potential as a relative value, by having a maximum sodium current value obtained from the graph of (b) of FIG. 13 be 1, and an obtained curve was analyzed by Boltzmann function to find a half-maximal activation (V1/2) and a slope factor (k). The view provided on the lower right of the graph in (a) of FIG. 14 represents that, in this experiment, "the depolarizing stimulus was changed by 10 mV each from -40 mV to +60 mV for 30 ms (milliseconds), with the holding potential being -100 mV, which holding potential is deeper than the resting membrane potential".
[0317] As a result of analyzing the voltage-gated activity of the calcium ion channel, it was found that (i) the mutant calcium ion channel "G266S" and "R1126H" show a significant hyperpolarization shift as compared to the normal channel, and that (ii) the mutant calcium ion channel "R1126H" and "Deletion2202-2205" significantly increased in the voltage-gated property as compared to the normal channel, by comparing the slope factor (k) (see (a) of FIG. 14 and Table 8). This means that the mutant calcium ion channel "G266S", "R1126H" and "Deletion2202-2205" are easily activated even in a low membrane potential, thereby tending to cause excess hyperexcitability of nerve cells.
[0318] Table 8 shows electrophysiologic properties of the calcium ion channel. Statistical comparison of the normal CACNA1A and the mutant CACNA1A were performed by the Student's t test. The asterisk (*) in Table 8 indicates that there is a significant difference between the normal CACNA1A and the mutant CACNA1A when a critical rate is under 5%, and the double asterisk (**) indicates that there is a significant difference between the normal CACNA1A and the mutant CACNA1A when the critical rate is under 1%.
TABLE-US-00009 TABLE 8 Electrophysiologic properties of calcium ion channel Activation V1/2 Inactivation (mV) k (mV) n V1/2 (mV) k (mV) n WT- 6.3 ± 4.3 ± 0.2 16 -16.9 ± 1.5 -4.5 ± 0.6 10 CACNA1A 1.3 G266S 1.0 ± 4.3 ± 0.4 11 -13.8 ± 1.6 -5.5 ± 0.3 10 1.2** R1126H 0.4 ± 3.3 ± 0.3* 10 -18.9 ± 0.6 -6.1 ± 0.7 8 1.6** R2201Q 6.4 ± 4.1 ± 0.2 8 -13.4 ± 1.7 -5.7 ± 0.4 10 1.5 Deletion2202- 1.3 ± 3.4 ± 0.2* 8 -13.3 ± 1.2 -4.7 ± 0.6 9 2205 1.4 R1126H + 2.6 ± 3.5 ± 0.2 10 -15.2 ± 0.9 -5.4 ± 0.1 10 R2201Q 1.1 V1/2, half-maximal voltage activation and inactivation; k, slope factor. Statistical coparison between WT-CACNA1A and mutant channels was performed by Student's t test (*P < 0.05 and **P < 0.01 versus WT-CACNA1A).
[0319] Illustrated in (b) of FIG. 14 is a time constant of channel voltage-gated activation, that is to say, a time required for each current to reach 66.7%. Moreover, (c) of FIG. 14 illustrates a time constant of voltage-gated activation at 20 mV. From (b) and (c) of FIG. 14, it was demonstrated that the mutant calcium ion channel "G266S" was significantly small in the time constant of voltage-gated activation at 20 mV, as compared to a normal channel. Since this point is considered as that the mutant calcium ion channel "G2665" is made so as to flow a lot of current within a short depolarization, this means that there is a trend of causing hyperexcitement in the nerve cells.
[0320] Illustrated in (d) of FIG. 14 is a voltage-gated inactivation curve of the calcium ion channel, which was measured upon changing a membrane potential to activate the calcium ion channel and thereafter providing a depolarizing stimulus to measure how much barium current was flown. Note that the view illustrated on the lower left of the graph illustrated in (d) of FIG. 14 shows that, in this experiment, "the depolarizing stimulus was changed by 20 mV each from -120 mV to +60 mV for 2 s (seconds), and subsequently be changed to 20 mV, with the holding potential being -100 mV, which holding potential is deeper than the resting membrane potential".
[0321] The voltage-gated inactivation curve of the calcium ion channel showed no recognizable significant difference, in either of the mutant channel or the normal channel.
[0322] Illustrated in (e) of FIG. 14 is a result of studying an inactivation time constant (τ). There are two kinds of inactivation: inactivation of a fast component and inactivation of a slow component. The "τfast" in the left graph of (e) of FIG. 14 is a constant representing a time required until the inactivation of the fast component reaches 33.3%, and the "τslow" in the right graph is a constant representing a time required until the inactivation of the slow component reaches 33.3%. These inactivation time constants were, more specifically, calculated by analyzing the inactivation curve with use of Clampfit 8.2 software.
[0323] As a result, there was no significant difference in the inactivation time constant between that of the normal calcium ion channel and that of the mutant calcium ion channel. Table 9 shows physiological properties of the mutant calcium ion channel. The arrow pointing upwards (↑) in Table 9 indicates that an increase in channel activity was recognized, and the hyphen "-" indicates that no change was recognized in the channel activity.
TABLE-US-00010 TABLE 9 Summary of electrophysiological properties of mutant calcium ion channel CACNA1A Biophysical Del R1126H + property G266S R1126H R2201Q 2202-2205 R2201Q Peak current density -- -- -- ↑ ↑↑ Activation V1/2 ↑ ↑ -- -- -- Activation slop -- ↑ -- ↑ -- factor Activation time ↑ -- -- -- -- constants Inactivation V1/2 -- -- -- -- -- Inactivation slope -- -- -- -- -- factor ↑, predicted gain of channel activity. --, no predicted change in channel activity.
[0324] It was found that the mutations other than "R2201Q" in the calcium ion channel were mutations of a gain of function kind, and tends to cause excitement of the nerve cells.
Example 6
Production of Dravet Syndrome Model Rat
[0325] From the foregoing findings, it was considered that having some kind of mutation on both of SCN1A and CACNA1A is important in the development of Dravet syndrome. Accordingly, a rat was produced which has both of the mutation on α1-subunit gene Scn1a of the voltage-gated sodium ion channel NaV1.1 and the mutation on α1-subunit gene Cacna1a of the voltage-gated calcium ion channel CaV2.1, to study the worsening of symptoms (human genes are represented as SCN1A and CACNA1A, and rat genes are represented as Scn1a and Cacna1a).
[0326] More specifically, a rat having a mutation on the Scn1a gene (F344-Scn1a.sup.Kyo811) and a rat having a mutation on the Cacna1a gene (GRY (groggy rat, Cacna1agry)) were used as parent rats. Each of these mice is described below.
[0327] <F344-Scn1a.sup.Kyo811>
[0328] A rat produced by ENU mutagenesis, having a missense mutation on a α1 subunit gene (Scn1a) of the voltage-gated sodium channel NaV1.1. Asparagine (N), which is an amino acid at position 1417, was mutated to histidine (H) (represented as "N1417H"). This rat served as a model animal of human generalized epilepsy febrile seizure plus (GEFS+). Background genealogy is F344/NS1c rat. This rat was provided from the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University.
[0329] <GRY (Groggy Rat, Cacna1agry)>
[0330] A mutant rat produced by administering methyl nitrosourea to Scl:Wistar, whose main symptoms are ataxia and absence-like seizure. This rat has an autosomal recessive mode of inheritance, and has a missense mutation on the α1-subunit of the voltage-gated calcium ion channel CaV2.1. Methionine (M), which is an amino acid at position 251, is mutated to lysine (K) (M251K). This rat was provided from the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University.
[0331] FIG. 11 is a view showing an amino acid sequence of a protein encoded by a human CACNA1A gene and an amino acid sequence of a protein encoded by a rat Cacna1a gene. The upper line of the amino acid sequence shown in FIG. 11 represents an amino acid sequence of the protein encoded by the rat Cacna1a gene (GenBank accession No. NM--012918) (SEQ ID NO.: 147), and the lower line is the amino acid sequence of the protein encoded by the human CACNA1A gene (GenBank accession No. NM--023035) (SEQ ID NO.: 3). Moreover, the squared amino acid "M" in FIG. 11 is an amino acid that is mutated from the amino acid "M" to an amino acid "K" in the human mutant CACNA1A (M249K) protein (SEQ ID NO.: 148) and the rat mutant Cacna1a (M251K) protein (SEQ ID NO.: 149).
[0332] As illustrated in FIG. 11, the mutation (M251K) on the α1 subunit of the rat voltage-gated calcium ion channel CaV2.1 corresponds to the mutation (M249K) on the al subunit of the human voltage-gated calcium ion channel CaV2.1.
[0333] The F344-Scn1a.sup.Kyo811 and GRY (groggy rat, Cacna1agry) as described above were mated to produce a rat having each of the gene mutations.
[0334] (1. Analysis on Functions of Mutant Voltage-Gated Sodium Ion Channel)
[0335] An analysis was performed with use of culture cells, on functions of a mutant sodium ion channel and normal sodium ion channel, before tests using the rats were performed. The rat having a mutation on the Scn1a gene (F344-Scn1a.sup.Kyo811) has asparagine (AAT), which is an amino acid at position 1417 of a protein encoded by the Scn1a gene, was changed to histidine (CAT) (N1417H). The asparagine at position 1417 is located in a pore formation region that is related to ionic permeation of sodium ion channel third domain. On this account, first, the function analysis of the mutant voltage-gated sodium ion channel included in F344-Scn1a.sup.Kyo811 was performed.
[0336] More specifically, an expression vector having a mutant SCN1A (N1417H) gene (SEQ ID NO.: 150) including a missense mutation was prepared with use of cDNA of human SCN1A gene. As control, an expression vector having a normal (wild-type) SCN1A gene (SEQ ID NO.: 2) was prepared.
[0337] FIG. 1 is a view showing an amino acid sequence of a protein encoded by the human SCN1A gene and an amino acid sequence of a protein encoded by the rat Scn1a gene. The upper line in the amino acid sequence shown in FIG. 1 represents an amino acid sequence of a protein that is encoded by the human SCN1A gene (SEQ ID NO.: 1), and the lower line represents an amino acid sequence of a protein that is encoded by the rat Scn1a gene (SEQ ID NO.: 144). Moreover, the squared amino acid "N" in FIG. 1 is an amino acid on which a mutation from an amino acid "N" to an amino acid "H" occurs, of the human mutant SCN1A (N1417H) protein (SEQ ID NO.: 145) and the rat mutant SCN1A (N1417H) protein (SEQ ID NO.: 146).
[0338] An analysis was performed with use of culture cells, on functions of the mutant sodium ion channel and the normal sodium ion channel. The α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1, which is a SCN1A gene product, was adjusted in its function by β1 subunit and β2 subunit that similarly make up the voltage-gated sodium ion channel NaV1.1. Hence, an expression vector having the SCN1A gene that encodes the α-subunit type 1 was coexpressed with an expression vector having the SCN1B gene that encodes the β1 subunit and the SCN2B gene that encodes the β2 subunit in a human renal cell HEK293, with use of a transfection reagent. The electrophysiologic properties were studied by patch clamping based on whole cell recording.
[0339] More specifically, recording of the sodium ion channel current was carried out at room temperature of 22° C. to 24° C., 24 hours to 48 hours after transfection. A patch electrode was prepared from borosilicate glass by use of multistage P-97 Flaming-Brown micropipette puller.
[0340] Composition of intracellular fluid was 110 mM CsF, 10 mM NaF, 20 mM CsCl, 2 mM EGTA, and 10 mM HEPES. On the other hand, the composition of extracellular fluid was 145 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES. Axopatch200B (Axon Instruments) was used as the amplifier.
[0341] Electrophysiologic properties of the mutation channel were compared with those of a normal channel, by studying voltage-gated channel activation, inactivation, recovery from inactivation, and duration current. The activation curve and the inactivation curve were analyzed by Boltzmann function, to find a half-maximal activation/inactivation (V1/2) and a slope factor (k). The recovery curve from the inactivation was analyzed by a two exponential function. Durable Na current was found by a difference in the duration current when depolarized at -10 mV for 100 ms, before and after addition of 10 μM of tetrodotoxin (TTX). Statistics used were unpaired Student's t test. Clampfit 8.2 software and OriginPro 7.0 (OriginLab) were used for data analysis.
[0342] FIGS. 2 to 4 are views illustrating results of performing function analysis of the sodium ion channel by patch clamping. The graphs of FIGS. 2 to 4 show the normal sodium ion channel as "WT" or "WT-SCN1A", and show the mutant sodium ion channel as "N1417H".
[0343] Illustrated in (a) of FIG. 2 is a typical example of a sodium current in response to a change in potential of the normal sodium ion channel and the mutant sodium ion channel. More specifically, a depolarizing stimulus was changed 10 mV each from -80 mV to +60 mV for depolarization, and sodium current that flowed in was measured. As a result, both of the normal sodium ion channel and the mutant sodium ion channel function as a channel, and there was no significant difference between the two.
[0344] Illustrated in (b) of FIG. 2 is a result of studying the inactivation time constant (τ). There are two types of inactivation; an inactivation of a fast component and an inactivation of a slow component. The "τ1" in (b) of FIG. 2 is indicative of a constant indicative of a time required for the inactivation of the fast component to reach 33.3%, and the "τ2" is indicative of a constant indicative of a time required for the inactivation of the slow component to reach 33.3%. These inactivation time constants, more specifically, were calculated by analyzing the inactive curve with use of the Clampfit 8.2 software. As a result, there was no significant difference in the inactivation time constant between that of the normal sodium ion channel and that of the mutant sodium ion channel.
[0345] Next, in order to specifically study the electrophysiologic properties of the sodium ion channel, a current-voltage relationship ((a) of FIG. 3), an activation of the sodium ion channel ((b) of FIG. 3), an inactivation of the sodium ion channel ((c) of FIG. 3), and recovery from the inactivation of the sodium ion channel ((d) of FIG. 3) were measured.
[0346] More specifically, the current-voltage relationship illustrated in (a) of FIG. 3 was obtained by (i) measuring a flowing sodium current for every membrane potential while having a holding potential, being deeper than a resting membrane potential, as -120 mV, and a depolarizing stimulus being changed by 10 mV each from -80 mV to +60 mV, and (ii) plotting the membrane potential on a horizontal axis and a current value on a vertical axis. The view illustrated on the lower left of the graph in (a) of FIG. 3 shows that in this experiment, "the depolarizing stimulus was changed by 10 mV each from -80 mV to +60 mV for 20 ms (milliseconds), with the holding potential being -120 mV, which holding potential is deeper than the resting membrane potential".
[0347] The activation curve illustrated in (b) of FIG. 3 shows a sodium current value flowing per membrane potential as a relative value, by having a maximum sodium current value obtained from the graph of (a) of FIG. 3 be 1, and an obtained curve was analyzed by Boltzmann function to find a half-maximal activation (V1/2) and a slope factor (k). The view provided on the lower right of the graph in (b) of FIG. 3 represents that in this experiment, "the depolarizing stimulus was changed by 10 mV each from -80 mV to +60 mV, for 20 ms (milliseconds), with the holding potential being -120 mV, which holding potential is deeper than the resting membrane potential".
[0348] The inactive curve illustrated in (c) of FIG. 3 was obtained by similarly changing the membrane potential to activate the channel and thereafter providing depolarizing stimulus and measuring how much the sodium current flows, to find the half-maximal inactivation (V1/2) and the slope factor (k). Note that the view provided on the lower left of the graph of (c) of FIG. 3 represents that in this experiment, "the depolarizing stimulus was changed by 10 mV each from -140 mV to +0 mV for 100 ms (milliseconds) and subsequently changed to -10 mV, with the holding potential being -120 mV".
[0349] The recovery curve from the inactivation illustrated in (d) of FIG. 3 was obtained as follows. When a depolarizing stimulus was provided with pulse 1 (P1), the channel became inactive upon opening. When the depolarizing stimulus was returned to the original -120 mV, the sodium ion channel returned to its resting state, and upon stimulation of pulse 2 (P2), the channel opened again. The recovery time of this pulse 1 and pulse 2 were changed to obtain the recovery curve from the inactivation. This curve was analyzed by a two exponential function. It was determined whether the function of the channel was made easily excited or in the opposite was made difficult to be excited, depending on whether the recovery was quicker or slower as compared to the normal channel. The view provided on the lower right of the graph of (d) of FIG. 3 indicates that in this experiment, "a holding potential was mV, -10 mV was provided for 100 ms (milliseconds) as the depolarizing stimulus and thereafter was returned to -120 mV, and after elapse of each of the times (milliseconds) shown on the x-axis, -10 mV was provided for 20 ms (milliseconds)".
[0350] As a result, no significant difference was recognized in the current-voltage relationship and the channel activation, between the normal sodium ion channel and the mutant sodium ion channel (see (a) and (b) of FIG. 3). Meanwhile, a significant test was performed regarding the channel inactivation, on a point that the normal sodium ion channel and the mutant sodium ion channel are inactivated by 50%, whereby resulted in finding that the mutant sodium ion channel had shifted significantly to the depolarization side (p<0.05) ((c) of FIG. 3).
[0351] As to the recovery from the channel inactivation, it was found that the recovery was significantly slow in the mutant sodium ion channel ((d) of FIG. 3). In (d) of FIG. 3, a part in which a period of recovery (Recovery period (ms)) from the inactivation was 1 ms to 8 ms corresponds to a "fast component", and a part in which the period of recovery from the inactivation was 10 ms to 100 ms corresponds to a "slow component".
[0352] More specifically, upon comparison between the normal sodium ion channel and an abnormal sodium ion channel based on a time required for the fast component in recovering from the inactivation to recover from the inactivation to 33.3%, it was found that the recovery was significantly slow for the mutant sodium ion channel (normal: τf=1.7±0.1 ms, n=14; mutant: τf=2.5±0.2 ms (P<0.01), n=12).
[0353] Similarly, upon comparison of the normal sodium ion channel with the abnormal sodium ion channel based on the time required for the slow component in recovering from the inactivation to recover from the inactivation to 33.3%, it was found that the mutant sodium ion channel was significantly slow in recovering (normal: τf=40.3±5.3 ms, n=14; mutant: τs=60.9±7.9 ms (P<0.05), n=12).
[0354] FIG. 4 shows that, even if the sodium ion channel was made inactivated after the potential was changed to activate the sodium ion channel, the baseline of the mutant sodium channel does not return back in the whole cell record, which indicates clearly that the sodium current was persistently flowing into the mutant sodium ion channel. The persistent sodium current is considered as an obstruction of an inactivation gate. From the view of (a) of FIG. 4, it was confirmed that even after the elapse of time, the inactivation was insufficient in the mutant sodium ion channel as compared to that of the normal sodium ion channel.
[0355] So as to find the persistent sodium current shown in (a) of FIG. 4, a relative value (%) was found by dividing, with a maximum current amount, a final current amount that flowed between 80 milliseconds to 100 milliseconds when a depolarizing stimulus of 100 milliseconds was given. Results thereof are shown in (b) of FIG. 4. From these results, it was found that the mutant sodium ion channel had properties that the persistent sodium current increases.
[0356] This data show that the function of the voltage-gated sodium ion channel NaV1.1 became abnormal by the mutation. Namely, this means that by having the mutation, the nerve cells are easily excessively excited, that is to say, more easily causes the occurrence of a convulsion.
[0357] Literature (Satoko Tokuda et. al., BRAINRESEARCH 1133 (2007) 168-177; Kenta Tanaka et. al., Neuroscience Letters 426 (2007) 75-80) discloses that the function of the voltage-gated calcium ion channel CaV2.1 of a rat becomes abnormal due to a mutation (M251K) on the α1 subunit of the voltage-gated calcium ion channel CaV2.1 of the rat.
[0358] Therefore, with a rat having the mutation on both the Scn1a gene and Cacna1a gene described later, it can be considered that the functions of both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1 are abnormal.
[0359] (2. Confirmation of Gene Mutation in Dravet Syndrome Model Rat)
[0360] The foregoing F344-Scn1a.sup.Kyo81 and the GRY (groggy rat, Cacna1agry) were mated as parent rats (P) to produce F1 (first filial generation) rats, and these F1 rats were mated to produce F2 (second filial generation) rats. FIG. 5 is a view showing genotypes of the parent rats (P), the F1 rats and the F2 rats. As illustrated in (a) of FIG. 5, the F1 rats have the heterozygous mutation on both the Scn1a gene and the Cacna1a gene (referred to as "Scn1a mutant (hetero)+Cacna1a mutant (hetero)"). Moreover, as illustrated in (b) of F1G. 5, rats showing 9 types of genotypes were born from the F2 rats. The genotypes of each of the rats were identified by extracting a tip tissue of the tail of the rats and extracting its DNA, to perform DNA sequencing with the extracted DNA and detect its gene mutation, or by detecting a digested pattern with use of a restriction enzyme.
[0361] (Method of Confirming Gene Mutation by DNA Sequencing)
[0362] Confirmation of gene mutation by DNA sequencing was performed as follows. First, a genomic DNA was amplified with use of a primer pair that sandwiches a mutation point (a nucleotide sequence of a Scn1a amplification primer pair is represented by SEQ ID NO.: 5 and SEQ ID NO.: 6, and a nucleotide sequence of a Cacna1a amplification primer pair is represented by SEQ ID NO.: 7 and SEQ ID NO.: 8), and thereafter, an obtained PCR product was purified with use of a PCR products pre-sequencing kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England). See the item "Sequence of primers" later described for the nucleotide sequence of the used primer pairs.
[0363] Next, sequence reaction was performed with use of a Big Dye Terminator FS ready-reaction kit (Applied Biosystems), to determine a nucleotide sequence with a fluorescence sequencer (ABI PRISM3100 sequencer; Applied Biosystems).
[0364] FIG. 6 is a view illustrating a method of identifying a genotype of the Scn1a gene and the Cacna1a gene of the F2 rats, by sequencing. As illustrated in FIG. 6, a wild-type Scn1a gene has a nucleotide at position 4249 be "A". In comparison, a mutant Scn1a gene (N1417H) has a nucleotide at position 4249 that is mutated from "A" to "C". As a result, a codon "AAT" that designates asparagine (N) being an amino acid at position 1417 in the wild-type Scn1a gene, is mutated to a codon "CAT" which designates histidine (H), in the mutant Scn1a gene (N1417H).
[0365] Moreover, the wild-type Cacna1a gene has a nucleotide at position 752 be "T". In comparison, the mutant Cacna1a gene (M251K) has a nucleotide at position 752 that is mutated from "T" to "A". As a result, a codon "ATG" that designates methionine, which is an amino acid at position 251, is mutated to a codon "AAG" that designates lysine.
[0366] (Method of Confirming Gene Mutation by Restriction Enzyme Digestion)
[0367] The method of confirming gene mutation by the restriction enzyme digestion was performed as follows. When detecting mutation in the Scn1a gene, a genomic DNA was amplified with use of a primer pair (SEQ ID NOs.: 5 and 6) that sandwich a mutation point in the Scn1a gene, and thereafter an obtained PCR product was reacted for three hours at 50° C., with use of a restriction enzyme BclI. Thereafter, the PCR product reacted with the restriction enzyme was subjected to electrophoresis with use of 4% agarose gel, and the size of the band was detected. FIG. 7 is a view illustrating a method of identifying the genotype of the Scn1a gene of the F2 rats, by restriction enzyme digestion.
[0368] As shown in (a) and (b) of FIG. 7, the wild-type Scn1a gene was not digested with BM so the size of the band remained as the size of the PCR product (nucleotide of 380 bp). On the other hand, the mutant Scn1a gene (N1417H) was digested with BM so two fragments (nucleotides of 276 bp and 104 bp) were detected. In a case of a heterozygous rat of the wild-type Scn1a gene and the mutant Scn1a gene (N1417H), three fragments (nucleotides of 380 bp, 276 bp, and 104 bp) were detected. Illustrated in (c) of FIG. 7 shows a result of electrophoresis.
[0369] In a case of detecting the mutation on the Cacna1a gene, a genomic DNA was amplified with use of a primer pair (SEQ ID NOs.: 7 and 8) that sandwich a mutation point of the Cacna1a gene, and thereafter, an obtained PCR product was reacted for hour at 37° C. with use of a restriction enzyme PciI. Thereafter, the PCR product reacted with the restriction enzyme was subjected to electrophoresis with use of 4% agarose gel, to detect the size of a band.
[0370] FIG. 8 is a view illustrating a method of identifying a genotype of the Cacna1a gene of the F2 rats, by restriction enzyme digestion. As illustrated in (a) and (b) of FIG. 8, a wild-type Cacna1a gene was not digested with PciI, so hence the size of the band remained as the size of the PCR product (nucleotide of 352 bp). On the other hand, the mutant Cacna1a gene (M251K) was digested with PciI, and thus two fragments (nucleotides of 219 by and 133 bp) were detected. With a heterozygous rat of the wild-type Cacna1a gene and an abnormal Cacna1a gene (M251K), three fragments (nucleotides of 352 bp, 219 bp, and 133 bp) were detected. Illustrated in (c) of FIG. 8 is a result of electrophoresis.
Example 7
Analysis of Dravet Syndrome Model Rat
[0371] A study was performed on what kind of (worsening) effect was given on the seizure when a mutation on the Cacna1a gene was added to a mutation on the Scn1a gene, with use of a Dravet syndrome model rat. More specifically, comparison was made regarding symptoms when a convulsion seizure was induced by heat load, between a rat having a homozygous mutation on the Scn1a gene (referred to as "Scn1a mutant (homo)+Cacna1a wild-type (homo)") and a rat having a homozygous mutation on the Scn1a gene and a heterozygous mutation on the Cacna1a gene (referred to as "Scn1a mutant (homo)+Cacna1a mutant (hetero)").
[0372] The Scn1a mutant (homo)+Cacna1a wild-type (homo) and the Scn1a mutant (homo)+Cacna1a mutant (hetero) both have a homozygous mutation on the Scn1a gene (N1417H). Hence, comparison is made between the wild-type Cacna1a gene and the mutant Cacna1a gene (M251K), under the condition of the homozygous mutation of the Scn1a gene.
[0373] Moreover, a rat having a wild-type Scn1a gene and a wild-type Cacna1a gene (referred to as "Scn1a wild-type (homo)+Cacna1a wild-type (homo)") and a rat having a wild-type homozygous mutation on the Scn1a gene and a heterozygous mutation on the Cacna1a gene (referred to as "Scn1a wild-type (homo)+Cacna1a mutation (hetero)") were used as control. The following lists the genotypes of the rats used in the experiment. The following numbers (1) to (4) correspond to the numbers in (b) of FIG. 5.
[0374] (1) Scn1awt/wtCacna1awt/wt (Scn1a wild-type (homo)+Cacna1a wild-type (homo)) 14 males
[0375] (2) Scn1amut/mut Cacna1awt/wt (Scn1a mutant (homo)+Cacna1a wild-type (homo)) 7 males
[0376] (3) Scn1amut/mut Cacna1awt/mut (Scn1a mutant (homo)+Cacna1a mutant (hetero)) 17 males
[0377] (4) Scn1awt/wt Cacna1awt/mut (Scn1a wild-type (homo)+Cacna1a mutant (hetero)) 12 males.
[0378] Hot bath load (45° C.) were given on male rats of 5 weeks old of the groups (1) to (4) described above, to compare their body temperatures at a time when a convulsion is induced, their duration of the convulsion, and their severity score of the convulsion. A rectal temperature at the time when the seizure started was measured, to serve as the body temperature at the time when the convulsion was induced. The seizure severity score of the convulsion were evaluated as follows: 0=no seizure, 1=facial convulsion, 2=clonic convulsion of both arms while maintaining posture, 3=sprint or jump, 4=generalized convulsion unable to maintain posture, and 5=death caused by persistent convulsion.
[0379] The results were as shown in FIG. 9. FIG. 9 is a view showing a result of the effect caused by the mutation on the Cacna1a gene in the Scn1a gene-mutated rat. In the graphs of (a) to (c) in FIG. 9, Scn1amut/mutCacna1awt/wt (the foregoing rat (2)) is shown as "Scn1a mutant (homo)". Scn1amut/mutCacna1awt/mut (the foregoing rat (3)) is shown as "Scn1a mutant (homo)+Cacna1a mutant (hetero)". Moreover, control Scn1awt/wtCacna1awt/wt (foregoing rat (1)) is shown as "WT", and control Scn1awt/wtCacna1awt/mut (foregoing rat (4)) is shown as "Cacna1a mutant (hetero)".
[0380] As a result of analysis, the group (3) rats (Scn1a mutant (homo)+Cacna1a mutant (hetero)) had no large difference in the body temperatures at the time of convulsion onset (convulsion threshold) ((a) of FIG. 9) and severity scores ((b) of FIG. 9), from those of the group (2) rats (Scn1a mutant (homo)+Cacna1a wild-type (homo)). However, it was found that the duration of the convulsion ((c) of FIG. 9) became significantly long. This result demonstrates that the mutation of the Cacna1a gene relates to the worsening of the symptoms of convulsion.
[0381] Furthermore, FIG. 10 shows a part of an electroencephalogram during a seizure of a group (3) rat (Scn1a mutant (homo)+Cacna1a mutant (hetero)). It was considered from this result that a rat having a mutation on the Scn1a gene and the Cacna1a gene could serve as a model rat of the intractable Dravet syndrome. The model rat is expected to be usefully used in the future for clarification of the onset mechanism of the intractable Dravet syndrome, development of medicament for Dravet syndrome, and like uses.
[0382] Moreover, these results are considered as supporting the gene analysis data of Example 1, that a variation of the CACNA1A gene was detected in addition to a mutation on the SCN1A gene, in a patient of Dravet syndrome which is an intractable epilepsy. Namely, the method according to the present invention of obtaining data for assessing the potential for development of Dravet syndrome can be said as a technique supported by the gene analysis results of the Examples, a mutant channel function analysis result, and animal experiment results.
CONCLUSION
[0383] The present invention was developed based on a molecular foundation of development of the intractable Dravet syndrome; the assessment method according to the present invention can be said as useful as an early detection method of Dravet syndrome patients. By use of the assessment method according to the present invention, it is possible to find Dravet syndrome, which has an unfavorable prognosis, in high accuracy and at an early stage. This allows for an epilepsy specialist to prepare a treatment management system for the patient of Dravet syndrome from an early stage. As a result, this leads to improvement in therapeutic intervention of the patient, reduction of mental load on the family, and reduction of economical burden. Moreover, it is possible to carry out appropriate treatment to the Dravet syndrome patient, so therefore is considered as contributive to the reduction of medical fees.
[0384] Furthermore, with use of the kit according to the present invention, it is possible to easily detect the mutation for both the SCN1A gene and CACNA1A gene. Consequently, the kit according to the present invention is useful for a general pediatrician to distinguish a patient of Dravet syndrome who requires treatment by a specialist out of the benign febrile epilepsies, during the initial stage of the disease under the age of one.
[0385] By use of the assessment method and the kit according to the present invention, it is possible to detect with high accuracy a patient of Dravet syndrome at the point in time of under the age of one, which was difficult to detect until now. Moreover, by examining gene abnormalities upon sending the blood taken to an examination center, it is possible to detect Dravet syndrome patients in high accuracy even for a remote personal hospital or the like.
[0386] Moreover, the model animal and cell according to the present invention may be usefully used in the clarification of an onset mechanism of the intractable Dravet syndrome, the development of medicament for Dravet syndrome, and like uses.
[0387] <Primer Sequences>
[0388] Table 10 shows a nucleotide sequence of a primer pair used for amplifying the Scn1a gene and amplifying the Cacna1a gene.
TABLE-US-00011 TABLE 10 Scn1a Sense 5'-TGA CTT TTC TTT CTC TCC GTT TG-3' SEQ ID amplification primer: NO.: 5 Antisense 5'-TGG CTG CAA TAA TCA CTT TGT T-3' SEQ ID primer: NO.: 6 Cacna1a Sense 5'-TCT CTG TCT CCC CAG GTT TAC-3' SEQ ID amplification primer: NO: 7 Antisense 5'-GTG GCT AAC ACA CAG CTT TGC-3' SEQ ID primer: NO.: 8
[0389] Tables 11 and 12 show nucleotide sequences of primer pairs used for detecting SCN1A gene genomes.
TABLE-US-00012 TABLE 11 Exon 1 Sense 5'-tcatggcacagttcctgtatc-3' SEQ ID amplification primer: NO.: 9 Antisense 5'-gcagtaggcaattagcagcaa-3' SEQ ID primer: NO.: 10 Exon 2 Sense 5'-tggggcactttagaaattgtg-3' SEQ ID amplification primer: NO.: 11 Antisense 5'-tgacaaagatgcaaaatgagag-3' SEQ ID primer: NO.: 12 Exon 3 Sense 5'-gcagtttgggcttttcaatg-3' SEQ ID amplification primer: NO.: 13 Antisense 5'-tgagcattgtcctcttgctg-3' SEQ ID primer: NO.: 14 Exon 4 Sense 5'-agggctacgtttcatttgtatg-3' SEQ ID amplification primer: NO.: 15 Antisense 5'-tgtgctaaattgaaatccagag-3' SEQ ID primer: NO.: 16 Exon 5 Sense 5'-CAGCTCTTCGCACTTTCAGA-3' SEQ ID amplification primer: NO.: 17 Antisense 5'-TCAAGCAGAGAAGGATGCTGA-3' SEQ ID primer: NO.: 18 Exon 6 Sense 5'-agcgttgcaaacattcttgg-3' SEQ ID amplification primer: NO.: 19 Antisense 5'-gggatatccagcccctcaag-3' SEQ ID primer: NO.: 20 Exon 7 Sense 5'-gacaaatacttgtgcctttgaatg-3' SEQ ID amplification primer: NO.: 21 Antisense 5'-acataatctcatactttatcaaaaacc-3' SEQ ID primer: NO.: 22 Exon 8 Sense 5'-gaaatggaggtgttgaaaatgc-3' SEQ ID amplification primer: NO.: 23 Antisense 5'-aatccttggcatcactctgc-3' SEQ ID primer: NO.: 24 Exon 9 Sense 5'-agtacagggtgctatgaccaac-3' SEQ ID amplification primer: NO.: 25 Antisense 5'-tcctcatacaaccacctgctc-3' SEQ ID primer: NO.: 26 Exon 10 Sense 5'-tctccaaaagccttcattagg-3' SEQ ID amplification primer: NO.: 27 Antisense 5'-ttctaattctccccctctctcc-3' SEQ ID primer: NO.: 28 Exon 11 Sense 5'-tcctcattctttaatcccaagg-3' SEQ ID amplification primer: NO.: 29 Antisense 5'-gccgttctgtagaaacactgg-3' SEQ ID primer: NO.: 30 Exon 12 Sense 5'-gtcagaaatatctgccatcacc-3' SEQ ID amplification primer: NO.: 31 Antisense 5'-gaatgcactattcccaactcac-3' SEQ ID primer: NO.: 32 Exon 13 Sense 5'-tgggctctatgtgtgtgtctg-3' SEQ ID amplification primer: NO.: 33 Antisense 5'-ggaagcatgaaggatggttg-3' SEQ ID primer: NO.: 34 Exon 14 Sense 5'-tacttcgcgtttccacaagg-3' SEQ ID amplification primer: NO.: 35 Antisense 5'-gctatgcaagaaccctgattg-3' SEQ ID primer: NO.: 36
TABLE-US-00013 TABLE 12 Exon 15 Sense 5'-atgagcctgagacggttagg-3' SEQ ID amplification primer: NO.: 37 Antisense 5'-atacatgtgccatgctggtg-3' SEQ ID primer: NO.: 38 Exon 16 Sense 5'-tgctgtggtgtttccttctc-3' SEQ ID amplification primer: NO.: 39 Antisense 5'-tgtattcataccttcccacacc-3' SEQ ID primer: NO.: 40 Exon 17 Sense 5'-aaaagggttagcacagacaatg-3' SEQ ID amplification primer: NO.: 41 Antisense 5'-attgggcagatataatcaaagc-3' SEQ ID primer: NO.: 42 Exon 18 Sense 5'-cacacagctgatgaatgtgc-3' SEQ ID amplification primer: NO.: 43 Antisense 5'-tgaagggctacactttctgg-3' SEQ ID primer: NO.: 44 Exon 19 Sense 5'-tctgccctcctattccaatg-3' SEQ ID amplification primer: NO.: 45 Antisense 5'-gcccttgtcttccagaaatg-3' SEQ ID primer: NO.: 46 Exon 20 Sense 5'-aaaaattacatcctttacatcaaactg-3' SEQ ID amplification primer: NO.: 47 Antisense 5'-ttttgcatgcatagattttcc-3' SEQ ID primer: NO.: 48 Exon 21 Sense 5'-tgaaccttgcttttacatatcc-3' SEQ ID amplification primer: NO.: 49 Antisense 5'-acccatctgggctcataaac-3' SEQ ID primer: NO.: 50 Exon 22 Sense 5'-tgtcttggtccaaaatctgtg-3' SEQ ID amplification primer: NO.: 51 Antisense 5'-ttggtcgtttatgctttattcg-3' SEQ ID primer: NO.: 52 Exon 23 Sense 5'-ccctaaaggccaatttcagg-3' SEQ ID amplification primer: NO.: 53 Antisense 5'-atttggcagagaaaacactcc-3' SEQ ID primer: NO.: 54 Exon 24 Sense 5'-gagatttgggggtgtttgtc-3' SEQ ID amplification primer: NO.: 55 Antisense 5'-ggattgtaatggggtgcttc-3' SEQ ID primer: NO.: 56 Exon 25 Sense 5'-caaaaatcagggccaatgac-3' SEQ ID amplification primer: NO.: 57 Antisense 5'-tgattgctgggatgatcttg-3' SEQ ID primer: NO.: 58 Exon 26(1) Sense 5'-aggactctgaaccttaccttgg-3' SEQ ID amplification primer: NO.: 59 Antisense 5'-ccatgaatcgctcttccatc-3' SEQ ID primer: NO.: 60 Exon 26(2) Sense 5'-tgtgggaacccatctgttg-3' SEQ ID amplification primer: NO.: 61 Antisense 5'-gtttgctgacaaggggtcac-3' SEQ ID primer: NO.: 62
[0390] Tables 13 and 14 show nucleotide sequences of primer pairs used for detecting the CACNA1A gene genome. In Tables 13 and 14, for example, E1F indicates an Exon 1 amplification sense primer, and E1Rv indicates an Exon 1 amplification antisense primer.
TABLE-US-00014 TABLE 13 Exon 1 CACNA1A-E1F: 5'-tctccgcagtcgtagctccag-3' SEQ ID NO.: 63 amplification CACNA1A-E1Rv: 5'-agagattctttcacactcctcc-3' SEQ ID NO.: 64 Exon 2 CACNA1A-E2F: 5'-tttagaagtcacctgatctggg-3' SEQ ID NO.: 65 amplification CACNA1A-E2Rv: 5'-gacagagcgagactctggttca-3' SEQ ID NO.: 66 Exon 3 CACNA1A-E3F: 5'-gacaagagaactctgcaagagg-3' SEQ ID NO.: 67 amplification CACNA1A-E3Rv: 5'-atacagctgagacatggaggtg-3' SEQ ID NO.: 68 Exon 4 CACNA1A-E4F: 5'-tttatcccgtgaggcaggtactg-3' SEQ ID NO.: 69 amplification CACNA1A-E4Rv: 5'-cctcctgagatgctctgcatag-3' SEQ ID NO.: 70 Exon 5 CACNA1A-E5F: 5'-tgtggtgcttccttcaccattg-3' SEQ ID NO.: 71 amplification CACNA1A-E5Rv: 5'-cagaggctatttcactcactgc-3' SEQ ID NO.: 72 Exon 6 CACNA1A-E6F: 5'-ccccaaagccaaacattgatctc-3' SEQ ID NO.: 73 amplification CACNA1A-E6Rv: 5'-actctgattgtccacacacactg-3' SEQ ID NO.: 74 Exon 7 CACNA1A-E7F: 5'-cagaaaacgttcctccatttccc-3' SEQ ID NO.: 75 amplification CACNA1A-E7Rv: 5'-aagcttcaatggcctctacttgg-3' SEQ ID NO.: 76 Exon 8 CACNA1A-E8F: 5'-gccatactctggcttttctatgc-3' SEQ ID NO.: 77 amplification CACNA1A-E8Rv: 5'-cgtgatgtcagatcctggcttc-3' SEQ ID NO.: 78 Exon 9 CACNA1A-E9F: 5'-gttggctattgctactgttgcg-3' SEQ ID NO.: 79 amplification CACNA1A-E9Rv: 5'-gatccttagaaccagtcacctg-3' SEQ ID NO.: 80 Exon 10 CACNA1A-E1OF: 5'-tgatagtgccaccttgaacctc-3' SEQ ID NO.: 81 amplification CACNA1A-E1ORv: 5'-tgatgtaatctgcccaggacac-3' SEQ ID NO.: 82 Exon 11 CACNA1A-E11F: 5'-ctgcaacagagaactatcagcc-3' SEQ ID NO.: 83 amplification CACNA1A-E11Rv: 5'-aagagaagtggaaaaagggtgtg-3' SEQ ID NO.: 84 Exon 12 CACNA1A-E12F: 5'-gtagttctagcatgttggaggc-3' SEQ ID NO.: 85 amplification CACNA1A-E12Rv: 5'-atctgtcattccaggcaagagc-3' SEQ ID NO.: 86 Exon 13~15 CACNA1A-E13F: 5'-atggatgaatgagggggtcaag-3' SEQ ID NO.: 87 amplification CACNA1A-E15Rv: 5'-agcaggcactttcatctgtgac-3' SEQ ID NO.: 88 Exon 13~15 CACNA1A-E13F2: 5'-tccatttggagggaggagtttg-3' SEQ ID NO.: 89 amplification CACNA1A-E15Rv: 5'-agcaggcactttcatctgtgac-3' SEQ ID NO.: 88 Exon 14~15 CACNA1A-E14F: 5'-cctccagaaagttgggaaagtg-3' SEQ ID NO.: 90 amplification CACNA1A-E15Rv: 5'-agcaggcactttcatctgtgac-3' SEQ ID NO.: 88 Exon 16~17 CACNA1A-E16F: 5'-aaggagaagccaacacggagtc-3' SEQ ID NO.: 91 amplification CACNA1A-E17Rv: 5'-ggtggtaactttgccagagaaac-3' SEQ ID NO.: 92 Exon 18 CACNA1A-E18F: 5'-agcaggtacccattccaattgg-3' SEQ ID NO.: 93 amplification CACNA1A-E18Rv: 5'-aatctgtgcctgggatagtgtg-3' SEQ ID NO.: 94 Exon 19 CACNA1A-E19F: 5'-cctgactcagatgctcacagac-3' SEQ ID NO.: 95 amplification CACNA1A-E19Rv: 5'-acacagcacgtgctactttggc-3' SEQ ID NO.: 96 (1) Exon 19 CACNA1A-E19F2: 5'-gaggacttcctcaggaaacag-3' SEQ ID NO.: 97 amplification CACNA1A-E19Rv: 5'-acacagcacgtgctactttggc-3' SEQ ID NO.: 96 (2) Exon 20 CACNA1A-E20F: 5'-agatggaatcttagctaggatcc-3' SEQ ID NO.: 98 amplification CACNA1A-E20Rv: 5'-aattatctcactgaaccctccac-3' SEQ ID NO.: 99 Exon 21 CACNA1A-E21F: 5'-agaaatgtcagccgcttcttgc-3' SEQ ID NO.: 100 amplification CACNA1A-E21Rv: 5'-ggtggtcaacactcactcattg-3' SEQ ID NO.: 101 Exon 22 CACNA1A-E22F: 5'-tttgttgtgtaggaggccttgg-3' SEQ ID NO.: 102 amplification CACNA1A-E22Rv: 5'-aacatcccaccctacctatgag-3' SEQ ID NO.: 103
TABLE-US-00015 TABLE 14 Exon 23 CACNA1A-E23F: 5'-cctgcgcaactgtatatagcag-3' SEQ ID NO.: 104 amplification CACNA1A-E23Rv: 5'-ctcaacctcctgatctcaagtg-3' SEQ ID NO.: 105 Exon 24 CACNA1A-E24F: 5'-cccaaagtttggatctaagagcc-3' SEQ ID NO.: 106 amplification CACNA1A-E24Rv: 5'-aaagccatcgaagctcttcctg-3' SEQ ID NO.: 107 Exon 25 CACNA1A-E25F: 5'-caggtgaaatggaccactcttc-3' SEQ ID NO.: 108 amplification CACNA1A-E25Rv: 5'-tccttgagcagtgtacaacctg-3' SEQ ID NO.: 109 Exon 26 CACNA1A-E26F: 5'-gaatgccaggattgagtccaac-3' SEQ ID NO.: 110 amplification CACNA1A-E26Rv: 5'-gaatgtgctggaaagtggagac-3' SEQ ID NO.: 111 Exon 27 CACNA1A-E27F: 5'-cactgcttcccaagcagtctag-3' SEQ ID NO.: 112 amplification CACNA1A-E27Rv: 5'-attacaggcgtgagccaccatg-3' SEQ ID NO.: 113 Exon 28 CACNA1A-E28F: 5'-tttccctctgttcctgttctgc-3' SEQ ID NO.: 114 amplification CACNA1A-E28Rv: 5'-ttcggttgggacaatgcttctg-3' SEQ ID NO.: 115 Exon 29 CACNA1A-E29F: 5'-ctcaagcaactgtagctgttgg-3' SEQ ID NO.: 116 amplification CACNA1A-E29Rv: 5'-ttatcagggtagaggcaggaac-3' SEQ ID NO.: 117 Exon 30 CACNA1A-E30F: 5'-gtgaaaagaagagcctagtccg-3' SEQ ID NO.: 118 amplification CACNA1A-E30Rv: 5'-atggtaacactcacaggttggg-3' SEQ ID NO.: 119 Exon 31 CACNA1A-E31F: 5'-gcccttcgaacaaccataactg-3' SEQ ID NO.: 120 amplification CACNA1A-E31Rv: 5'-cctacagccaagctttggttac-3' SEQ ID NO.: 121 Exon 32 CACNA1A-E32F: 5'-cccattggttttttggcactgg-3' SEQ ID NO.: 122 amplification CACNA1A-E32Rv: 5'-ggacagacagacagaggagag-3' SEQ ID NO.: 123 Exon 33~35 CACNA1A-E33F: 5'-tgttggttggcttcatgtaggg-3' SEQ ID NO.: 124 amplification CACNA1A-E35Rv: 5'-cagaattatcagagcaggtccc-3' SEQ ID NO.: 125 Exon 36 CACNA1A-E36F: 5'-tctcagctcccagtaaaaggag-3' SEQ ID NO.: 126 amplification CACNA1A-E36Rv: 5'-caacagtgctgagtttgagacg-3' SEQ ID NO.: 127 Exon 37 CACNA1A-E37F: 5'-ggcctctgtgtacatgtctttg-3' SEQ ID NO.: 128 amplification CACNA1A-E37Rv: 5'-gggtatgcaagggtgatgattc-3' SEQ ID NO.: 129 Exon 38 CACNA1A-E38F: 5'-tgtttctccccacctctcttc-3' SEQ ID NO.: 130 amplification CACNA1A-E38Rv: 5'-aaaaaaacccagtgcctggacg-3' SEQ ID NO.: 131 Exon 39 CACNA1A-E39F: 5'-agaaactgagtactgggacagg-3' SEQ ID NO.: 132 amplification CACNA1A-E39Rv: 5'-ggaagagtgaatgaagatccgg-3' SEQ ID NO.: 133 Exon 40~41 CACNA1A-E40F: 5'-aaagattggggtctcgttctcg-3' SEQ ID NO.: 134 amplification CACNA1A-E41Rv: 5'-ccctcatattccagttggttcc-3' SEQ ID NO.: 135 Exon 42~44 CACNA1A-E42F: 5'-gtgtgtgtgtgtgtatactggg-3' SEQ ID NO.: 136 amplification CACNA1A-E44Rv: 5'-cagactgcttcagagactgaag-3' SEQ ID NO.: 137 Exon 45 CACNA1A-E45F: 5'-ccgatttctcttgatgccagtg-3' SEQ ID NO.: 138 amplification CACNA1A-E45Rv: 5'-agggtgcgattgccaaagaaag-3' SEQ ID NO.: 139 Exon 46~47 CACNA1A-E46F: 5'-acccagagccctgattgatcag-3' SEQ ID NO.: 140 amplification CACNA1A-E47Rv: 5'-ttggatggggtatccccttctc-3' SEQ ID NO.: 141 Exon 48 CACNA1A-E48F: 5'-tctcttcctcccaatcccgtg-3' SEQ ID NO.: 142 amplification CACNA1A-E48Rv: 5'-tgcccaggagggtctcttttg-3' SEQ ID NO.: 143
INDUSTRIAL APPLICABILITY
[0391] As described above, by detecting the presence of a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1, it is possible to obtain data for assessing a potential for development of Dravet syndrome of a subject who has not yet been subjected to onset of Dravet syndrome, with high accuracy. Hence, it is possible to distinguish a patient of Dravet syndrome that requires treatment by a specialist, out of benign febrile seizure patents, at an initial stage of disease under the age of one. Hence, it is possible to use not only in the field of diagnosis medical treatment such as medical devices, diagnosis kits and the like, but broadly in the health science and medical field industry.
[0392] Moreover, in the present invention, by introducing a mutation on both of α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1, it is possible to produce a model animal of Dravet syndrome. Such a model animal of Dravet syndrome can be used for development of medicament and treatment methods of Dravet syndrome. Hence, the present invention can be widely used in the industry of life science fields including the pharmaceutical field.
Sequence CWU
1
15212009PRTHomo sapiens 1Met Glu Gln Thr Val Leu Val Pro Pro Gly Pro Asp
Ser Phe Asn Phe1 5 10
15Phe Thr Arg Glu Ser Leu Ala Ala Ile Glu Arg Arg Ile Ala Glu Glu
20 25 30Lys Ala Lys Asn Pro Lys Pro
Asp Lys Lys Asp Asp Asp Glu Asn Gly 35 40
45Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Asn Leu Pro Phe
Ile 50 55 60Tyr Gly Asp Ile Pro Pro
Glu Met Val Ser Glu Pro Leu Glu Asp Leu65 70
75 80Asp Pro Tyr Tyr Ile Asn Lys Lys Thr Phe Ile
Val Leu Asn Lys Gly 85 90
95Lys Ala Ile Phe Arg Phe Ser Ala Thr Ser Ala Leu Tyr Ile Leu Thr
100 105 110Pro Phe Asn Pro Leu Arg
Lys Ile Ala Ile Lys Ile Leu Val His Ser 115 120
125Leu Phe Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys
Val Phe 130 135 140Met Thr Met Ser Asn
Pro Pro Asp Trp Thr Lys Asn Val Glu Tyr Thr145 150
155 160Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu
Ile Lys Ile Ile Ala Arg 165 170
175Gly Phe Cys Leu Glu Asp Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp
180 185 190Leu Asp Phe Thr Val
Ile Thr Phe Ala Tyr Val Thr Glu Phe Val Asp 195
200 205Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val
Leu Arg Ala Leu 210 215 220Lys Thr Ile
Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu225
230 235 240Ile Gln Ser Val Lys Lys Leu
Ser Asp Val Met Ile Leu Thr Val Phe 245
250 255Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu
Phe Met Gly Asn 260 265 270Leu
Arg Asn Lys Cys Ile Gln Trp Pro Pro Thr Asn Ala Ser Leu Glu 275
280 285Glu His Ser Ile Glu Lys Asn Ile Thr
Val Asn Tyr Asn Gly Thr Leu 290 295
300Ile Asn Glu Thr Val Phe Glu Phe Asp Trp Lys Ser Tyr Ile Gln Asp305
310 315 320Ser Arg Tyr His
Tyr Phe Leu Glu Gly Phe Leu Asp Ala Leu Leu Cys 325
330 335Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro
Glu Gly Tyr Met Cys Val 340 345
350Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser Phe Asp Thr Phe
355 360 365Ser Trp Ala Phe Leu Ser Leu
Phe Arg Leu Met Thr Gln Asp Phe Trp 370 375
380Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly Lys Thr Tyr
Met385 390 395 400Ile Phe
Phe Val Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn
405 410 415Leu Ile Leu Ala Val Val Ala
Met Ala Tyr Glu Glu Gln Asn Gln Ala 420 425
430Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe Gln Gln
Met Ile 435 440 445Glu Gln Leu Lys
Lys Gln Gln Glu Ala Ala Gln Gln Ala Ala Thr Ala 450
455 460Thr Ala Ser Glu His Ser Arg Glu Pro Ser Ala Ala
Gly Arg Leu Ser465 470 475
480Asp Ser Ser Ser Glu Ala Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu
485 490 495Arg Arg Asn Arg Arg
Lys Lys Arg Lys Gln Lys Glu Gln Ser Gly Gly 500
505 510Glu Glu Lys Asp Glu Asp Glu Phe Gln Lys Ser Glu
Ser Glu Asp Ser 515 520 525Ile Arg
Arg Lys Gly Phe Arg Phe Ser Ile Glu Gly Asn Arg Leu Thr 530
535 540Tyr Glu Lys Arg Tyr Ser Ser Pro His Gln Ser
Leu Leu Ser Ile Arg545 550 555
560Gly Ser Leu Phe Ser Pro Arg Arg Asn Ser Arg Thr Ser Leu Phe Ser
565 570 575Phe Arg Gly Arg
Ala Lys Asp Val Gly Ser Glu Asn Asp Phe Ala Asp 580
585 590Asp Glu His Ser Thr Phe Glu Asp Asn Glu Ser
Arg Arg Asp Ser Leu 595 600 605Phe
Val Pro Arg Arg His Gly Glu Arg Arg Asn Ser Asn Leu Ser Gln 610
615 620Thr Ser Arg Ser Ser Arg Met Leu Ala Val
Phe Pro Ala Asn Gly Lys625 630 635
640Met His Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Val Gly
Gly 645 650 655Pro Ser Val
Pro Thr Ser Pro Val Gly Gln Leu Leu Pro Glu Val Ile 660
665 670Ile Asp Lys Pro Ala Thr Asp Asp Asn Gly
Thr Thr Thr Glu Thr Glu 675 680
685Met Arg Lys Arg Arg Ser Ser Ser Phe His Val Ser Met Asp Phe Leu 690
695 700Glu Asp Pro Ser Gln Arg Gln Arg
Ala Met Ser Ile Ala Ser Ile Leu705 710
715 720Thr Asn Thr Val Glu Glu Leu Glu Glu Ser Arg Gln
Lys Cys Pro Pro 725 730
735Cys Trp Tyr Lys Phe Ser Asn Ile Phe Leu Ile Trp Asp Cys Ser Pro
740 745 750Tyr Trp Leu Lys Val Lys
His Val Val Asn Leu Val Val Met Asp Pro 755 760
765Phe Val Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr
Leu Phe 770 775 780Met Ala Met Glu His
Tyr Pro Met Thr Asp His Phe Asn Asn Val Leu785 790
795 800Thr Val Gly Asn Leu Val Phe Thr Gly Ile
Phe Thr Ala Glu Met Phe 805 810
815Leu Lys Ile Ile Ala Met Asp Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp
820 825 830Asn Ile Phe Asp Gly
Phe Ile Val Thr Leu Ser Leu Val Glu Leu Gly 835
840 845Leu Ala Asn Val Glu Gly Leu Ser Val Leu Arg Ser
Phe Arg Leu Leu 850 855 860Arg Val Phe
Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile865
870 875 880Lys Ile Ile Gly Asn Ser Val
Gly Ala Leu Gly Asn Leu Thr Leu Val 885
890 895Leu Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly
Met Gln Leu Phe 900 905 910Gly
Lys Ser Tyr Lys Asp Cys Val Cys Lys Ile Ala Ser Asp Cys Gln 915
920 925Leu Pro Arg Trp His Met Asn Asp Phe
Phe His Ser Phe Leu Ile Val 930 935
940Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met945
950 955 960Glu Val Ala Gly
Gln Ala Met Cys Leu Thr Val Phe Met Met Val Met 965
970 975Val Ile Gly Asn Leu Val Val Leu Asn Leu
Phe Leu Ala Leu Leu Leu 980 985
990Ser Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp Asp Asn Glu
995 1000 1005Met Asn Asn Leu Gln Ile
Ala Val Asp Arg Met His Lys Gly Val 1010 1015
1020Ala Tyr Val Lys Arg Lys Ile Tyr Glu Phe Ile Gln Gln Ser
Phe 1025 1030 1035Ile Arg Lys Gln Lys
Ile Leu Asp Glu Ile Lys Pro Leu Asp Asp 1040 1045
1050Leu Asn Asn Lys Lys Asp Ser Cys Met Ser Asn His Thr
Thr Glu 1055 1060 1065Ile Gly Lys Asp
Leu Asp Tyr Leu Lys Asp Val Asn Gly Thr Thr 1070
1075 1080Ser Gly Ile Gly Thr Gly Ser Ser Val Glu Lys
Tyr Ile Ile Asp 1085 1090 1095Glu Ser
Asp Tyr Met Ser Phe Ile Asn Asn Pro Ser Leu Thr Val 1100
1105 1110Thr Val Pro Ile Ala Val Gly Glu Ser Asp
Phe Glu Asn Leu Asn 1115 1120 1125Thr
Glu Asp Phe Ser Ser Glu Ser Asp Leu Glu Glu Ser Lys Glu 1130
1135 1140Lys Leu Asn Glu Ser Ser Ser Ser Ser
Glu Gly Ser Thr Val Asp 1145 1150
1155Ile Gly Ala Pro Val Glu Glu Gln Pro Val Val Glu Pro Glu Glu
1160 1165 1170Thr Leu Glu Pro Glu Ala
Cys Phe Thr Glu Gly Cys Val Gln Arg 1175 1180
1185Phe Lys Cys Cys Gln Ile Asn Val Glu Glu Gly Arg Gly Lys
Gln 1190 1195 1200Trp Trp Asn Leu Arg
Arg Thr Cys Phe Arg Ile Val Glu His Asn 1205 1210
1215Trp Phe Glu Thr Phe Ile Val Phe Met Ile Leu Leu Ser
Ser Gly 1220 1225 1230Ala Leu Ala Phe
Glu Asp Ile Tyr Ile Asp Gln Arg Lys Thr Ile 1235
1240 1245Lys Thr Met Leu Glu Tyr Ala Asp Lys Val Phe
Thr Tyr Ile Phe 1250 1255 1260Ile Leu
Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Tyr Gln Thr 1265
1270 1275Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp
Phe Leu Ile Val Asp 1280 1285 1290Val
Ser Leu Val Ser Leu Thr Ala Asn Ala Leu Gly Tyr Ser Glu 1295
1300 1305Leu Gly Ala Ile Lys Ser Leu Arg Thr
Leu Arg Ala Leu Arg Pro 1310 1315
1320Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn
1325 1330 1335Ala Leu Leu Gly Ala Ile
Pro Ser Ile Met Asn Val Leu Leu Val 1340 1345
1350Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn
Leu 1355 1360 1365Phe Ala Gly Lys Phe
Tyr His Cys Ile Asn Thr Thr Thr Gly Asp 1370 1375
1380Arg Phe Asp Ile Glu Asp Val Asn Asn His Thr Asp Cys
Leu Lys 1385 1390 1395Leu Ile Glu Arg
Asn Glu Thr Ala Arg Trp Lys Asn Val Lys Val 1400
1405 1410Asn Phe Asp Asn Val Gly Phe Gly Tyr Leu Ser
Leu Leu Gln Val 1415 1420 1425Ala Thr
Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp 1430
1435 1440Ser Arg Asn Val Glu Leu Gln Pro Lys Tyr
Glu Glu Ser Leu Tyr 1445 1450 1455Met
Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe 1460
1465 1470Thr Leu Asn Leu Phe Ile Gly Val Ile
Ile Asp Asn Phe Asn Gln 1475 1480
1485Gln Lys Lys Lys Phe Gly Gly Gln Asp Ile Phe Met Thr Glu Glu
1490 1495 1500Gln Lys Lys Tyr Tyr Asn
Ala Met Lys Lys Leu Gly Ser Lys Lys 1505 1510
1515Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe Gln Gly
Met 1520 1525 1530Val Phe Asp Phe Val
Thr Arg Gln Val Phe Asp Ile Ser Ile Met 1535 1540
1545Ile Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu
Thr Asp 1550 1555 1560Asp Gln Ser Glu
Tyr Val Thr Thr Ile Leu Ser Arg Ile Asn Leu 1565
1570 1575Val Phe Ile Val Leu Phe Thr Gly Glu Cys Val
Leu Lys Leu Ile 1580 1585 1590Ser Leu
Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn Ile Phe Asp 1595
1600 1605Phe Val Val Val Ile Leu Ser Ile Val Gly
Met Phe Leu Ala Glu 1610 1615 1620Leu
Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile 1625
1630 1635Arg Leu Ala Arg Ile Gly Arg Ile Leu
Arg Leu Ile Lys Gly Ala 1640 1645
1650Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro
1655 1660 1665Ala Leu Phe Asn Ile Gly
Leu Leu Leu Phe Leu Val Met Phe Ile 1670 1675
1680Tyr Ala Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Arg
Glu 1685 1690 1695Val Gly Ile Asp Asp
Met Phe Asn Phe Glu Thr Phe Gly Asn Ser 1700 1705
1710Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp
Asp Gly 1715 1720 1725Leu Leu Ala Pro
Ile Leu Asn Ser Lys Pro Pro Asp Cys Asp Pro 1730
1735 1740Asn Lys Val Asn Pro Gly Ser Ser Val Lys Gly
Asp Cys Gly Asn 1745 1750 1755Pro Ser
Val Gly Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile Ser 1760
1765 1770Phe Leu Val Val Val Asn Met Tyr Ile Ala
Val Ile Leu Glu Asn 1775 1780 1785Phe
Ser Val Ala Thr Glu Glu Ser Ala Glu Pro Leu Ser Glu Asp 1790
1795 1800Asp Phe Glu Met Phe Tyr Glu Val Trp
Glu Lys Phe Asp Pro Asp 1805 1810
1815Ala Thr Gln Phe Met Glu Phe Glu Lys Leu Ser Gln Phe Ala Ala
1820 1825 1830Ala Leu Glu Pro Pro Leu
Asn Leu Pro Gln Pro Asn Lys Leu Gln 1835 1840
1845Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile
His 1850 1855 1860Cys Leu Asp Ile Leu
Phe Ala Phe Thr Lys Arg Val Leu Gly Glu 1865 1870
1875Ser Gly Glu Met Asp Ala Leu Arg Ile Gln Met Glu Glu
Arg Phe 1880 1885 1890Met Ala Ser Asn
Pro Ser Lys Val Ser Tyr Gln Pro Ile Thr Thr 1895
1900 1905Thr Leu Lys Arg Lys Gln Glu Glu Val Ser Ala
Val Ile Ile Gln 1910 1915 1920Arg Ala
Tyr Arg Arg His Leu Leu Lys Arg Thr Val Lys Gln Ala 1925
1930 1935Ser Phe Thr Tyr Asn Lys Asn Lys Ile Lys
Gly Gly Ala Asn Leu 1940 1945 1950Leu
Ile Lys Glu Asp Met Ile Ile Asp Arg Ile Asn Glu Asn Ser 1955
1960 1965Ile Thr Glu Lys Thr Asp Leu Thr Met
Ser Thr Ala Ala Cys Pro 1970 1975
1980Pro Ser Tyr Asp Arg Val Thr Lys Pro Ile Val Glu Lys His Glu
1985 1990 1995Gln Glu Gly Lys Asp Glu
Lys Ala Lys Gly Lys 2000 200526030DNAHomo sapiens
2atggagcaaa cagtgcttgt accaccagga cctgacagct tcaacttctt caccagagaa
60tctcttgcgg ctattgaaag acgcattgca gaagaaaagg caaagaatcc caaaccagac
120aaaaaagatg acgacgaaaa tggcccaaag ccaaatagtg acttggaagc tggaaagaac
180cttccattta tttatggaga cattcctcca gagatggtgt cagagcccct ggaggacctg
240gacccctact atatcaataa gaaaactttt atagtattga ataaagggaa ggccatcttc
300cggttcagtg ccacctctgc cctgtacatt ttaactccct tcaatcctct taggaaaata
360gctattaaga ttttggtaca ttcattattc agcatgctaa ttatgtgcac tattttgaca
420aactgtgtgt ttatgacaat gagtaaccct cctgattgga caaagaatgt agaatacacc
480ttcacaggaa tatatacttt tgaatcactt ataaaaatta ttgcaagggg attctgttta
540gaagatttta ctttccttcg ggatccatgg aactggctcg atttcactgt cattacattt
600gcgtacgtca cagagtttgt ggacctgggc aatgtctcgg cattgagaac attcagagtt
660ctccgagcat tgaagacgat ttcagtcatt ccaggcctga aaaccattgt gggagccctg
720atccagtctg tgaagaagct ctcagatgta atgatcctga ctgtgttctg tctgagcgta
780tttgctctaa ttgggctgca gctgttcatg ggcaacctga ggaataaatg tatacaatgg
840cctcccacca atgcttcctt ggaggaacat agtatagaaa agaatataac tgtgaattat
900aatggtacac ttataaatga aactgtcttt gagtttgact ggaagtcata tattcaagat
960tcaagatatc attatttcct ggagggtttt ttagatgcac tactatgtgg aaatagctct
1020gatgcaggcc aatgtccaga gggatatatg tgtgtgaaag ctggtagaaa tcccaattat
1080ggctacacaa gctttgatac cttcagttgg gcttttttgt ccttgtttcg actaatgact
1140caggacttct gggaaaatct ttatcaactg acattacgtg ctgctgggaa aacgtacatg
1200atattttttg tattggtcat tttcttgggc tcattctacc taataaattt gatcctggct
1260gtggtggcca tggcctacga ggaacagaat caggccacct tggaagaagc agaacagaaa
1320gaggccgaat ttcagcagat gattgaacag cttaaaaagc aacaggaggc agctcagcag
1380gcagcaacgg caactgcctc agaacattcc agagagccca gtgcagcagg caggctctca
1440gacagctcat ctgaagcctc taagttgagt tccaagagtg ctaaggaaag aagaaatcgg
1500aggaagaaaa gaaaacagaa agagcagtct ggtggggaag agaaagatga ggatgaattc
1560caaaaatctg aatctgagga cagcatcagg aggaaaggtt ttcgcttctc cattgaaggg
1620aaccgattga catatgaaaa gaggtactcc tccccacacc agtctttgtt gagcatccgt
1680ggctccctat tttcaccaag gcgaaatagc agaacaagcc ttttcagctt tagagggcga
1740gcaaaggatg tgggatctga gaacgacttc gcagatgatg agcacagcac ctttgaggat
1800aacgagagcc gtagagattc cttgtttgtg ccccgacgac acggagagag acgcaacagc
1860aacctgagtc agaccagtag gtcatcccgg atgctggcag tgtttccagc gaatgggaag
1920atgcacagca ctgtggattg caatggtgtg gtttccttgg ttggtggacc ttcagttcct
1980acatcgcctg ttggacagct tctgccagag gtgataatag ataagccagc tactgatgac
2040aatggaacaa ccactgaaac tgaaatgaga aagagaaggt caagttcttt ccacgtttcc
2100atggactttc tagaagatcc ttcccaaagg caacgagcaa tgagtatagc cagcattcta
2160acaaatacag tagaagaact tgaagaatcc aggcagaaat gcccaccctg ttggtataaa
2220ttttccaaca tattcttaat ctgggactgt tctccatatt ggttaaaagt gaaacatgtt
2280gtcaacctgg ttgtgatgga cccatttgtt gacctggcca tcaccatctg tattgtctta
2340aatactcttt tcatggccat ggagcactat ccaatgacgg accatttcaa taatgtgctt
2400acagtaggaa acttggtttt cactgggatc tttacagcag aaatgtttct gaaaattatt
2460gccatggatc cttactatta tttccaagaa ggctggaata tctttgacgg ttttattgtg
2520acgcttagcc tggtagaact tggactcgcc aatgtggaag gattatctgt tctccgttca
2580tttcgattgc tgcgagtttt caagttggca aaatcttggc caacgttaaa tatgctaata
2640aagatcatcg gcaattccgt gggggctctg ggaaatttaa ccctcgtctt ggccatcatc
2700gtcttcattt ttgccgtggt cggcatgcag ctctttggta aaagctacaa agattgtgtc
2760tgcaagatcg ccagtgattg tcaactccca cgctggcaca tgaatgactt cttccactcc
2820ttcctgattg tgttccgcgt gctgtgtggg gagtggatag agaccatgtg ggactgtatg
2880gaggttgctg gtcaagccat gtgccttact gtcttcatga tggtcatggt gattggaaac
2940ctagtggtcc tgaatctctt tctggccttg cttctgagct catttagtgc agacaacctt
3000gcagccactg atgatgataa tgaaatgaat aatctccaaa ttgctgtgga taggatgcac
3060aaaggagtag cttatgtgaa aagaaaaata tatgaattta ttcaacagtc cttcattagg
3120aaacaaaaga ttttagatga aattaaacca cttgatgatc taaacaacaa gaaagacagt
3180tgtatgtcca atcatacaac agaaattggg aaagatcttg actatcttaa agatgtaaat
3240ggaactacaa gtggtatagg aactggcagc agtgttgaaa aatacattat tgatgaaagt
3300gattacatgt cattcataaa caaccccagt cttactgtga ctgtaccaat tgctgtagga
3360gaatctgact ttgaaaattt aaacacggaa gactttagta gtgaatcgga tctggaagaa
3420agcaaagaga aactgaatga aagcagtagc tcatcagaag gtagcactgt ggacatcggc
3480gcacctgtag aagaacagcc cgtagtggaa cctgaagaaa ctcttgaacc agaagcttgt
3540ttcactgaag gctgtgtaca aagattcaag tgttgtcaaa tcaatgtgga agaaggcaga
3600ggaaaacaat ggtggaacct gagaaggacg tgtttccgaa tagttgaaca taactggttt
3660gagaccttca ttgttttcat gattctcctt agtagtggtg ctctggcatt tgaagatata
3720tatattgatc agcgaaagac gattaagacg atgttggaat atgctgacaa ggttttcact
3780tacattttca ttctggaaat gcttctaaaa tgggtggcat atggctatca aacatatttc
3840accaatgcct ggtgttggct ggacttctta attgttgatg tttcattggt cagtttaaca
3900gcaaatgcct tgggttactc agaacttgga gccatcaaat ctctcaggac actaagagct
3960ctgagacctc taagagcctt atctcgattt gaagggatga gggtggttgt gaatgccctt
4020ttaggagcaa ttccatccat catgaatgtg cttctggttt gtcttatatt ctggctaatt
4080ttcagcatca tgggcgtaaa tttgtttgct ggcaaattct accactgtat taacaccaca
4140actggtgaca ggtttgacat cgaagacgtg aataatcata ctgattgcct aaaactaata
4200gaaagaaatg agactgctcg atggaaaaat gtgaaagtaa actttgataa tgtaggattt
4260gggtatctct ctttgcttca agttgccaca ttcaaaggat ggatggatat aatgtatgca
4320gcagttgatt ccagaaatgt ggaactccag cctaagtatg aagaaagtct gtacatgtat
4380ctttactttg ttattttcat catctttggg tccttcttca ccttgaacct gtttattggt
4440gtcatcatag ataatttcaa ccagcagaaa aagaagtttg gaggtcaaga catctttatg
4500acagaagaac agaagaaata ctataatgca atgaaaaaat taggatcgaa aaaaccgcaa
4560aagcctatac ctcgaccagg aaacaaattt caaggaatgg tctttgactt cgtaaccaga
4620caagtttttg acataagcat catgattctc atctgtctta acatggtcac aatgatggtg
4680gaaacagatg accagagtga atatgtgact accattttgt cacgcatcaa tctggtgttc
4740attgtgctat ttactggaga gtgtgtactg aaactcatct ctctacgcca ttattatttt
4800accattggat ggaatatttt tgattttgtg gttgtcattc tctccattgt aggtatgttt
4860cttgccgagc tgatagaaaa gtatttcgtg tcccctaccc tgttccgagt gatccgtctt
4920gctaggattg gccgaatcct acgtctgatc aaaggagcaa aggggatccg cacgctgctc
4980tttgctttga tgatgtccct tcctgcgttg tttaacatcg gcctcctact cttcctagtc
5040atgttcatct acgccatctt tgggatgtcc aactttgcct atgttaagag ggaagttggg
5100atcgatgaca tgttcaactt tgagaccttt ggcaacagca tgatctgcct attccaaatt
5160acaacctctg ctggctggga tggattgcta gcacccattc tcaacagtaa gccacccgac
5220tgtgacccta ataaagttaa ccctggaagc tcagttaagg gagactgtgg gaacccatct
5280gttggaattt tcttttttgt cagttacatc atcatatcct tcctggttgt ggtgaacatg
5340tacatcgcgg tcatcctgga gaacttcagt gttgctactg aagaaagtgc agagcctctg
5400agtgaggatg actttgagat gttctatgag gtttgggaga agtttgatcc cgatgcaact
5460cagttcatgg aatttgaaaa attatctcag tttgcagctg cgcttgaacc gcctctcaat
5520ctgccacaac caaacaaact ccagctcatt gccatggatt tgcccatggt gagtggtgac
5580cggatccact gtcttgatat cttatttgct tttacaaagc gggttctagg agagagtgga
5640gagatggatg ctctacgaat acagatggaa gagcgattca tggcttccaa tccttccaag
5700gtctcctatc agccaatcac tactacttta aaacgaaaac aagaggaagt atctgctgtc
5760attattcagc gtgcttacag acgccacctt ttaaagcgaa ctgtaaaaca agcttccttt
5820acgtacaata aaaacaaaat caaaggtggg gctaatcttc ttataaaaga agacatgata
5880attgacagaa taaatgaaaa ctctattaca gaaaaaactg atctgaccat gtccactgca
5940gcttgtccac cttcctatga ccgggtgaca aagccaattg tggaaaaaca tgagcaagaa
6000ggcaaagatg aaaaagccaa agggaaataa
603032512PRTHomo sapiens 3Met Ala Arg Phe Gly Asp Glu Met Pro Ala Arg Tyr
Gly Gly Gly Gly1 5 10
15Ser Gly Ala Ala Ala Gly Val Val Val Gly Ser Gly Gly Gly Arg Gly
20 25 30Ala Gly Gly Ser Arg Gln Gly
Gly Gln Pro Gly Ala Gln Arg Met Tyr 35 40
45Lys Gln Ser Met Ala Gln Arg Ala Arg Thr Met Ala Leu Tyr Asn
Pro 50 55 60Ile Pro Val Arg Gln Asn
Cys Leu Thr Val Asn Arg Ser Leu Phe Leu65 70
75 80Phe Ser Glu Asp Asn Val Val Arg Lys Tyr Ala
Lys Lys Ile Thr Glu 85 90
95Trp Pro Pro Phe Glu Tyr Met Ile Leu Ala Thr Ile Ile Ala Asn Cys
100 105 110Ile Val Leu Ala Leu Glu
Gln His Leu Pro Asp Asp Asp Lys Thr Pro 115 120
125Met Ser Glu Arg Leu Asp Asp Thr Glu Pro Tyr Phe Ile Gly
Ile Phe 130 135 140Cys Phe Glu Ala Gly
Ile Lys Ile Ile Ala Leu Gly Phe Ala Phe His145 150
155 160Lys Gly Ser Tyr Leu Arg Asn Gly Trp Asn
Val Met Asp Phe Val Val 165 170
175Val Leu Thr Gly Ile Leu Ala Thr Val Gly Thr Glu Phe Asp Leu Arg
180 185 190Thr Leu Arg Ala Val
Arg Val Leu Arg Pro Leu Lys Leu Val Ser Gly 195
200 205Ile Pro Ser Leu Gln Val Val Leu Lys Ser Ile Met
Lys Ala Met Ile 210 215 220Pro Leu Leu
Gln Ile Gly Leu Leu Leu Phe Phe Ala Ile Leu Ile Phe225
230 235 240Ala Ile Ile Gly Leu Glu Phe
Tyr Met Gly Lys Phe His Thr Thr Cys 245
250 255Phe Glu Glu Gly Thr Asp Asp Ile Gln Gly Glu Ser
Pro Ala Pro Cys 260 265 270Gly
Thr Glu Glu Pro Ala Arg Thr Cys Pro Asn Gly Thr Lys Cys Gln 275
280 285Pro Tyr Trp Glu Gly Pro Asn Asn Gly
Ile Thr Gln Phe Asp Asn Ile 290 295
300Leu Phe Ala Val Leu Thr Val Phe Gln Cys Ile Thr Met Glu Gly Trp305
310 315 320Thr Asp Leu Leu
Tyr Asn Ser Asn Asp Ala Ser Gly Asn Thr Trp Asn 325
330 335Trp Leu Tyr Phe Ile Pro Leu Ile Ile Ile
Gly Ser Phe Phe Met Leu 340 345
350Asn Leu Val Leu Gly Val Leu Ser Gly Glu Phe Ala Lys Glu Arg Glu
355 360 365Arg Val Glu Asn Arg Arg Ala
Phe Leu Lys Leu Arg Arg Gln Gln Gln 370 375
380Ile Glu Arg Glu Leu Asn Gly Tyr Met Glu Trp Ile Ser Lys Ala
Glu385 390 395 400Glu Val
Ile Leu Ala Glu Asp Glu Thr Asp Gly Glu Gln Arg His Pro
405 410 415Phe Asp Gly Ala Leu Arg Arg
Thr Thr Ile Lys Lys Ser Lys Thr Asp 420 425
430Leu Leu Asn Pro Glu Glu Ala Glu Asp Gln Leu Ala Asp Ile
Ala Ser 435 440 445Val Gly Ser Pro
Phe Ala Arg Ala Ser Ile Lys Ser Ala Lys Leu Glu 450
455 460Asn Ser Thr Phe Phe His Lys Lys Glu Arg Arg Met
Arg Phe Tyr Ile465 470 475
480Arg Arg Met Val Lys Thr Gln Ala Phe Tyr Trp Thr Val Leu Ser Leu
485 490 495Val Ala Leu Asn Thr
Leu Cys Val Ala Ile Val His Tyr Asn Gln Pro 500
505 510Glu Trp Leu Ser Asp Phe Leu Tyr Tyr Ala Glu Phe
Ile Phe Leu Gly 515 520 525Leu Phe
Met Ser Glu Met Phe Ile Lys Met Tyr Gly Leu Gly Thr Arg 530
535 540Pro Tyr Phe His Ser Ser Phe Asn Cys Phe Asp
Cys Gly Val Ile Ile545 550 555
560Gly Ser Ile Phe Glu Val Ile Trp Ala Val Ile Lys Pro Gly Thr Ser
565 570 575Phe Gly Ile Ser
Val Leu Arg Ala Leu Arg Leu Leu Arg Ile Phe Lys 580
585 590Val Thr Lys Tyr Trp Ala Ser Leu Arg Asn Leu
Val Val Ser Leu Leu 595 600 605Asn
Ser Met Lys Ser Ile Ile Ser Leu Leu Phe Leu Leu Phe Leu Phe 610
615 620Ile Val Val Phe Ala Leu Leu Gly Met Gln
Leu Phe Gly Gly Gln Phe625 630 635
640Asn Phe Asp Glu Gly Thr Pro Pro Thr Asn Phe Asp Thr Phe Pro
Ala 645 650 655Ala Ile Met
Thr Val Phe Gln Ile Leu Thr Gly Glu Asp Trp Asn Glu 660
665 670Val Met Tyr Asp Gly Ile Lys Ser Gln Gly
Gly Val Gln Gly Gly Met 675 680
685Val Phe Ser Ile Tyr Phe Ile Val Leu Thr Leu Phe Gly Asn Tyr Thr 690
695 700Leu Leu Asn Val Phe Leu Ala Ile
Ala Val Asp Asn Leu Ala Asn Ala705 710
715 720Gln Glu Leu Thr Lys Val Glu Ala Asp Glu Gln Glu
Glu Glu Glu Ala 725 730
735Ala Asn Gln Lys Leu Ala Leu Gln Lys Ala Lys Glu Val Ala Glu Val
740 745 750Ser Pro Leu Ser Ala Ala
Asn Met Ser Ile Ala Val Lys Glu Gln Gln 755 760
765Lys Asn Gln Lys Pro Ala Lys Ser Val Trp Glu Gln Arg Thr
Ser Glu 770 775 780Met Arg Lys Gln Asn
Leu Leu Ala Ser Arg Glu Ala Leu Tyr Asn Glu785 790
795 800Met Asp Pro Asp Glu Arg Trp Lys Ala Ala
Tyr Thr Arg His Leu Arg 805 810
815Pro Asp Met Lys Thr His Leu Asp Arg Pro Leu Val Val Asp Pro Gln
820 825 830Glu Asn Arg Asn Asn
Asn Thr Asn Lys Ser Arg Ala Ala Glu Pro Thr 835
840 845Val Asp Gln Arg Leu Gly Gln Gln Arg Ala Glu Asp
Phe Leu Arg Lys 850 855 860Gln Ala Arg
Tyr His Asp Arg Ala Arg Asp Pro Ser Gly Ser Ala Gly865
870 875 880Leu Asp Ala Arg Arg Pro Trp
Ala Gly Ser Gln Glu Ala Glu Leu Ser 885
890 895Arg Glu Gly Pro Tyr Gly Arg Glu Ser Asp His His
Ala Arg Glu Gly 900 905 910Ser
Leu Glu Gln Pro Gly Phe Trp Glu Gly Glu Ala Glu Arg Gly Lys 915
920 925Ala Gly Asp Pro His Arg Arg His Val
His Arg Gln Gly Gly Ser Arg 930 935
940Glu Ser Arg Ser Gly Ser Pro Arg Thr Gly Ala Asp Gly Glu His Arg945
950 955 960Arg His Arg Ala
His Arg Arg Pro Gly Glu Glu Gly Pro Glu Asp Lys 965
970 975Ala Glu Arg Arg Ala Arg His Arg Glu Gly
Ser Arg Pro Ala Arg Gly 980 985
990Gly Glu Gly Glu Gly Glu Gly Pro Asp Gly Gly Glu Arg Arg Arg Arg
995 1000 1005His Arg His Gly Ala Pro
Ala Thr Tyr Glu Gly Asp Ala Arg Arg 1010 1015
1020Glu Asp Lys Glu Arg Arg His Arg Arg Arg Lys Glu Asn Gln
Gly 1025 1030 1035Ser Gly Val Pro Val
Ser Gly Pro Asn Leu Ser Thr Thr Arg Pro 1040 1045
1050Ile Gln Gln Asp Leu Gly Arg Gln Asp Pro Pro Leu Ala
Glu Asp 1055 1060 1065Ile Asp Asn Met
Lys Asn Asn Lys Leu Ala Thr Ala Glu Ser Ala 1070
1075 1080Ala Pro His Gly Ser Leu Gly His Ala Gly Leu
Pro Gln Ser Pro 1085 1090 1095Ala Lys
Met Gly Asn Ser Thr Asp Pro Gly Pro Met Leu Ala Ile 1100
1105 1110Pro Ala Met Ala Thr Asn Pro Gln Asn Ala
Ala Ser Arg Arg Thr 1115 1120 1125Pro
Asn Asn Pro Gly Asn Pro Ser Asn Pro Gly Pro Pro Lys Thr 1130
1135 1140Pro Glu Asn Ser Leu Ile Val Thr Asn
Pro Ser Gly Thr Gln Thr 1145 1150
1155Asn Ser Ala Lys Thr Ala Arg Lys Pro Asp His Thr Thr Val Asp
1160 1165 1170Ile Pro Pro Ala Cys Pro
Pro Pro Leu Asn His Thr Val Val Gln 1175 1180
1185Val Asn Lys Asn Ala Asn Pro Asp Pro Leu Pro Lys Lys Glu
Glu 1190 1195 1200Glu Lys Lys Glu Glu
Glu Glu Asp Asp Arg Gly Glu Asp Gly Pro 1205 1210
1215Lys Pro Met Pro Pro Tyr Ser Ser Met Phe Ile Leu Ser
Thr Thr 1220 1225 1230Asn Pro Leu Arg
Arg Leu Cys His Tyr Ile Leu Asn Leu Arg Tyr 1235
1240 1245Phe Glu Met Cys Ile Leu Met Val Ile Ala Met
Ser Ser Ile Ala 1250 1255 1260Leu Ala
Ala Glu Asp Pro Val Gln Pro Asn Ala Pro Arg Asn Asn 1265
1270 1275Val Leu Arg Tyr Phe Asp Tyr Val Phe Thr
Gly Val Phe Thr Phe 1280 1285 1290Glu
Met Val Ile Lys Met Ile Asp Leu Gly Leu Val Leu His Gln 1295
1300 1305Gly Ala Tyr Phe Arg Asp Leu Trp Asn
Ile Leu Asp Phe Ile Val 1310 1315
1320Val Ser Gly Ala Leu Val Ala Phe Ala Phe Thr Gly Asn Ser Lys
1325 1330 1335Gly Lys Asp Ile Asn Thr
Ile Lys Ser Leu Arg Val Leu Arg Val 1340 1345
1350Leu Arg Pro Leu Lys Thr Ile Lys Arg Leu Pro Lys Leu Lys
Ala 1355 1360 1365Val Phe Asp Cys Val
Val Asn Ser Leu Lys Asn Val Phe Asn Ile 1370 1375
1380Leu Ile Val Tyr Met Leu Phe Met Phe Ile Phe Ala Val
Val Ala 1385 1390 1395Val Gln Leu Phe
Lys Gly Lys Phe Phe His Cys Thr Asp Glu Ser 1400
1405 1410Lys Glu Phe Glu Lys Asp Cys Arg Gly Lys Tyr
Leu Leu Tyr Glu 1415 1420 1425Lys Asn
Glu Val Lys Ala Arg Asp Arg Glu Trp Lys Lys Tyr Glu 1430
1435 1440Phe His Tyr Asp Asn Val Leu Trp Ala Leu
Leu Thr Leu Phe Thr 1445 1450 1455Val
Ser Thr Gly Glu Gly Trp Pro Gln Val Leu Lys His Ser Val 1460
1465 1470Asp Ala Thr Phe Glu Asn Gln Gly Pro
Ser Pro Gly Tyr Arg Met 1475 1480
1485Glu Met Ser Ile Phe Tyr Val Val Tyr Phe Val Val Phe Pro Phe
1490 1495 1500Phe Phe Val Asn Ile Phe
Val Ala Leu Ile Ile Ile Thr Phe Gln 1505 1510
1515Glu Gln Gly Asp Lys Met Met Glu Glu Tyr Ser Leu Glu Lys
Asn 1520 1525 1530Glu Arg Ala Cys Ile
Asp Phe Ala Ile Ser Ala Lys Pro Leu Thr 1535 1540
1545Arg His Met Pro Gln Asn Lys Gln Ser Phe Gln Tyr Arg
Met Trp 1550 1555 1560Gln Phe Val Val
Ser Pro Pro Phe Glu Tyr Thr Ile Met Ala Met 1565
1570 1575Ile Ala Leu Asn Thr Ile Val Leu Met Met Lys
Phe Tyr Gly Ala 1580 1585 1590Ser Val
Ala Tyr Glu Asn Ala Leu Arg Val Phe Asn Ile Val Phe 1595
1600 1605Thr Ser Leu Phe Ser Leu Glu Cys Val Leu
Lys Val Met Ala Phe 1610 1615 1620Gly
Ile Leu Asn Tyr Phe Arg Asp Ala Trp Asn Ile Phe Asp Phe 1625
1630 1635Val Thr Val Leu Gly Ser Ile Thr Asp
Ile Leu Val Thr Glu Phe 1640 1645
1650Gly Asn Pro Asn Asn Phe Ile Asn Leu Ser Phe Leu Arg Leu Phe
1655 1660 1665Arg Ala Ala Arg Leu Ile
Lys Leu Leu Arg Gln Gly Tyr Thr Ile 1670 1675
1680Arg Ile Leu Leu Trp Thr Phe Val Gln Ser Phe Lys Ala Leu
Pro 1685 1690 1695Tyr Val Cys Leu Leu
Ile Ala Met Leu Phe Phe Ile Tyr Ala Ile 1700 1705
1710Ile Gly Met Gln Val Phe Gly Asn Ile Gly Ile Asp Val
Glu Asp 1715 1720 1725Glu Asp Ser Asp
Glu Asp Glu Phe Gln Ile Thr Glu His Asn Asn 1730
1735 1740Phe Arg Thr Phe Phe Gln Ala Leu Met Leu Leu
Phe Arg Ser Ala 1745 1750 1755Thr Gly
Glu Ala Trp His Asn Ile Met Leu Ser Cys Leu Ser Gly 1760
1765 1770Lys Pro Cys Asp Lys Asn Ser Gly Ile Leu
Thr Arg Glu Cys Gly 1775 1780 1785Asn
Glu Phe Ala Tyr Phe Tyr Phe Val Ser Phe Ile Phe Leu Cys 1790
1795 1800Ser Phe Leu Met Leu Asn Leu Phe Val
Ala Val Ile Met Asp Asn 1805 1810
1815Phe Glu Tyr Leu Thr Arg Asp Ser Ser Ile Leu Gly Pro His His
1820 1825 1830Leu Asp Glu Tyr Val Arg
Val Trp Ala Glu Tyr Asp Pro Ala Ala 1835 1840
1845Trp Gly Arg Met Pro Tyr Leu Asp Met Tyr Gln Met Leu Arg
His 1850 1855 1860Met Ser Pro Pro Leu
Gly Leu Gly Lys Lys Cys Pro Ala Arg Val 1865 1870
1875Ala Tyr Lys Arg Leu Leu Arg Met Asp Leu Pro Val Ala
Asp Asp 1880 1885 1890Asn Thr Val His
Phe Asn Ser Thr Leu Met Ala Leu Ile Arg Thr 1895
1900 1905Ala Leu Asp Ile Lys Ile Ala Lys Gly Gly Ala
Asp Lys Gln Gln 1910 1915 1920Met Asp
Ala Glu Leu Arg Lys Glu Met Met Ala Ile Trp Pro Asn 1925
1930 1935Leu Ser Gln Lys Thr Leu Asp Leu Leu Val
Thr Pro His Lys Ser 1940 1945 1950Thr
Asp Leu Thr Val Gly Lys Ile Tyr Ala Ala Met Met Ile Met 1955
1960 1965Glu Tyr Tyr Arg Gln Ser Lys Ala Lys
Lys Leu Gln Ala Met Arg 1970 1975
1980Glu Glu Gln Asp Arg Thr Pro Leu Met Phe Gln Arg Met Glu Pro
1985 1990 1995Pro Ser Pro Thr Gln Glu
Gly Gly Pro Gly Gln Asn Ala Leu Pro 2000 2005
2010Ser Thr Gln Leu Asp Pro Gly Gly Ala Leu Met Ala His Glu
Ser 2015 2020 2025Gly Leu Lys Glu Ser
Pro Ser Trp Val Thr Gln Arg Ala Gln Glu 2030 2035
2040Met Phe Gln Lys Thr Gly Thr Trp Ser Pro Glu Gln Gly
Pro Pro 2045 2050 2055Thr Asp Met Pro
Asn Ser Gln Pro Asn Ser Gln Ser Val Glu Met 2060
2065 2070Arg Glu Met Gly Arg Asp Gly Tyr Ser Asp Ser
Glu His Tyr Leu 2075 2080 2085Pro Met
Glu Gly Gln Gly Arg Ala Ala Ser Met Pro Arg Leu Pro 2090
2095 2100Ala Glu Asn Gln Arg Arg Arg Gly Arg Pro
Arg Gly Asn Asn Leu 2105 2110 2115Ser
Thr Ile Ser Asp Thr Ser Pro Met Lys Arg Ser Ala Ser Val 2120
2125 2130Leu Gly Pro Lys Ala Arg Arg Leu Asp
Asp Tyr Ser Leu Glu Arg 2135 2140
2145Val Pro Pro Glu Glu Asn Gln Arg His His Gln Arg Arg Arg Asp
2150 2155 2160Arg Ser His Arg Ala Ser
Glu Arg Ser Leu Gly Arg Tyr Thr Asp 2165 2170
2175Val Asp Thr Gly Leu Gly Thr Asp Leu Ser Met Thr Thr Gln
Ser 2180 2185 2190Gly Asp Leu Pro Ser
Lys Glu Arg Asp Gln Glu Arg Gly Arg Pro 2195 2200
2205Lys Asp Arg Lys His Arg Gln His His His His His His
His His 2210 2215 2220His His Pro Pro
Pro Pro Asp Lys Asp Arg Tyr Ala Gln Glu Arg 2225
2230 2235Pro Asp His Gly Arg Ala Arg Ala Arg Asp Gln
Arg Trp Ser Arg 2240 2245 2250Ser Pro
Ser Glu Gly Arg Glu His Met Ala His Arg Gln Gly Ser 2255
2260 2265Ser Ser Val Ser Gly Ser Pro Ala Pro Ser
Thr Ser Gly Thr Ser 2270 2275 2280Thr
Pro Arg Arg Gly Arg Arg Gln Leu Pro Gln Thr Pro Ser Thr 2285
2290 2295Pro Arg Pro His Val Ser Tyr Ser Pro
Val Ile Arg Lys Ala Gly 2300 2305
2310Gly Ser Gly Pro Pro Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
2315 2320 2325Gln Gln Gln Ala Val Ala
Arg Pro Gly Arg Ala Ala Thr Ser Gly 2330 2335
2340Pro Arg Arg Tyr Pro Gly Pro Thr Ala Glu Pro Leu Ala Gly
Asp 2345 2350 2355Arg Pro Pro Thr Gly
Gly His Ser Ser Gly Arg Ser Pro Arg Met 2360 2365
2370Glu Arg Arg Val Pro Gly Pro Ala Arg Ser Glu Ser Pro
Arg Ala 2375 2380 2385Cys Arg His Gly
Gly Ala Arg Trp Pro Ala Ser Gly Pro His Val 2390
2395 2400Ser Glu Gly Pro Pro Gly Pro Arg His His Gly
Tyr Tyr Arg Gly 2405 2410 2415Ser Asp
Tyr Asp Glu Ala Asp Gly Pro Gly Ser Gly Gly Gly Glu 2420
2425 2430Glu Ala Met Ala Gly Ala Tyr Asp Ala Pro
Pro Pro Val Arg His 2435 2440 2445Ala
Ser Ser Gly Ala Thr Gly Arg Ser Pro Arg Thr Pro Arg Ala 2450
2455 2460Ser Gly Pro Ala Cys Ala Ser Pro Ser
Arg His Gly Arg Arg Leu 2465 2470
2475Pro Asn Gly Tyr Tyr Pro Ala His Gly Leu Ala Arg Pro Arg Gly
2480 2485 2490Pro Gly Ser Arg Lys Gly
Leu His Glu Pro Tyr Ser Glu Ser Asp 2495 2500
2505Asp Asp Trp Cys 251047539DNAHomo sapiens 4atggcccgct
tcggagacga gatgccggcc cgctacgggg gaggaggctc cggggcagcc 60gccggggtgg
tcgtgggcag cggaggcggg cgaggagccg ggggcagccg gcagggcggg 120cagcccgggg
cgcaaaggat gtacaagcag tcaatggcgc agagagcgcg gaccatggca 180ctctacaacc
ccatccccgt ccgacagaac tgcctcacgg ttaaccggtc tctcttcctc 240ttcagcgaag
acaacgtggt gagaaaatac gccaaaaaga tcaccgaatg gcctcccttt 300gaatatatga
ttttagccac catcatagcg aattgcatcg tcctcgcact ggagcagcat 360ctgcctgatg
atgacaagac cccgatgtct gaacggctgg atgacacaga accatacttc 420attggaattt
tttgtttcga ggctggaatt aaaatcattg cccttgggtt tgccttccac 480aaaggctcct
acttgaggaa tggctggaat gtcatggact ttgtggtggt gctaacgggc 540atcttggcga
cagttgggac ggagtttgac ctacggacgc tgagggcagt tcgagtgctg 600cggccgctca
agctggtgtc tggaatccca agtttacaag tcgtcctgaa gtcgatcatg 660aaggcgatga
tccctttgct gcagatcggc ctcctcctat tttttgcaat ccttattttt 720gcaatcatag
ggttagaatt ttatatggga aaatttcata ccacctgctt tgaagagggg 780acagatgaca
ttcagggtga gtctccggct ccatgtggga cagaagagcc cgcccgcacc 840tgccccaatg
ggaccaaatg tcagccctac tgggaagggc ccaacaacgg gatcactcag 900ttcgacaaca
tcctgtttgc agtgctgact gttttccagt gcataaccat ggaagggtgg 960actgatctcc
tctacaatag caacgatgcc tcagggaaca cttggaactg gttgtacttc 1020atccccctca
tcatcatcgg ctcctttttt atgctgaacc ttgtgctggg tgtgctgtca 1080ggggagtttg
ccaaagaaag ggaacgggtg gagaaccggc gggcttttct gaagctgagg 1140cggcaacaac
agattgaacg tgagctcaat gggtacatgg agtggatctc aaaagcagaa 1200gaggtgatcc
tcgccgagga tgaaactgac ggggagcaga ggcatccctt tgatggagct 1260ctgcggagaa
ccaccataaa gaaaagcaag acagatttgc tcaaccccga agaggctgag 1320gatcagctgg
ctgatatagc ctctgtgggt tctcccttcg cccgagccag cattaaaagt 1380gccaagctgg
agaactcgac cttttttcac aaaaaggaga ggaggatgcg tttctacatc 1440cgccgcatgg
tcaaaactca ggccttctac tggactgtac tcagtttggt agctctcaac 1500acgctgtgtg
ttgctattgt tcactacaac cagcccgagt ggctctccga cttcctttac 1560tatgcagaat
tcattttctt aggactcttt atgtccgaaa tgtttataaa aatgtacggg 1620cttgggacgc
ggccttactt ccactcttcc ttcaactgct ttgactgtgg ggttatcatt 1680gggagcatct
tcgaggtcat ctgggctgtc ataaaacctg gcacatcctt tggaatcagc 1740gtgttacgag
ccctcaggtt attgcgtatt ttcaaagtca caaagtactg ggcatctctc 1800agaaacctgg
tcgtctctct cctcaactcc atgaagtcca tcatcagcct gttgtttctc 1860cttttcctgt
tcattgtcgt cttcgccctt ttgggaatgc aactcttcgg cggccagttt 1920aatttcgatg
aagggactcc tcccaccaac ttcgatactt ttccagcagc aataatgacg 1980gtgtttcaga
tcctgacggg cgaagactgg aacgaggtca tgtacgacgg gatcaagtct 2040caggggggcg
tgcagggcgg catggtgttc tccatctatt tcattgtact gacgctcttt 2100gggaactaca
ccctcctgaa tgtgttcttg gccatcgctg tggacaatct ggccaacgcc 2160caggagctca
ccaaggtgga ggcggacgag caagaggaag aagaagcagc gaaccagaaa 2220cttgccctac
agaaagccaa ggaggtggca gaagtgagtc ctctgtccgc ggccaacatg 2280tctatagctg
tgaaagagca acagaagaat caaaagccag ccaagtccgt gtgggagcag 2340cggaccagtg
agatgcgaaa gcagaacttg ctggccagcc gggaggccct gtataacgaa 2400atggacccgg
acgagcgctg gaaggctgcc tacacgcggc acctgcggcc agacatgaag 2460acgcacttgg
accggccgct ggtggtggac ccgcaggaga accgcaacaa caacaccaac 2520aagagccggg
cggccgagcc caccgtggac cagcgcctcg gccagcagcg cgccgaggac 2580ttcctcagga
aacaggcccg ctaccacgat cgggcccggg accccagcgg ctcggcgggc 2640ctggacgcac
ggaggccctg ggcgggaagc caggaggccg agctgagccg ggagggaccc 2700tacggccgcg
agtcggacca ccacgcccgg gagggcagcc tggagcaacc cgggttctgg 2760gagggcgagg
ccgagcgagg caaggccggg gacccccacc ggaggcacgt gcaccggcag 2820gggggcagca
gggagagccg cagcgggtcc ccgcgcacgg gcgcggacgg ggagcatcga 2880cgtcatcgcg
cgcaccgcag gcccggggag gagggtccgg aggacaaggc ggagcggagg 2940gcgcggcacc
gcgagggcag ccggccggcc cggggcggcg agggcgaggg cgagggcccc 3000gacgggggcg
agcgcaggag aaggcaccgg catggcgctc cagccacgta cgagggggac 3060gcgcggaggg
aggacaagga gcggaggcat cggaggagga aagagaacca gggctccggg 3120gtccctgtgt
cgggccccaa cctgtcaacc acccggccaa tccagcagga cctgggccgc 3180caagacccac
ccctggcaga ggatattgac aacatgaaga acaacaagct ggccaccgcg 3240gagtcggccg
ctccccacgg cagccttggc cacgccggcc tgccccagag cccagccaag 3300atgggaaaca
gcaccgaccc cggccccatg ctggccatcc ctgccatggc caccaacccc 3360cagaacgccg
ccagccgccg gacgcccaac aacccgggga acccatccaa tcccggcccc 3420cccaagaccc
ccgagaatag ccttatcgtc accaacccca gcggcaccca gaccaattca 3480gctaagactg
ccaggaaacc cgaccacacc acagtggaca tccccccagc ctgcccaccc 3540cccctcaacc
acaccgtcgt acaagtgaac aaaaacgcca acccagaccc actgccaaaa 3600aaagaggaag
agaagaagga ggaggaggaa gacgaccgtg gggaagacgg ccctaagcca 3660atgcctccct
atagctccat gttcatcctg tccacgacca acccccttcg ccgcctgtgc 3720cattacatcc
tgaacctgcg ctactttgag atgtgcatcc tcatggtcat tgccatgagc 3780agcatcgccc
tggccgccga ggaccctgtg cagcccaacg cacctcggaa caacgtgctg 3840cgatactttg
actacgtttt tacaggcgtc tttacctttg agatggtgat caagatgatt 3900gacctggggc
tcgtcctgca tcagggtgcc tacttccgtg acctctggaa tattctcgac 3960ttcatagtgg
tcagtggggc cctggtagcc tttgccttca ctggcaatag caaaggaaaa 4020gacatcaaca
cgattaaatc cctccgagtc ctccgggtgc tacgacctct taaaaccatc 4080aagcggctgc
caaagctcaa ggctgtgttt gactgtgtgg tgaactcact taaaaacgtc 4140ttcaacatcc
tcatcgtcta catgctattc atgttcatct tcgccgtggt ggctgtgcag 4200ctcttcaagg
ggaaattctt ccactgcact gacgagtcca aagagtttga gaaagattgt 4260cgaggcaaat
acctcctcta cgagaagaat gaggtgaagg cgcgagaccg ggagtggaag 4320aagtatgaat
tccattacga caatgtgctg tgggctctgc tgaccctctt caccgtgtcc 4380acgggagaag
gctggccaca ggtcctcaag cattcggtgg acgccacctt tgagaaccag 4440ggccccagcc
ccgggtaccg catggagatg tccattttct acgtcgtcta ctttgtggtg 4500ttccccttct
tctttgtcaa tatctttgtg gccttgatca tcatcacctt ccaggagcaa 4560ggggacaaga
tgatggagga atacagcctg gagaaaaatg agagggcctg cattgatttc 4620gccatcagcg
ccaagccgct gacccgacac atgccgcaga acaagcagag cttccagtac 4680cgcatgtggc
agttcgtggt gtctccgcct ttcgagtaca cgatcatggc catgatcgcc 4740ctcaacacca
tcgtgcttat gatgaagttc tatggggctt ctgttgctta tgaaaatgcc 4800ctgcgggtgt
tcaacatcgt cttcacctcc ctcttctctc tggaatgtgt gctgaaagtc 4860atggcttttg
ggattctgaa ttatttccgc gatgcctgga acatcttcga ctttgtgact 4920gttctgggca
gcatcaccga tatcctcgtg actgagtttg ggaatccgaa taacttcatc 4980aacctgagct
ttctccgcct cttccgagct gcccggctca tcaaacttct ccgtcagggt 5040tacaccatcc
gcattcttct ctggaccttt gtgcagtcct tcaaggccct gccttatgtc 5100tgtctgctga
tcgccatgct cttcttcatc tatgccatca ttgggatgca ggtgtttggt 5160aacattggca
tcgacgtgga ggacgaggac agtgatgaag atgagttcca aatcactgag 5220cacaataact
tccggacctt cttccaggcc ctcatgcttc tcttccggag tgccaccggg 5280gaagcttggc
acaacatcat gctttcctgc ctcagcggga aaccgtgtga taagaactct 5340ggcatcctga
ctcgagagtg tggcaatgaa tttgcttatt tttactttgt ttccttcatc 5400ttcctctgct
cgtttctgat gctgaatctc tttgtcgccg tcatcatgga caactttgag 5460tacctcaccc
gagactcctc catcctgggc ccccaccacc tggatgagta cgtgcgtgtc 5520tgggccgagt
atgaccccgc agcttggggc cgcatgcctt acctggacat gtatcagatg 5580ctgagacaca
tgtctccgcc cctgggtctg gggaagaagt gtccggccag agtggcttac 5640aagcggcttc
tgcggatgga cctgcccgtc gcagatgaca acaccgtcca cttcaattcc 5700accctcatgg
ctctgatccg cacagccctg gacatcaaga ttgccaaggg aggagccgac 5760aaacagcaga
tggacgctga gctgcggaag gagatgatgg cgatttggcc caatctgtcc 5820cagaagacgc
tagacctgct ggtcacacct cacaagtcca cggacctcac cgtggggaag 5880atctacgcag
ccatgatgat catggagtac taccggcaga gcaaggccaa gaagctgcag 5940gccatgcgcg
aggagcagga ccggacaccc ctcatgttcc agcgcatgga gcccccgtcc 6000ccaacgcagg
aagggggacc tggccagaac gccctcccct ccacccagct ggacccagga 6060ggagccctga
tggctcacga aagcggcctc aaggagagcc cgtcctgggt gacccagcgt 6120gcccaggaga
tgttccagaa gacgggcaca tggagtccgg aacaaggccc ccctaccgac 6180atgcccaaca
gccagcctaa ctctcagtcc gtggagatgc gagagatggg cagagatggc 6240tactccgaca
gcgagcacta cctccccatg gaaggccagg gccgggctgc ctccatgccc 6300cgcctccctg
cagagaacca gaggagaagg ggccggccac gtgggaataa cctcagtacc 6360atctcagaca
ccagccccat gaagcgttca gcctccgtgc tgggccccaa ggcccgacgc 6420ctggacgatt
actcgctgga gcgggtcccg cccgaggaga accagcggca ccaccagcgg 6480cgccgcgacc
gcagccaccg cgcctctgag cgctccctgg gccgctacac cgatgtggac 6540acaggcttgg
ggacagacct gagcatgacc acccaatccg gggacctgcc gtcgaaggag 6600cgggaccagg
agcggggccg gcccaaggat cggaagcatc gacagcacca ccaccaccac 6660caccaccacc
accatccccc gccccccgac aaggaccgct atgcccagga acggccggac 6720cacggccggg
cacgggctcg ggaccagcgc tggtcccgct cgcccagcga gggccgagag 6780cacatggcgc
accggcaggg cagtagttcc gtaagtggaa gcccagcccc ctcaacatct 6840ggtaccagca
ctccgcggcg gggccgccgc cagctccccc agaccccctc caccccccgg 6900ccacacgtgt
cctattcccc tgtgatccgt aaggccggcg gctcggggcc cccgcagcag 6960cagcagcagc
agcagcagca gcagcagcag caggcggtgg ccaggccggg ccgggcggcc 7020accagcggcc
ctcggaggta cccaggcccc acggccgagc ctctggccgg agatcggccg 7080cccacggggg
gccacagcag cggccgctcg cccaggatgg agaggcgggt cccaggcccg 7140gcccggagcg
agtcccccag ggcctgtcga cacggcgggg cccggtggcc ggcatctggc 7200ccgcacgtgt
ccgaggggcc cccgggtccc cggcaccatg gctactaccg gggctccgac 7260tacgacgagg
ccgatggccc gggcagcggg ggcggcgagg aggccatggc cggggcctac 7320gacgcgccac
cccccgtacg acacgcgtcc tcgggcgcca ccgggcgctc gcccaggact 7380ccccgggcct
cgggcccggc ctgcgcctcg ccttctcggc acggccggcg actccccaac 7440ggctactacc
cggcgcacgg actggccagg ccccgcgggc cgggctccag gaagggcctg 7500cacgaaccct
acagcgagag tgacgatgat tggtgctaa
7539523DNAArtificial SequenceDescription of Artificial Sequence
Synthesized Oligonucleotide 5tgacttttct ttctctccgt ttg
23622DNAArtificial SequenceDescription of
Artificial Sequence Synthesized Oligonucleotide 6tggctgcaat
aatcactttg tt
22721DNAArtificial SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 7tctctgtctc cccaggttta c
21821DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 8gtggctaaca cacagctttg c
21921DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 9tcatggcaca gttcctgtat c
211021DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 10gcagtaggca attagcagca a
211121DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 11tggggcactt tagaaattgt g
211222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 12tgacaaagat gcaaaatgag ag
221320DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 13gcagtttggg cttttcaatg
201420DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 14tgagcattgt cctcttgctg
201522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 15agggctacgt ttcatttgta tg
221622DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 16tgtgctaaat tgaaatccag ag
221720DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 17cagctcttcg cactttcaga
201821DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 18tcaagcagag aaggatgctg a
211920DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 19agcgttgcaa acattcttgg
202020DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 20gggatatcca gcccctcaag
202124DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 21gacaaatact tgtgcctttg aatg
242227DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 22acataatctc atactttatc aaaaacc
272322DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 23gaaatggagg tgttgaaaat gc
222420DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 24aatccttggc atcactctgc
202522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 25agtacagggt gctatgacca ac
222621DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 26tcctcataca accacctgct c
212721DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 27tctccaaaag ccttcattag g
212822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 28ttctaattct ccccctctct cc
222922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 29tcctcattct ttaatcccaa gg
223021DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 30gccgttctgt agaaacactg g
213122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 31gtcagaaata tctgccatca cc
223222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 32gaatgcacta ttcccaactc ac
223321DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 33tgggctctat gtgtgtgtct g
213420DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 34ggaagcatga aggatggttg
203520DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 35tacttcgcgt ttccacaagg
203621DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 36gctatgcaag aaccctgatt g
213720DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 37atgagcctga gacggttagg
203820DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 38atacatgtgc catgctggtg
203920DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 39tgctgtggtg tttccttctc
204022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 40tgtattcata ccttcccaca cc
224122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 41aaaagggtta gcacagacaa tg
224222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 42attgggcaga tataatcaaa gc
224320DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 43cacacagctg atgaatgtgc
204420DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 44tgaagggcta cactttctgg
204520DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 45tctgccctcc tattccaatg
204620DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 46gcccttgtct tccagaaatg
204727DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 47aaaaattaca tcctttacat caaactg
274821DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 48ttttgcatgc atagattttc c
214922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 49tgaaccttgc ttttacatat cc
225020DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 50acccatctgg gctcataaac
205121DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 51tgtcttggtc caaaatctgt g
215222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 52ttggtcgttt atgctttatt cg
225320DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 53ccctaaaggc caatttcagg
205421DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 54atttggcaga gaaaacactc c
215520DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 55gagatttggg ggtgtttgtc
205620DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 56ggattgtaat ggggtgcttc
205720DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 57caaaaatcag ggccaatgac
205820DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 58tgattgctgg gatgatcttg
205922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 59aggactctga accttacctt gg
226020DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 60ccatgaatcg ctcttccatc
206119DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 61tgtgggaacc catctgttg
196220DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 62gtttgctgac aaggggtcac
206321DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 63tctccgcagt cgtagctcca g
216422DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 64agagattctt tcacactcct cc
226522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 65tttagaagtc acctgatctg gg
226622DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 66gacagagcga gactctggtt ca
226722DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 67gacaagagaa ctctgcaaga gg
226822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 68atacagctga gacatggagg tg
226923DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 69tttatcccgt gaggcaggta ctg
237022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 70cctcctgaga tgctctgcat ag
227122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 71tgtggtgctt ccttcaccat tg
227222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 72cagaggctat ttcactcact gc
227323DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 73ccccaaagcc aaacattgat ctc
237423DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 74actctgattg tccacacaca ctg
237523DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 75cagaaaacgt tcctccattt ccc
237623DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 76aagcttcaat ggcctctact tgg
237723DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 77gccatactct ggcttttcta tgc
237822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 78cgtgatgtca gatcctggct tc
227922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 79gttggctatt gctactgttg cg
228022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 80gatccttaga accagtcacc tg
228122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 81tgatagtgcc accttgaacc tc
228222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 82tgatgtaatc tgcccaggac ac
228322DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 83ctgcaacaga gaactatcag cc
228423DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 84aagagaagtg gaaaaagggt gtg
238522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 85gtagttctag catgttggag gc
228622DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 86atctgtcatt ccaggcaaga gc
228722DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 87atggatgaat gagggggtca ag
228822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 88agcaggcact ttcatctgtg ac
228922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 89tccatttgga gggaggagtt tg
229022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 90cctccagaaa gttgggaaag tg
229122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 91aaggagaagc caacacggag tc
229223DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 92ggtggtaact ttgccagaga aac
239322DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 93agcaggtacc cattccaatt gg
229422DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 94aatctgtgcc tgggatagtg tg
229522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 95cctgactcag atgctcacag ac
229622DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 96acacagcacg tgctactttg gc
229721DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 97gaggacttcc tcaggaaaca g
219823DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 98agatggaatc ttagctagga tcc
239923DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 99aattatctca ctgaaccctc cac
2310022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 100agaaatgtca gccgcttctt gc
2210122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 101ggtggtcaac actcactcat tg
2210222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 102tttgttgtgt aggaggcctt gg
2210322DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 103aacatcccac cctacctatg ag
2210422DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 104cctgcgcaac tgtatatagc ag
2210522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 105ctcaacctcc tgatctcaag tg
2210623DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 106cccaaagttt ggatctaaga gcc
2310722DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 107aaagccatcg aagctcttcc tg
2210822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 108caggtgaaat ggaccactct tc
2210922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 109tccttgagca gtgtacaacc tg
2211022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 110gaatgccagg attgagtcca ac
2211122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 111gaatgtgctg gaaagtggag ac
2211222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 112cactgcttcc caagcagtct ag
2211322DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 113attacaggcg tgagccacca tg
2211422DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 114tttccctctg ttcctgttct gc
2211522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 115ttcggttggg acaatgcttc tg
2211622DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 116ctcaagcaac tgtagctgtt gg
2211722DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 117ttatcagggt agaggcagga ac
2211822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 118gtgaaaagaa gagcctagtc cg
2211922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 119atggtaacac tcacaggttg gg
2212022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 120gcccttcgaa caaccataac tg
2212122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 121cctacagcca agctttggtt ac
2212222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 122cccattggtt ttttggcact gg
2212321DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 123ggacagacag acagaggaga g
2112422DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 124tgttggttgg cttcatgtag gg
2212522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 125cagaattatc agagcaggtc cc
2212622DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 126tctcagctcc cagtaaaagg ag
2212722DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 127caacagtgct gagtttgaga cg
2212822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 128ggcctctgtg tacatgtctt tg
2212922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 129gggtatgcaa gggtgatgat tc
2213021DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 130tgtttctccc cacctctctt c
2113122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 131aaaaaaaccc agtgcctgga cg
2213222DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 132agaaactgag tactgggaca gg
2213322DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 133ggaagagtga atgaagatcc gg
2213422DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 134aaagattggg gtctcgttct cg
2213522DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 135ccctcatatt ccagttggtt cc
2213622DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 136gtgtgtgtgt gtgtatactg gg
2213722DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 137cagactgctt cagagactga ag
2213822DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 138ccgatttctc ttgatgccag tg
2213922DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 139agggtgcgat tgccaaagaa ag
2214022DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 140acccagagcc ctgattgatc ag
2214122DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 141ttggatgggg tatccccttc tc
2214221DNAArtificial SequenceDescription of Artificial
Sequence Synthesized Oligonucleotide 142tctcttcctc ccaatcccgt g
2114321DNAArtificial
SequenceDescription of Artificial Sequence Synthesized
Oligonucleotide 143tgcccaggag ggtctctttt g
211442009PRTRattus norvegicus 144Met Glu Gln Thr Val Leu
Val Pro Pro Gly Pro Asp Ser Phe Asn Phe1 5
10 15Phe Thr Arg Glu Ser Leu Ala Ala Ile Glu Arg Arg
Ile Ala Glu Glu 20 25 30Lys
Ala Lys Asn Pro Lys Pro Asp Lys Lys Asp Asp Asp Glu Asn Gly 35
40 45Pro Lys Pro Asn Ser Asp Leu Glu Ala
Gly Lys Asn Leu Pro Phe Ile 50 55
60Tyr Gly Asp Ile Pro Pro Glu Met Val Ser Glu Pro Leu Glu Asp Leu65
70 75 80Asp Pro Tyr Tyr Ile
Asn Lys Lys Thr Phe Ile Val Leu Asn Lys Gly 85
90 95Lys Ala Ile Phe Arg Phe Ser Ala Thr Ser Ala
Leu Tyr Ile Leu Thr 100 105
110Pro Phe Asn Pro Leu Arg Lys Ile Ala Ile Lys Ile Leu Val His Ser
115 120 125Leu Phe Ser Met Leu Ile Met
Cys Thr Ile Leu Thr Asn Cys Val Phe 130 135
140Met Thr Met Ser Asn Pro Pro Asp Trp Thr Lys Asn Val Glu Tyr
Thr145 150 155 160Phe Thr
Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys Ile Ile Ala Arg
165 170 175Gly Phe Cys Leu Glu Asp Phe
Thr Phe Leu Arg Asp Pro Trp Asn Trp 180 185
190Leu Asp Phe Thr Val Ile Thr Phe Ala Tyr Val Thr Glu Phe
Val Asp 195 200 205Leu Gly Asn Val
Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu 210
215 220Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile
Val Gly Ala Leu225 230 235
240Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val Phe
245 250 255Cys Leu Ser Val Phe
Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn 260
265 270Leu Arg Asn Lys Cys Val Gln Trp Pro Pro Thr Asn
Ala Ser Leu Glu 275 280 285Glu His
Ser Ile Glu Lys Asn Val Thr Thr Asp Tyr Asn Gly Thr Leu 290
295 300Val Asn Glu Thr Val Phe Glu Phe Asp Trp Lys
Ser Tyr Ile Gln Asp305 310 315
320Ser Arg Tyr His Tyr Phe Leu Glu Gly Val Leu Asp Ala Leu Leu Cys
325 330 335Gly Asn Ser Ser
Asp Ala Gly Gln Cys Pro Glu Gly Tyr Met Cys Val 340
345 350Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr
Ser Phe Asp Thr Phe 355 360 365Ser
Trp Ala Phe Leu Ser Leu Phe Arg Leu Met Thr Gln Asp Phe Trp 370
375 380Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala
Ala Gly Lys Thr Tyr Met385 390 395
400Ile Phe Phe Val Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile
Asn 405 410 415Leu Ile Leu
Ala Val Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala 420
425 430Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala
Glu Phe Gln Gln Met Leu 435 440
445Glu Gln Leu Lys Lys Gln Gln Glu Ala Ala Gln Gln Ala Ala Ala Ala 450
455 460Thr Ala Ser Glu His Ser Arg Glu
Pro Ser Ala Ala Gly Arg Leu Ser465 470
475 480Asp Ser Ser Ser Glu Ala Ser Lys Leu Ser Ser Lys
Ser Ala Lys Glu 485 490
495Arg Arg Asn Arg Arg Lys Lys Arg Lys Gln Lys Glu Gln Ser Gly Gly
500 505 510Glu Glu Lys Asp Asp Asp
Glu Phe His Lys Ser Glu Ser Glu Asp Ser 515 520
525Ile Arg Arg Lys Gly Phe Arg Phe Ser Ile Glu Gly Asn Arg
Leu Thr 530 535 540Tyr Glu Lys Arg Tyr
Ser Ser Pro His Gln Ser Leu Leu Ser Ile Arg545 550
555 560Gly Ser Leu Phe Ser Pro Arg Arg Asn Ser
Arg Thr Ser Leu Phe Ser 565 570
575Phe Arg Gly Arg Ala Lys Asp Val Gly Ser Glu Asn Asp Phe Ala Asp
580 585 590Asp Glu His Ser Thr
Phe Glu Asp Asn Glu Ser Arg Arg Asp Ser Leu 595
600 605Phe Val Pro Arg Arg His Gly Glu Arg Arg Asn Ser
Asn Leu Ser Gln 610 615 620Thr Ser Arg
Ser Ser Arg Met Leu Ala Gly Leu Pro Ala Asn Gly Lys625
630 635 640Met His Ser Thr Val Asp Cys
Asn Gly Val Val Ser Leu Val Gly Gly 645
650 655Pro Ser Val Pro Thr Ser Pro Val Gly Gln Leu Leu
Pro Glu Val Ile 660 665 670Ile
Asp Lys Pro Ala Thr Asp Asp Asn Gly Thr Thr Thr Glu Thr Glu 675
680 685Met Arg Lys Arg Arg Ser Ser Ser Phe
His Val Ser Met Asp Phe Leu 690 695
700Glu Asp Pro Ser Gln Arg Gln Arg Ala Met Ser Ile Ala Ser Ile Leu705
710 715 720Thr Asn Thr Val
Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Pro Pro 725
730 735Cys Trp Tyr Lys Phe Ser Asn Ile Phe Leu
Ile Trp Asp Cys Ser Pro 740 745
750Tyr Trp Leu Lys Val Lys His Ile Val Asn Leu Val Val Met Asp Pro
755 760 765Phe Val Asp Leu Ala Ile Thr
Ile Cys Ile Val Leu Asn Thr Leu Phe 770 775
780Met Ala Met Glu His Tyr Pro Met Thr Glu His Phe Asn His Val
Leu785 790 795 800Thr Val
Gly Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met Phe
805 810 815Leu Lys Ile Ile Ala Met Asp
Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp 820 825
830Asn Ile Phe Asp Gly Phe Ile Val Thr Leu Ser Leu Val Glu
Leu Gly 835 840 845Leu Ala Asn Val
Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu Leu 850
855 860Arg Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu
Asn Met Leu Ile865 870 875
880Lys Ile Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val
885 890 895Leu Ala Ile Ile Val
Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe 900
905 910Gly Lys Ser Tyr Lys Asp Cys Val Cys Lys Ile Ala
Thr Asp Cys Lys 915 920 925Leu Pro
Arg Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val 930
935 940Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Thr
Met Trp Asp Cys Met945 950 955
960Glu Val Ala Gly Gln Ala Met Cys Leu Thr Val Phe Met Met Val Met
965 970 975Val Ile Arg Asn
Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu 980
985 990Ser Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr
Asp Asp Asp Asn Glu 995 1000
1005Met Asn Asn Leu Gln Ile Ala Val Asp Arg Met His Lys Gly Val
1010 1015 1020Ala Tyr Val Lys Arg Lys
Ile Tyr Glu Phe Ile Gln Gln Ser Phe 1025 1030
1035Val Arg Lys Gln Lys Ile Leu Asp Glu Ile Lys Pro Leu Asp
Asp 1040 1045 1050Leu Asn Asn Arg Lys
Asp Asn Cys Thr Ser Asn His Thr Thr Glu 1055 1060
1065Ile Gly Lys Asp Leu Asp Cys Leu Lys Asp Val Asn Gly
Thr Thr 1070 1075 1080Ser Gly Ile Gly
Thr Gly Ser Ser Val Glu Lys Tyr Ile Ile Asp 1085
1090 1095Glu Ser Asp Tyr Met Ser Phe Ile Asn Asn Pro
Ser Leu Thr Val 1100 1105 1110Thr Val
Pro Ile Ala Val Gly Glu Ser Asp Phe Glu Asn Leu Asn 1115
1120 1125Thr Glu Asp Phe Ser Ser Glu Ser Asp Leu
Glu Glu Ser Lys Glu 1130 1135 1140Lys
Leu Asn Glu Ser Ser Ser Ser Ser Glu Gly Ser Thr Val Asp 1145
1150 1155Ile Gly Ala Pro Ala Glu Glu Gln Pro
Val Met Glu Pro Glu Glu 1160 1165
1170Thr Leu Glu Pro Glu Ala Cys Phe Thr Glu Gly Cys Val Gln Arg
1175 1180 1185Phe Lys Cys Cys Gln Ile
Ser Val Glu Glu Gly Arg Gly Lys Gln 1190 1195
1200Trp Trp Asn Leu Arg Arg Thr Cys Phe Arg Ile Val Glu His
Asn 1205 1210 1215Trp Phe Glu Thr Phe
Ile Val Phe Met Ile Leu Leu Ser Ser Gly 1220 1225
1230Ala Leu Ala Phe Glu Asp Ile Tyr Ile Asp Gln Arg Lys
Thr Ile 1235 1240 1245Lys Thr Met Leu
Glu Tyr Ala Asp Lys Val Phe Thr Tyr Ile Phe 1250
1255 1260Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr
Gly Tyr Gln Thr 1265 1270 1275Tyr Phe
Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val Asp 1280
1285 1290Val Ser Leu Val Ser Leu Thr Ala Asn Ala
Leu Gly Tyr Ser Glu 1295 1300 1305Leu
Gly Ala Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro 1310
1315 1320Leu Arg Ala Leu Ser Arg Phe Glu Gly
Met Arg Val Val Val Asn 1325 1330
1335Ala Leu Leu Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val
1340 1345 1350Cys Leu Ile Phe Trp Leu
Ile Phe Ser Ile Met Gly Val Asn Leu 1355 1360
1365Phe Ala Gly Lys Phe Tyr His Cys Val Asn Thr Thr Thr Gly
Asp 1370 1375 1380Thr Phe Glu Ile Thr
Glu Val Asn Asn His Ser Asp Cys Leu Lys 1385 1390
1395Leu Ile Glu Arg Asn Glu Thr Ala Arg Trp Lys Asn Val
Lys Val 1400 1405 1410Asn Phe Asp Asn
Val Gly Phe Gly Tyr Leu Ser Leu Leu Gln Val 1415
1420 1425Ala Thr Phe Lys Gly Trp Met Asp Ile Met Tyr
Ala Ala Val Asp 1430 1435 1440Ser Arg
Asn Val Glu Leu Gln Pro Lys Tyr Glu Glu Ser Leu Tyr 1445
1450 1455Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile
Phe Gly Ser Phe Phe 1460 1465 1470Thr
Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln 1475
1480 1485Gln Lys Lys Lys Phe Gly Gly Gln Asp
Ile Phe Met Thr Glu Glu 1490 1495
1500Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser Lys Lys
1505 1510 1515Pro Gln Lys Pro Ile Pro
Arg Pro Gly Asn Lys Phe Gln Gly Met 1520 1525
1530Val Phe Asp Phe Val Thr Arg Gln Val Phe Asp Ile Ser Ile
Met 1535 1540 1545Ile Leu Ile Cys Leu
Asn Met Val Thr Met Met Val Glu Thr Asp 1550 1555
1560Asp Gln Ser Asp Tyr Val Thr Ser Ile Leu Ser Arg Ile
Asn Leu 1565 1570 1575Val Phe Ile Val
Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile 1580
1585 1590Ser Leu Arg His Tyr Tyr Phe Thr Ile Gly Trp
Asn Ile Phe Asp 1595 1600 1605Phe Val
Val Val Ile Leu Ser Ile Val Gly Met Phe Leu Ala Glu 1610
1615 1620Leu Ile Glu Lys Tyr Phe Val Ser Pro Thr
Leu Phe Arg Val Ile 1625 1630 1635Arg
Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala 1640
1645 1650Lys Gly Ile Arg Thr Leu Leu Phe Ala
Leu Met Met Ser Leu Pro 1655 1660
1665Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile
1670 1675 1680Tyr Ala Ile Phe Gly Met
Ser Asn Phe Ala Tyr Val Lys Arg Glu 1685 1690
1695Val Gly Ile Asp Asp Met Phe Asn Phe Glu Thr Phe Gly Asn
Ser 1700 1705 1710Met Ile Cys Leu Phe
Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly 1715 1720
1725Leu Leu Ala Pro Ile Leu Asn Ser Lys Pro Pro Asp Cys
Asp Pro 1730 1735 1740Asn Lys Val Asn
Pro Gly Ser Ser Val Lys Gly Asp Cys Gly Asn 1745
1750 1755Pro Ser Val Gly Ile Phe Phe Phe Val Ser Tyr
Ile Ile Ile Ser 1760 1765 1770Phe Leu
Val Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn 1775
1780 1785Phe Ser Val Ala Thr Glu Glu Ser Ala Glu
Pro Leu Ser Glu Asp 1790 1795 1800Asp
Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp 1805
1810 1815Ala Thr Gln Phe Met Glu Phe Glu Lys
Leu Ser Gln Phe Ala Ala 1820 1825
1830Ala Leu Glu Pro Pro Leu Asn Leu Pro Gln Pro Asn Lys Leu Gln
1835 1840 1845Leu Ile Ala Met Asp Leu
Pro Met Val Ser Gly Asp Arg Ile His 1850 1855
1860Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly
Glu 1865 1870 1875Ser Gly Glu Met Asp
Ala Leu Arg Ile Gln Met Glu Glu Arg Phe 1880 1885
1890Met Ala Ser Asn Pro Ser Lys Val Ser Tyr Gln Pro Ile
Thr Thr 1895 1900 1905Thr Leu Lys Arg
Lys Gln Glu Glu Val Ser Ala Val Ile Ile Gln 1910
1915 1920Arg Ala Tyr Arg Arg His Leu Leu Lys Arg Thr
Val Lys Gln Ala 1925 1930 1935Ser Phe
Thr Tyr Asn Lys Asn Lys Leu Lys Gly Gly Ala Asn Leu 1940
1945 1950Leu Val Lys Glu Asp Met Ile Ile Asp Arg
Ile Asn Glu Asn Ser 1955 1960 1965Ile
Thr Glu Lys Thr Asp Leu Thr Met Ser Thr Ala Ala Cys Pro 1970
1975 1980Pro Ser Tyr Asp Arg Val Thr Lys Pro
Ile Val Glu Lys His Glu 1985 1990
1995Gln Glu Gly Lys Asp Glu Lys Ala Lys Gly Lys 2000
20051452009PRTHomo sapiens 145Met Glu Gln Thr Val Leu Val Pro Pro Gly Pro
Asp Ser Phe Asn Phe1 5 10
15Phe Thr Arg Glu Ser Leu Ala Ala Ile Glu Arg Arg Ile Ala Glu Glu
20 25 30Lys Ala Lys Asn Pro Lys Pro
Asp Lys Lys Asp Asp Asp Glu Asn Gly 35 40
45Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Asn Leu Pro Phe
Ile 50 55 60Tyr Gly Asp Ile Pro Pro
Glu Met Val Ser Glu Pro Leu Glu Asp Leu65 70
75 80Asp Pro Tyr Tyr Ile Asn Lys Lys Thr Phe Ile
Val Leu Asn Lys Gly 85 90
95Lys Ala Ile Phe Arg Phe Ser Ala Thr Ser Ala Leu Tyr Ile Leu Thr
100 105 110Pro Phe Asn Pro Leu Arg
Lys Ile Ala Ile Lys Ile Leu Val His Ser 115 120
125Leu Phe Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys
Val Phe 130 135 140Met Thr Met Ser Asn
Pro Pro Asp Trp Thr Lys Asn Val Glu Tyr Thr145 150
155 160Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu
Ile Lys Ile Ile Ala Arg 165 170
175Gly Phe Cys Leu Glu Asp Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp
180 185 190Leu Asp Phe Thr Val
Ile Thr Phe Ala Tyr Val Thr Glu Phe Val Asp 195
200 205Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val
Leu Arg Ala Leu 210 215 220Lys Thr Ile
Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu225
230 235 240Ile Gln Ser Val Lys Lys Leu
Ser Asp Val Met Ile Leu Thr Val Phe 245
250 255Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu
Phe Met Gly Asn 260 265 270Leu
Arg Asn Lys Cys Ile Gln Trp Pro Pro Thr Asn Ala Ser Leu Glu 275
280 285Glu His Ser Ile Glu Lys Asn Ile Thr
Val Asn Tyr Asn Gly Thr Leu 290 295
300Ile Asn Glu Thr Val Phe Glu Phe Asp Trp Lys Ser Tyr Ile Gln Asp305
310 315 320Ser Arg Tyr His
Tyr Phe Leu Glu Gly Phe Leu Asp Ala Leu Leu Cys 325
330 335Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro
Glu Gly Tyr Met Cys Val 340 345
350Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser Phe Asp Thr Phe
355 360 365Ser Trp Ala Phe Leu Ser Leu
Phe Arg Leu Met Thr Gln Asp Phe Trp 370 375
380Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly Lys Thr Tyr
Met385 390 395 400Ile Phe
Phe Val Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn
405 410 415Leu Ile Leu Ala Val Val Ala
Met Ala Tyr Glu Glu Gln Asn Gln Ala 420 425
430Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe Gln Gln
Met Ile 435 440 445Glu Gln Leu Lys
Lys Gln Gln Glu Ala Ala Gln Gln Ala Ala Thr Ala 450
455 460Thr Ala Ser Glu His Ser Arg Glu Pro Ser Ala Ala
Gly Arg Leu Ser465 470 475
480Asp Ser Ser Ser Glu Ala Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu
485 490 495Arg Arg Asn Arg Arg
Lys Lys Arg Lys Gln Lys Glu Gln Ser Gly Gly 500
505 510Glu Glu Lys Asp Glu Asp Glu Phe Gln Lys Ser Glu
Ser Glu Asp Ser 515 520 525Ile Arg
Arg Lys Gly Phe Arg Phe Ser Ile Glu Gly Asn Arg Leu Thr 530
535 540Tyr Glu Lys Arg Tyr Ser Ser Pro His Gln Ser
Leu Leu Ser Ile Arg545 550 555
560Gly Ser Leu Phe Ser Pro Arg Arg Asn Ser Arg Thr Ser Leu Phe Ser
565 570 575Phe Arg Gly Arg
Ala Lys Asp Val Gly Ser Glu Asn Asp Phe Ala Asp 580
585 590Asp Glu His Ser Thr Phe Glu Asp Asn Glu Ser
Arg Arg Asp Ser Leu 595 600 605Phe
Val Pro Arg Arg His Gly Glu Arg Arg Asn Ser Asn Leu Ser Gln 610
615 620Thr Ser Arg Ser Ser Arg Met Leu Ala Val
Phe Pro Ala Asn Gly Lys625 630 635
640Met His Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Val Gly
Gly 645 650 655Pro Ser Val
Pro Thr Ser Pro Val Gly Gln Leu Leu Pro Glu Val Ile 660
665 670Ile Asp Lys Pro Ala Thr Asp Asp Asn Gly
Thr Thr Thr Glu Thr Glu 675 680
685Met Arg Lys Arg Arg Ser Ser Ser Phe His Val Ser Met Asp Phe Leu 690
695 700Glu Asp Pro Ser Gln Arg Gln Arg
Ala Met Ser Ile Ala Ser Ile Leu705 710
715 720Thr Asn Thr Val Glu Glu Leu Glu Glu Ser Arg Gln
Lys Cys Pro Pro 725 730
735Cys Trp Tyr Lys Phe Ser Asn Ile Phe Leu Ile Trp Asp Cys Ser Pro
740 745 750Tyr Trp Leu Lys Val Lys
His Val Val Asn Leu Val Val Met Asp Pro 755 760
765Phe Val Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr
Leu Phe 770 775 780Met Ala Met Glu His
Tyr Pro Met Thr Asp His Phe Asn Asn Val Leu785 790
795 800Thr Val Gly Asn Leu Val Phe Thr Gly Ile
Phe Thr Ala Glu Met Phe 805 810
815Leu Lys Ile Ile Ala Met Asp Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp
820 825 830Asn Ile Phe Asp Gly
Phe Ile Val Thr Leu Ser Leu Val Glu Leu Gly 835
840 845Leu Ala Asn Val Glu Gly Leu Ser Val Leu Arg Ser
Phe Arg Leu Leu 850 855 860Arg Val Phe
Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile865
870 875 880Lys Ile Ile Gly Asn Ser Val
Gly Ala Leu Gly Asn Leu Thr Leu Val 885
890 895Leu Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly
Met Gln Leu Phe 900 905 910Gly
Lys Ser Tyr Lys Asp Cys Val Cys Lys Ile Ala Ser Asp Cys Gln 915
920 925Leu Pro Arg Trp His Met Asn Asp Phe
Phe His Ser Phe Leu Ile Val 930 935
940Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met945
950 955 960Glu Val Ala Gly
Gln Ala Met Cys Leu Thr Val Phe Met Met Val Met 965
970 975Val Ile Gly Asn Leu Val Val Leu Asn Leu
Phe Leu Ala Leu Leu Leu 980 985
990Ser Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp Asp Asn Glu
995 1000 1005Met Asn Asn Leu Gln Ile
Ala Val Asp Arg Met His Lys Gly Val 1010 1015
1020Ala Tyr Val Lys Arg Lys Ile Tyr Glu Phe Ile Gln Gln Ser
Phe 1025 1030 1035Ile Arg Lys Gln Lys
Ile Leu Asp Glu Ile Lys Pro Leu Asp Asp 1040 1045
1050Leu Asn Asn Lys Lys Asp Ser Cys Met Ser Asn His Thr
Thr Glu 1055 1060 1065Ile Gly Lys Asp
Leu Asp Tyr Leu Lys Asp Val Asn Gly Thr Thr 1070
1075 1080Ser Gly Ile Gly Thr Gly Ser Ser Val Glu Lys
Tyr Ile Ile Asp 1085 1090 1095Glu Ser
Asp Tyr Met Ser Phe Ile Asn Asn Pro Ser Leu Thr Val 1100
1105 1110Thr Val Pro Ile Ala Val Gly Glu Ser Asp
Phe Glu Asn Leu Asn 1115 1120 1125Thr
Glu Asp Phe Ser Ser Glu Ser Asp Leu Glu Glu Ser Lys Glu 1130
1135 1140Lys Leu Asn Glu Ser Ser Ser Ser Ser
Glu Gly Ser Thr Val Asp 1145 1150
1155Ile Gly Ala Pro Val Glu Glu Gln Pro Val Val Glu Pro Glu Glu
1160 1165 1170Thr Leu Glu Pro Glu Ala
Cys Phe Thr Glu Gly Cys Val Gln Arg 1175 1180
1185Phe Lys Cys Cys Gln Ile Asn Val Glu Glu Gly Arg Gly Lys
Gln 1190 1195 1200Trp Trp Asn Leu Arg
Arg Thr Cys Phe Arg Ile Val Glu His Asn 1205 1210
1215Trp Phe Glu Thr Phe Ile Val Phe Met Ile Leu Leu Ser
Ser Gly 1220 1225 1230Ala Leu Ala Phe
Glu Asp Ile Tyr Ile Asp Gln Arg Lys Thr Ile 1235
1240 1245Lys Thr Met Leu Glu Tyr Ala Asp Lys Val Phe
Thr Tyr Ile Phe 1250 1255 1260Ile Leu
Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Tyr Gln Thr 1265
1270 1275Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp
Phe Leu Ile Val Asp 1280 1285 1290Val
Ser Leu Val Ser Leu Thr Ala Asn Ala Leu Gly Tyr Ser Glu 1295
1300 1305Leu Gly Ala Ile Lys Ser Leu Arg Thr
Leu Arg Ala Leu Arg Pro 1310 1315
1320Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn
1325 1330 1335Ala Leu Leu Gly Ala Ile
Pro Ser Ile Met Asn Val Leu Leu Val 1340 1345
1350Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn
Leu 1355 1360 1365Phe Ala Gly Lys Phe
Tyr His Cys Ile Asn Thr Thr Thr Gly Asp 1370 1375
1380Arg Phe Asp Ile Glu Asp Val Asn Asn His Thr Asp Cys
Leu Lys 1385 1390 1395Leu Ile Glu Arg
Asn Glu Thr Ala Arg Trp Lys Asn Val Lys Val 1400
1405 1410Asn Phe Asp His Val Gly Phe Gly Tyr Leu Ser
Leu Leu Gln Val 1415 1420 1425Ala Thr
Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp 1430
1435 1440Ser Arg Asn Val Glu Leu Gln Pro Lys Tyr
Glu Glu Ser Leu Tyr 1445 1450 1455Met
Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe 1460
1465 1470Thr Leu Asn Leu Phe Ile Gly Val Ile
Ile Asp Asn Phe Asn Gln 1475 1480
1485Gln Lys Lys Lys Phe Gly Gly Gln Asp Ile Phe Met Thr Glu Glu
1490 1495 1500Gln Lys Lys Tyr Tyr Asn
Ala Met Lys Lys Leu Gly Ser Lys Lys 1505 1510
1515Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe Gln Gly
Met 1520 1525 1530Val Phe Asp Phe Val
Thr Arg Gln Val Phe Asp Ile Ser Ile Met 1535 1540
1545Ile Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu
Thr Asp 1550 1555 1560Asp Gln Ser Glu
Tyr Val Thr Thr Ile Leu Ser Arg Ile Asn Leu 1565
1570 1575Val Phe Ile Val Leu Phe Thr Gly Glu Cys Val
Leu Lys Leu Ile 1580 1585 1590Ser Leu
Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn Ile Phe Asp 1595
1600 1605Phe Val Val Val Ile Leu Ser Ile Val Gly
Met Phe Leu Ala Glu 1610 1615 1620Leu
Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile 1625
1630 1635Arg Leu Ala Arg Ile Gly Arg Ile Leu
Arg Leu Ile Lys Gly Ala 1640 1645
1650Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro
1655 1660 1665Ala Leu Phe Asn Ile Gly
Leu Leu Leu Phe Leu Val Met Phe Ile 1670 1675
1680Tyr Ala Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Arg
Glu 1685 1690 1695Val Gly Ile Asp Asp
Met Phe Asn Phe Glu Thr Phe Gly Asn Ser 1700 1705
1710Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp
Asp Gly 1715 1720 1725Leu Leu Ala Pro
Ile Leu Asn Ser Lys Pro Pro Asp Cys Asp Pro 1730
1735 1740Asn Lys Val Asn Pro Gly Ser Ser Val Lys Gly
Asp Cys Gly Asn 1745 1750 1755Pro Ser
Val Gly Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile Ser 1760
1765 1770Phe Leu Val Val Val Asn Met Tyr Ile Ala
Val Ile Leu Glu Asn 1775 1780 1785Phe
Ser Val Ala Thr Glu Glu Ser Ala Glu Pro Leu Ser Glu Asp 1790
1795 1800Asp Phe Glu Met Phe Tyr Glu Val Trp
Glu Lys Phe Asp Pro Asp 1805 1810
1815Ala Thr Gln Phe Met Glu Phe Glu Lys Leu Ser Gln Phe Ala Ala
1820 1825 1830Ala Leu Glu Pro Pro Leu
Asn Leu Pro Gln Pro Asn Lys Leu Gln 1835 1840
1845Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile
His 1850 1855 1860Cys Leu Asp Ile Leu
Phe Ala Phe Thr Lys Arg Val Leu Gly Glu 1865 1870
1875Ser Gly Glu Met Asp Ala Leu Arg Ile Gln Met Glu Glu
Arg Phe 1880 1885 1890Met Ala Ser Asn
Pro Ser Lys Val Ser Tyr Gln Pro Ile Thr Thr 1895
1900 1905Thr Leu Lys Arg Lys Gln Glu Glu Val Ser Ala
Val Ile Ile Gln 1910 1915 1920Arg Ala
Tyr Arg Arg His Leu Leu Lys Arg Thr Val Lys Gln Ala 1925
1930 1935Ser Phe Thr Tyr Asn Lys Asn Lys Ile Lys
Gly Gly Ala Asn Leu 1940 1945 1950Leu
Ile Lys Glu Asp Met Ile Ile Asp Arg Ile Asn Glu Asn Ser 1955
1960 1965Ile Thr Glu Lys Thr Asp Leu Thr Met
Ser Thr Ala Ala Cys Pro 1970 1975
1980Pro Ser Tyr Asp Arg Val Thr Lys Pro Ile Val Glu Lys His Glu
1985 1990 1995Gln Glu Gly Lys Asp Glu
Lys Ala Lys Gly Lys 2000 20051462009PRTRattus
norvegicus 146Met Glu Gln Thr Val Leu Val Pro Pro Gly Pro Asp Ser Phe Asn
Phe1 5 10 15Phe Thr Arg
Glu Ser Leu Ala Ala Ile Glu Arg Arg Ile Ala Glu Glu 20
25 30Lys Ala Lys Asn Pro Lys Pro Asp Lys Lys
Asp Asp Asp Glu Asn Gly 35 40
45Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Asn Leu Pro Phe Ile 50
55 60Tyr Gly Asp Ile Pro Pro Glu Met Val
Ser Glu Pro Leu Glu Asp Leu65 70 75
80Asp Pro Tyr Tyr Ile Asn Lys Lys Thr Phe Ile Val Leu Asn
Lys Gly 85 90 95Lys Ala
Ile Phe Arg Phe Ser Ala Thr Ser Ala Leu Tyr Ile Leu Thr 100
105 110Pro Phe Asn Pro Leu Arg Lys Ile Ala
Ile Lys Ile Leu Val His Ser 115 120
125Leu Phe Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Val Phe
130 135 140Met Thr Met Ser Asn Pro Pro
Asp Trp Thr Lys Asn Val Glu Tyr Thr145 150
155 160Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys
Ile Ile Ala Arg 165 170
175Gly Phe Cys Leu Glu Asp Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp
180 185 190Leu Asp Phe Thr Val Ile
Thr Phe Ala Tyr Val Thr Glu Phe Val Asp 195 200
205Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg
Ala Leu 210 215 220Lys Thr Ile Ser Val
Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu225 230
235 240Ile Gln Ser Val Lys Lys Leu Ser Asp Val
Met Ile Leu Thr Val Phe 245 250
255Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn
260 265 270Leu Arg Asn Lys Cys
Val Gln Trp Pro Pro Thr Asn Ala Ser Leu Glu 275
280 285Glu His Ser Ile Glu Lys Asn Val Thr Thr Asp Tyr
Asn Gly Thr Leu 290 295 300Val Asn Glu
Thr Val Phe Glu Phe Asp Trp Lys Ser Tyr Ile Gln Asp305
310 315 320Ser Arg Tyr His Tyr Phe Leu
Glu Gly Val Leu Asp Ala Leu Leu Cys 325
330 335Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly
Tyr Met Cys Val 340 345 350Lys
Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser Phe Asp Thr Phe 355
360 365Ser Trp Ala Phe Leu Ser Leu Phe Arg
Leu Met Thr Gln Asp Phe Trp 370 375
380Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met385
390 395 400Ile Phe Phe Val
Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn 405
410 415Leu Ile Leu Ala Val Val Ala Met Ala Tyr
Glu Glu Gln Asn Gln Ala 420 425
430Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe Gln Gln Met Leu
435 440 445Glu Gln Leu Lys Lys Gln Gln
Glu Ala Ala Gln Gln Ala Ala Ala Ala 450 455
460Thr Ala Ser Glu His Ser Arg Glu Pro Ser Ala Ala Gly Arg Leu
Ser465 470 475 480Asp Ser
Ser Ser Glu Ala Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu
485 490 495Arg Arg Asn Arg Arg Lys Lys
Arg Lys Gln Lys Glu Gln Ser Gly Gly 500 505
510Glu Glu Lys Asp Asp Asp Glu Phe His Lys Ser Glu Ser Glu
Asp Ser 515 520 525Ile Arg Arg Lys
Gly Phe Arg Phe Ser Ile Glu Gly Asn Arg Leu Thr 530
535 540Tyr Glu Lys Arg Tyr Ser Ser Pro His Gln Ser Leu
Leu Ser Ile Arg545 550 555
560Gly Ser Leu Phe Ser Pro Arg Arg Asn Ser Arg Thr Ser Leu Phe Ser
565 570 575Phe Arg Gly Arg Ala
Lys Asp Val Gly Ser Glu Asn Asp Phe Ala Asp 580
585 590Asp Glu His Ser Thr Phe Glu Asp Asn Glu Ser Arg
Arg Asp Ser Leu 595 600 605Phe Val
Pro Arg Arg His Gly Glu Arg Arg Asn Ser Asn Leu Ser Gln 610
615 620Thr Ser Arg Ser Ser Arg Met Leu Ala Gly Leu
Pro Ala Asn Gly Lys625 630 635
640Met His Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Val Gly Gly
645 650 655Pro Ser Val Pro
Thr Ser Pro Val Gly Gln Leu Leu Pro Glu Val Ile 660
665 670Ile Asp Lys Pro Ala Thr Asp Asp Asn Gly Thr
Thr Thr Glu Thr Glu 675 680 685Met
Arg Lys Arg Arg Ser Ser Ser Phe His Val Ser Met Asp Phe Leu 690
695 700Glu Asp Pro Ser Gln Arg Gln Arg Ala Met
Ser Ile Ala Ser Ile Leu705 710 715
720Thr Asn Thr Val Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Pro
Pro 725 730 735Cys Trp Tyr
Lys Phe Ser Asn Ile Phe Leu Ile Trp Asp Cys Ser Pro 740
745 750Tyr Trp Leu Lys Val Lys His Ile Val Asn
Leu Val Val Met Asp Pro 755 760
765Phe Val Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe 770
775 780Met Ala Met Glu His Tyr Pro Met
Thr Glu His Phe Asn His Val Leu785 790
795 800Thr Val Gly Asn Leu Val Phe Thr Gly Ile Phe Thr
Ala Glu Met Phe 805 810
815Leu Lys Ile Ile Ala Met Asp Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp
820 825 830Asn Ile Phe Asp Gly Phe
Ile Val Thr Leu Ser Leu Val Glu Leu Gly 835 840
845Leu Ala Asn Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg
Leu Leu 850 855 860Arg Val Phe Lys Leu
Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile865 870
875 880Lys Ile Ile Gly Asn Ser Val Gly Ala Leu
Gly Asn Leu Thr Leu Val 885 890
895Leu Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe
900 905 910Gly Lys Ser Tyr Lys
Asp Cys Val Cys Lys Ile Ala Thr Asp Cys Lys 915
920 925Leu Pro Arg Trp His Met Asn Asp Phe Phe His Ser
Phe Leu Ile Val 930 935 940Phe Arg Val
Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met945
950 955 960Glu Val Ala Gly Gln Ala Met
Cys Leu Thr Val Phe Met Met Val Met 965
970 975Val Ile Arg Asn Leu Val Val Leu Asn Leu Phe Leu
Ala Leu Leu Leu 980 985 990Ser
Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp Asp Asn Glu 995
1000 1005Met Asn Asn Leu Gln Ile Ala Val
Asp Arg Met His Lys Gly Val 1010 1015
1020Ala Tyr Val Lys Arg Lys Ile Tyr Glu Phe Ile Gln Gln Ser Phe
1025 1030 1035Val Arg Lys Gln Lys Ile
Leu Asp Glu Ile Lys Pro Leu Asp Asp 1040 1045
1050Leu Asn Asn Arg Lys Asp Asn Cys Thr Ser Asn His Thr Thr
Glu 1055 1060 1065Ile Gly Lys Asp Leu
Asp Cys Leu Lys Asp Val Asn Gly Thr Thr 1070 1075
1080Ser Gly Ile Gly Thr Gly Ser Ser Val Glu Lys Tyr Ile
Ile Asp 1085 1090 1095Glu Ser Asp Tyr
Met Ser Phe Ile Asn Asn Pro Ser Leu Thr Val 1100
1105 1110Thr Val Pro Ile Ala Val Gly Glu Ser Asp Phe
Glu Asn Leu Asn 1115 1120 1125Thr Glu
Asp Phe Ser Ser Glu Ser Asp Leu Glu Glu Ser Lys Glu 1130
1135 1140Lys Leu Asn Glu Ser Ser Ser Ser Ser Glu
Gly Ser Thr Val Asp 1145 1150 1155Ile
Gly Ala Pro Ala Glu Glu Gln Pro Val Met Glu Pro Glu Glu 1160
1165 1170Thr Leu Glu Pro Glu Ala Cys Phe Thr
Glu Gly Cys Val Gln Arg 1175 1180
1185Phe Lys Cys Cys Gln Ile Ser Val Glu Glu Gly Arg Gly Lys Gln
1190 1195 1200Trp Trp Asn Leu Arg Arg
Thr Cys Phe Arg Ile Val Glu His Asn 1205 1210
1215Trp Phe Glu Thr Phe Ile Val Phe Met Ile Leu Leu Ser Ser
Gly 1220 1225 1230Ala Leu Ala Phe Glu
Asp Ile Tyr Ile Asp Gln Arg Lys Thr Ile 1235 1240
1245Lys Thr Met Leu Glu Tyr Ala Asp Lys Val Phe Thr Tyr
Ile Phe 1250 1255 1260Ile Leu Glu Met
Leu Leu Lys Trp Val Ala Tyr Gly Tyr Gln Thr 1265
1270 1275Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe
Leu Ile Val Asp 1280 1285 1290Val Ser
Leu Val Ser Leu Thr Ala Asn Ala Leu Gly Tyr Ser Glu 1295
1300 1305Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu
Arg Ala Leu Arg Pro 1310 1315 1320Leu
Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn 1325
1330 1335Ala Leu Leu Gly Ala Ile Pro Ser Ile
Met Asn Val Leu Leu Val 1340 1345
1350Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu
1355 1360 1365Phe Ala Gly Lys Phe Tyr
His Cys Val Asn Thr Thr Thr Gly Asp 1370 1375
1380Thr Phe Glu Ile Thr Glu Val Asn Asn His Ser Asp Cys Leu
Lys 1385 1390 1395Leu Ile Glu Arg Asn
Glu Thr Ala Arg Trp Lys Asn Val Lys Val 1400 1405
1410Asn Phe Asp His Val Gly Phe Gly Tyr Leu Ser Leu Leu
Gln Val 1415 1420 1425Ala Thr Phe Lys
Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp 1430
1435 1440Ser Arg Asn Val Glu Leu Gln Pro Lys Tyr Glu
Glu Ser Leu Tyr 1445 1450 1455Met Tyr
Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe 1460
1465 1470Thr Leu Asn Leu Phe Ile Gly Val Ile Ile
Asp Asn Phe Asn Gln 1475 1480 1485Gln
Lys Lys Lys Phe Gly Gly Gln Asp Ile Phe Met Thr Glu Glu 1490
1495 1500Gln Lys Lys Tyr Tyr Asn Ala Met Lys
Lys Leu Gly Ser Lys Lys 1505 1510
1515Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe Gln Gly Met
1520 1525 1530Val Phe Asp Phe Val Thr
Arg Gln Val Phe Asp Ile Ser Ile Met 1535 1540
1545Ile Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Thr
Asp 1550 1555 1560Asp Gln Ser Asp Tyr
Val Thr Ser Ile Leu Ser Arg Ile Asn Leu 1565 1570
1575Val Phe Ile Val Leu Phe Thr Gly Glu Cys Val Leu Lys
Leu Ile 1580 1585 1590Ser Leu Arg His
Tyr Tyr Phe Thr Ile Gly Trp Asn Ile Phe Asp 1595
1600 1605Phe Val Val Val Ile Leu Ser Ile Val Gly Met
Phe Leu Ala Glu 1610 1615 1620Leu Ile
Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile 1625
1630 1635Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg
Leu Ile Lys Gly Ala 1640 1645 1650Lys
Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro 1655
1660 1665Ala Leu Phe Asn Ile Gly Leu Leu Leu
Phe Leu Val Met Phe Ile 1670 1675
1680Tyr Ala Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Arg Glu
1685 1690 1695Val Gly Ile Asp Asp Met
Phe Asn Phe Glu Thr Phe Gly Asn Ser 1700 1705
1710Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp
Gly 1715 1720 1725Leu Leu Ala Pro Ile
Leu Asn Ser Lys Pro Pro Asp Cys Asp Pro 1730 1735
1740Asn Lys Val Asn Pro Gly Ser Ser Val Lys Gly Asp Cys
Gly Asn 1745 1750 1755Pro Ser Val Gly
Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile Ser 1760
1765 1770Phe Leu Val Val Val Asn Met Tyr Ile Ala Val
Ile Leu Glu Asn 1775 1780 1785Phe Ser
Val Ala Thr Glu Glu Ser Ala Glu Pro Leu Ser Glu Asp 1790
1795 1800Asp Phe Glu Met Phe Tyr Glu Val Trp Glu
Lys Phe Asp Pro Asp 1805 1810 1815Ala
Thr Gln Phe Met Glu Phe Glu Lys Leu Ser Gln Phe Ala Ala 1820
1825 1830Ala Leu Glu Pro Pro Leu Asn Leu Pro
Gln Pro Asn Lys Leu Gln 1835 1840
1845Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile His
1850 1855 1860Cys Leu Asp Ile Leu Phe
Ala Phe Thr Lys Arg Val Leu Gly Glu 1865 1870
1875Ser Gly Glu Met Asp Ala Leu Arg Ile Gln Met Glu Glu Arg
Phe 1880 1885 1890Met Ala Ser Asn Pro
Ser Lys Val Ser Tyr Gln Pro Ile Thr Thr 1895 1900
1905Thr Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Val Ile
Ile Gln 1910 1915 1920Arg Ala Tyr Arg
Arg His Leu Leu Lys Arg Thr Val Lys Gln Ala 1925
1930 1935Ser Phe Thr Tyr Asn Lys Asn Lys Leu Lys Gly
Gly Ala Asn Leu 1940 1945 1950Leu Val
Lys Glu Asp Met Ile Ile Asp Arg Ile Asn Glu Asn Ser 1955
1960 1965Ile Thr Glu Lys Thr Asp Leu Thr Met Ser
Thr Ala Ala Cys Pro 1970 1975 1980Pro
Ser Tyr Asp Arg Val Thr Lys Pro Ile Val Glu Lys His Glu 1985
1990 1995Gln Glu Gly Lys Asp Glu Lys Ala Lys
Gly Lys 2000 20051472368PRTRattus norvegicus 147Met
Ala Arg Phe Gly Asp Glu Met Pro Gly Arg Tyr Gly Ala Gly Gly1
5 10 15Gly Gly Ser Gly Pro Ala Ala
Gly Val Val Val Gly Ala Ala Gly Gly 20 25
30Arg Gly Ala Gly Gly Ser Arg Gln Gly Gly Gln Pro Gly Ala
Gln Arg 35 40 45Met Tyr Lys Gln
Ser Met Ala Gln Arg Ala Arg Thr Met Ala Leu Tyr 50 55
60Asn Pro Ile Pro Val Arg Gln Asn Cys Leu Thr Val Asn
Arg Ser Leu65 70 75
80Phe Leu Phe Ser Glu Asp Asn Val Val Arg Lys Tyr Ala Lys Lys Ile
85 90 95Thr Glu Trp Pro Pro Phe
Glu Tyr Met Ile Leu Ala Thr Ile Ile Ala 100
105 110Asn Cys Ile Val Leu Ala Leu Glu Gln His Leu Pro
Asp Asp Asp Lys 115 120 125Thr Pro
Met Ser Glu Arg Leu Asp Asp Thr Glu Pro Tyr Phe Ile Gly 130
135 140Ile Phe Cys Phe Glu Ala Gly Ile Lys Ile Val
Ala Leu Gly Phe Ala145 150 155
160Phe His Lys Gly Ser Tyr Leu Arg Asn Gly Trp Asn Val Met Asp Phe
165 170 175Val Val Val Leu
Thr Gly Ile Leu Ala Thr Val Gly Thr Glu Phe Asp 180
185 190Leu Arg Thr Leu Arg Ala Val Arg Val Leu Arg
Pro Leu Lys Leu Val 195 200 205Ser
Gly Ile Pro Ser Leu Gln Val Val Leu Lys Ser Ile Met Lys Ala 210
215 220Met Ile Pro Leu Leu Gln Ile Gly Leu Leu
Leu Phe Phe Ala Ile Leu225 230 235
240Ile Phe Ala Ile Ile Gly Leu Glu Phe Tyr Met Gly Lys Phe His
Thr 245 250 255Thr Cys Phe
Glu Glu Gly Thr Asp Asp Ile Gln Gly Glu Ser Pro Ala 260
265 270Pro Cys Gly Thr Glu Glu Pro Ala Arg Thr
Cys Pro Asn Gly Thr Lys 275 280
285Cys Gln Pro Tyr Trp Glu Gly Pro Asn Asn Gly Ile Thr Gln Phe Asp 290
295 300Asn Ile Leu Phe Ala Val Leu Thr
Val Phe Gln Cys Ile Thr Met Glu305 310
315 320Gly Trp Thr Asp Leu Leu Tyr Asn Ser Asn Asp Ala
Ser Gly Asn Thr 325 330
335Trp Asn Trp Leu Tyr Phe Ile Pro Leu Ile Ile Ile Gly Ser Phe Phe
340 345 350Met Leu Asn Leu Val Leu
Gly Val Leu Ser Gly Glu Phe Ala Lys Glu 355 360
365Arg Glu Arg Val Glu Asn Arg Arg Ala Phe Leu Lys Leu Arg
Arg Gln 370 375 380Gln Gln Ile Glu Arg
Glu Leu Asn Gly Tyr Met Glu Trp Ile Ser Lys385 390
395 400Ala Glu Glu Val Ile Leu Ala Glu Asp Glu
Thr Asp Val Glu Gln Arg 405 410
415His Pro Phe Asp Gly Ala Leu Arg Arg Ala Thr Leu Lys Lys Ser Lys
420 425 430Thr Asp Leu Leu Asn
Pro Glu Glu Ala Glu Asp Gln Leu Ala Asp Ile 435
440 445Ala Ser Val Gly Ser Pro Phe Ala Arg Ala Ser Ile
Lys Ser Ala Lys 450 455 460Leu Glu Asn
Ser Thr Phe Phe His Lys Lys Glu Arg Arg Met Arg Phe465
470 475 480Tyr Ile Arg Arg Met Val Lys
Thr Gln Ala Phe Tyr Trp Thr Val Leu 485
490 495Ser Leu Val Ala Leu Asn Thr Leu Trp Leu Ala Ile
Val His Tyr Asn 500 505 510Gln
Pro Glu Trp Leu Ser Asp Phe Leu Tyr Tyr Ala Glu Phe Ile Phe 515
520 525Leu Gly Leu Phe Met Ser Glu Met Phe
Ile Lys Met Tyr Gly Leu Gly 530 535
540Thr Arg Pro Tyr Phe His Ser Ser Phe Asn Cys Phe Asp Cys Gly Val545
550 555 560Ile Ile Gly Ser
Ile Phe Glu Val Ile Trp Ala Val Ile Lys Pro Gly 565
570 575Thr Ser Phe Gly Ile Ser Val Leu Arg Ala
Leu Arg Leu Leu Arg Ile 580 585
590Phe Lys Val Thr Lys Tyr Trp Ala Ser Leu Arg Asn Leu Val Val Ser
595 600 605Leu Leu Asn Ser Met Lys Ser
Ile Ile Ser Leu Leu Phe Leu Leu Phe 610 615
620Leu Phe Ile Val Val Phe Ala Leu Leu Gly Met Gln Leu Phe Gly
Gly625 630 635 640Gln Phe
Asn Phe Asp Glu Gly Thr Pro Pro Thr Asn Phe Asp Thr Phe
645 650 655Pro Ala Ala Ile Met Thr Val
Phe Gln Ile Leu Thr Gly Glu Asp Trp 660 665
670Asn Glu Val Met Tyr Asp Glu Ile Lys Ser Gln Gly Gly Val
Gln Gly 675 680 685Gly Met Val Phe
Ser Ile Tyr Phe Ile Val Leu Thr Leu Phe Gly Asn 690
695 700Tyr Thr Leu Leu Asn Val Phe Leu Ala Ile Ala Val
Asp Asn Leu Ala705 710 715
720Asn Ala Gln Glu Leu Thr Lys Asp Glu Gln Glu Glu Glu Glu Ala Ala
725 730 735Asn Gln Lys Leu Ala
Leu Gln Lys Ala Lys Glu Val Ala Glu Val Ser 740
745 750Pro Leu Ser Ala Ala Asn Met Ser Ile Ala Val Lys
Glu Gln Gln Lys 755 760 765Asn Gln
Lys Pro Ala Lys Ser Val Trp Glu Gln Arg Thr Ser Glu Met 770
775 780Arg Lys Gln Asn Leu Leu Ala Ser Arg Glu Ala
Leu Tyr Gly Asp Ala785 790 795
800Ala Glu Arg Trp Pro Thr Thr Tyr Ala Arg Pro Leu Arg Pro Asp Val
805 810 815Lys Thr His Leu
Asp Arg Pro Leu Val Val Asp Pro Gln Glu Asn Arg 820
825 830Asn Asn Asn Thr Asn Lys Ser Arg Ala Pro Glu
Ala Leu Arg Gln Thr 835 840 845Ala
Arg Pro Arg Glu Ser Ala Arg Asp Pro Asp Ala Arg Arg Ala Trp 850
855 860Pro Ser Ser Pro Glu Arg Ala Pro Gly Arg
Glu Gly Pro Tyr Gly Arg865 870 875
880Glu Ser Glu Pro Gln Gln Arg Glu His Ala Pro Pro Arg Glu His
Val 885 890 895Pro Trp Asp
Ala Asp Pro Glu Arg Ala Lys Ala Gly Asp Ala Pro Arg 900
905 910Arg His Thr His Arg Pro Val Ala Glu Gly
Glu Pro Arg Arg His Arg 915 920
925Ala Arg Arg Arg Pro Gly Asp Glu Pro Asp Asp Arg Pro Glu Arg Arg 930
935 940Pro Arg Pro Arg Asp Ala Thr Arg
Pro Ala Arg Ala Ala Asp Gly Glu945 950
955 960Gly Asp Asp Gly Glu Arg Lys Arg Arg His Arg His
Gly Pro Pro Ala 965 970
975His Asp Asp Arg Glu Arg Arg His Arg Arg Arg Lys Glu Ser Gln Gly
980 985 990Ser Gly Val Pro Met Ser
Gly Pro Asn Leu Ser Thr Thr Arg Pro Ile 995 1000
1005Gln Gln Asp Leu Gly Arg Gln Asp Leu Pro Leu Ala
Glu Asp Leu 1010 1015 1020Asp Asn Met
Lys Asn Asn Lys Leu Ala Thr Gly Glu Pro Ala Ser 1025
1030 1035Pro His Asp Ser Leu Gly His Ser Gly Leu Pro
Pro Ser Pro Ala 1040 1045 1050Lys Ile
Gly Asn Ser Thr Asn Pro Gly Pro Ala Leu Ala Thr Asn 1055
1060 1065Pro Gln Asn Ala Ala Ser Arg Arg Thr Pro
Asn Asn Pro Gly Asn 1070 1075 1080Pro
Ser Asn Pro Gly Pro Pro Lys Thr Pro Glu Asn Ser Leu Ile 1085
1090 1095Val Thr Asn Pro Ser Ser Thr Gln Pro
Asn Ser Ala Lys Thr Ala 1100 1105
1110Arg Lys Pro Glu His Met Ala Val Glu Ile Pro Pro Ala Cys Pro
1115 1120 1125Pro Leu Asn His Thr Val
Val Gln Val Asn Lys Asn Ala Asn Pro 1130 1135
1140Asp Pro Leu Pro Lys Lys Glu Glu Glu Lys Lys Glu Glu Glu
Glu 1145 1150 1155Ala Asp Pro Gly Glu
Asp Gly Pro Lys Pro Met Pro Pro Tyr Ser 1160 1165
1170Ser Met Phe Ile Leu Ser Thr Thr Asn Pro Leu Arg Arg
Leu Cys 1175 1180 1185His Tyr Ile Leu
Asn Leu Arg Tyr Phe Glu Met Cys Ile Leu Met 1190
1195 1200Val Ile Ala Met Ser Ser Ile Ala Leu Ala Ala
Glu Asp Pro Val 1205 1210 1215Gln Pro
Asn Ala Pro Arg Asn Asn Val Leu Arg Tyr Phe Asp Tyr 1220
1225 1230Val Phe Thr Gly Val Phe Thr Phe Glu Met
Val Ile Lys Met Ile 1235 1240 1245Asp
Leu Gly Leu Val Leu His Gln Gly Ala Tyr Phe Arg Asp Leu 1250
1255 1260Trp Asn Ile Leu Asp Phe Ile Val Val
Ser Gly Ala Leu Val Ala 1265 1270
1275Phe Ala Phe Thr Gly Asn Ser Lys Gly Lys Asp Ile Asn Thr Ile
1280 1285 1290Lys Ser Leu Arg Val Leu
Arg Val Leu Arg Pro Leu Lys Thr Ile 1295 1300
1305Lys Arg Leu Pro Lys Leu Lys Ala Val Phe Asp Cys Val Val
Asn 1310 1315 1320Ser Leu Lys Asn Val
Phe Asn Ile Leu Ile Val Tyr Met Leu Phe 1325 1330
1335Met Phe Ile Phe Ala Val Val Ala Val Gln Leu Phe Lys
Gly Lys 1340 1345 1350Phe Phe His Cys
Thr Asp Glu Ser Lys Glu Phe Glu Arg Asp Cys 1355
1360 1365Arg Gly Lys Tyr Leu Leu Tyr Glu Lys Asn Glu
Val Lys Ala Arg 1370 1375 1380Asp Arg
Glu Trp Lys Lys Tyr Asp Phe His Tyr Asp Asn Val Leu 1385
1390 1395Trp Ala Leu Leu Thr Leu Phe Thr Val Ser
Thr Gly Glu Gly Trp 1400 1405 1410Pro
Gln Val Leu Lys His Ser Val Asp Ala Thr Phe Glu Asn Gln 1415
1420 1425Gly Pro Ser Pro Gly Tyr Arg Met Glu
Met Ser Ile Phe Tyr Val 1430 1435
1440Val Tyr Phe Val Val Phe Pro Phe Phe Phe Val Asn Ile Phe Val
1445 1450 1455Ala Leu Ile Ile Ile Thr
Phe Gln Glu Gln Gly Asp Lys Met Met 1460 1465
1470Glu Glu Tyr Ser Leu Glu Lys Asn Glu Arg Ala Cys Ile Asp
Phe 1475 1480 1485Ala Ile Ser Ala Lys
Pro Leu Thr Arg His Met Pro Gln Asn Lys 1490 1495
1500Gln Ser Phe Gln Tyr Arg Met Trp Gln Phe Val Val Ser
Pro Pro 1505 1510 1515Phe Glu Tyr Thr
Ile Met Ala Met Ile Ala Leu Asn Thr Ile Val 1520
1525 1530Leu Met Met Lys Phe Tyr Gly Ala Ser Val Ala
Tyr Glu Asn Ala 1535 1540 1545Leu Arg
Val Phe Asn Ile Val Phe Thr Ser Leu Phe Ser Leu Glu 1550
1555 1560Cys Val Leu Lys Val Met Ala Phe Gly Ile
Leu Asn Tyr Phe Arg 1565 1570 1575Asp
Ala Trp Asn Ile Phe Asp Phe Val Thr Val Leu Gly Ser Ile 1580
1585 1590Thr Asp Ile Leu Val Thr Glu Phe Gly
Asn Asn Phe Ile Asn Leu 1595 1600
1605Ser Phe Leu Arg Leu Phe Arg Ala Ala Arg Leu Ile Lys Leu Leu
1610 1615 1620Arg Gln Gly Tyr Thr Ile
Arg Ile Leu Leu Trp Thr Phe Val Gln 1625 1630
1635Ser Phe Lys Ala Leu Pro Tyr Val Cys Leu Leu Ile Ala Met
Leu 1640 1645 1650Phe Phe Ile Tyr Ala
Ile Ile Gly Met Gln Val Phe Gly Asn Ile 1655 1660
1665Gly Ile Asp Gly Glu Asp Glu Asp Ser Asp Glu Asp Glu
Phe Gln 1670 1675 1680Ile Thr Glu His
Asn Asn Phe Arg Thr Phe Phe Gln Ala Leu Met 1685
1690 1695Leu Leu Phe Arg Ser Ala Thr Gly Glu Ala Trp
His Asn Ile Met 1700 1705 1710Leu Ser
Cys Leu Ser Gly Lys Pro Cys Asp Lys Asn Ser Gly Ile 1715
1720 1725Gln Lys Pro Glu Cys Gly Asn Glu Phe Ala
Tyr Phe Tyr Phe Val 1730 1735 1740Ser
Phe Ile Phe Leu Cys Ser Phe Leu Met Leu Asn Leu Phe Val 1745
1750 1755Ala Val Ile Met Asp Asn Phe Glu Tyr
Leu Thr Arg Asp Ser Ser 1760 1765
1770Ile Leu Gly Pro His His Leu Asp Glu Tyr Val Arg Val Trp Ala
1775 1780 1785Glu Tyr Asp Pro Ala Ala
Cys Gly Arg Ile His Tyr Lys Asp Met 1790 1795
1800Tyr Ser Leu Leu Arg Val Ile Ser Pro Pro Leu Gly Leu Gly
Lys 1805 1810 1815Lys Cys Pro His Arg
Val Ala Cys Lys Arg Leu Leu Arg Met Asp 1820 1825
1830Leu Pro Val Ala Asp Asp Asn Thr Val His Phe Asn Ser
Thr Leu 1835 1840 1845Met Ala Leu Ile
Arg Thr Ala Leu Asp Ile Lys Ile Ala Lys Gly 1850
1855 1860Gly Ala Asp Lys Gln Gln Met Asp Ala Glu Leu
Arg Lys Glu Met 1865 1870 1875Met Ala
Ile Trp Pro Asn Leu Ser Gln Lys Thr Leu Asp Leu Leu 1880
1885 1890Val Thr Pro His Lys Ser Thr Asp Leu Thr
Val Gly Lys Ile Tyr 1895 1900 1905Ala
Ala Met Met Ile Met Glu Tyr Tyr Arg Gln Ser Lys Ala Lys 1910
1915 1920Lys Leu Gln Ala Met Arg Glu Glu Gln
Asn Arg Thr Pro Leu Met 1925 1930
1935Phe Gln Arg Met Glu Pro Pro Ser Pro Thr Gln Glu Gly Gly Pro
1940 1945 1950Ser Gln Asn Ala Leu Pro
Ser Thr Gln Leu Asp Pro Gly Gly Gly 1955 1960
1965Leu Met Ala Gln Glu Ser Ser Met Lys Glu Ser Pro Ser Trp
Val 1970 1975 1980Thr Gln Arg Ala Gln
Glu Met Phe Gln Lys Thr Gly Thr Trp Ser 1985 1990
1995Pro Glu Arg Gly Pro Pro Ile Asp Met Pro Asn Ser Gln
Pro Asn 2000 2005 2010Ser Gln Ser Val
Glu Met Arg Glu Met Gly Thr Asp Gly Tyr Ser 2015
2020 2025Asp Ser Glu His Tyr Leu Pro Met Glu Gly Gln
Thr Arg Ala Ala 2030 2035 2040Ser Met
Pro Arg Leu Pro Ala Glu Asn Gln Arg Arg Arg Gly Arg 2045
2050 2055Pro Arg Gly Asn Asn Leu Ser Thr Ile Ser
Asp Thr Ser Pro Met 2060 2065 2070Lys
Arg Ser Ala Ser Val Leu Gly Pro Lys Ala Arg Arg Leu Asp 2075
2080 2085Asp Tyr Ser Leu Glu Arg Val Pro Pro
Glu Glu Asn Gln Arg Tyr 2090 2095
2100His Gln Arg Arg Arg Asp Arg Gly His Arg Thr Ser Glu Arg Ser
2105 2110 2115Leu Gly Arg Tyr Thr Asp
Val Asp Thr Gly Leu Gly Thr Asp Leu 2120 2125
2130Ser Met Thr Thr Gln Ser Gly Asp Leu Pro Ser Lys Asp Arg
Asp 2135 2140 2145Gln Asp Arg Gly Arg
Pro Lys Asp Arg Lys His Arg Pro His His 2150 2155
2160His His His His His His His His Pro Pro Ala Pro Asp
Arg Glu 2165 2170 2175Arg Tyr Ala Gln
Glu Arg Pro Asp Thr Gly Arg Ala Arg Ala Arg 2180
2185 2190Glu Gln Arg Trp Ser Arg Ser Pro Ser Glu Gly
Arg Glu His Ala 2195 2200 2205Thr His
Arg Gln Gly Ser Ser Ser Val Ser Gly Ser Pro Ala Pro 2210
2215 2220Ser Thr Ser Gly Thr Ser Thr Pro Arg Arg
Gly Arg Arg Gln Leu 2225 2230 2235Pro
Gln Thr Pro Cys Thr Pro Arg Pro Leu Val Ser Tyr Ser Pro 2240
2245 2250Ala Pro Arg Arg Pro Ala Ala Arg Arg
Met Ala Gly Pro Pro Ala 2255 2260
2265Pro Pro Gly Gly Ser Pro Arg Gly Cys Arg Arg Ala Pro Arg Trp
2270 2275 2280Pro Ala His Ala Pro Glu
Gly Pro Arg Pro Arg Gly Ala Asp Tyr 2285 2290
2295Thr Glu Pro Asp Ser Pro Arg Glu Pro Pro Gly Gly Ala His
Glu 2300 2305 2310Pro Ala Pro Arg Ser
Pro Arg Thr Pro Arg Ala Ala Gly Cys Ala 2315 2320
2325Ser Pro Arg His Gly Arg Arg Leu Pro Asn Gly Tyr Tyr
Ala Gly 2330 2335 2340His Gly Ala Pro
Arg Pro Arg Thr Ala Arg Arg Gly Ala His Asp 2345
2350 2355Ala Tyr Ser Glu Ser Glu Asp Asp Trp Cys
2360 23651482512PRTHomo sapiens 148Met Ala Arg Phe Gly
Asp Glu Met Pro Ala Arg Tyr Gly Gly Gly Gly1 5
10 15Ser Gly Ala Ala Ala Gly Val Val Val Gly Ser
Gly Gly Gly Arg Gly 20 25
30Ala Gly Gly Ser Arg Gln Gly Gly Gln Pro Gly Ala Gln Arg Met Tyr
35 40 45Lys Gln Ser Met Ala Gln Arg Ala
Arg Thr Met Ala Leu Tyr Asn Pro 50 55
60Ile Pro Val Arg Gln Asn Cys Leu Thr Val Asn Arg Ser Leu Phe Leu65
70 75 80Phe Ser Glu Asp Asn
Val Val Arg Lys Tyr Ala Lys Lys Ile Thr Glu 85
90 95Trp Pro Pro Phe Glu Tyr Met Ile Leu Ala Thr
Ile Ile Ala Asn Cys 100 105
110Ile Val Leu Ala Leu Glu Gln His Leu Pro Asp Asp Asp Lys Thr Pro
115 120 125Met Ser Glu Arg Leu Asp Asp
Thr Glu Pro Tyr Phe Ile Gly Ile Phe 130 135
140Cys Phe Glu Ala Gly Ile Lys Ile Ile Ala Leu Gly Phe Ala Phe
His145 150 155 160Lys Gly
Ser Tyr Leu Arg Asn Gly Trp Asn Val Met Asp Phe Val Val
165 170 175Val Leu Thr Gly Ile Leu Ala
Thr Val Gly Thr Glu Phe Asp Leu Arg 180 185
190Thr Leu Arg Ala Val Arg Val Leu Arg Pro Leu Lys Leu Val
Ser Gly 195 200 205Ile Pro Ser Leu
Gln Val Val Leu Lys Ser Ile Met Lys Ala Met Ile 210
215 220Pro Leu Leu Gln Ile Gly Leu Leu Leu Phe Phe Ala
Ile Leu Ile Phe225 230 235
240Ala Ile Ile Gly Leu Glu Phe Tyr Lys Gly Lys Phe His Thr Thr Cys
245 250 255Phe Glu Glu Gly Thr
Asp Asp Ile Gln Gly Glu Ser Pro Ala Pro Cys 260
265 270Gly Thr Glu Glu Pro Ala Arg Thr Cys Pro Asn Gly
Thr Lys Cys Gln 275 280 285Pro Tyr
Trp Glu Gly Pro Asn Asn Gly Ile Thr Gln Phe Asp Asn Ile 290
295 300Leu Phe Ala Val Leu Thr Val Phe Gln Cys Ile
Thr Met Glu Gly Trp305 310 315
320Thr Asp Leu Leu Tyr Asn Ser Asn Asp Ala Ser Gly Asn Thr Trp Asn
325 330 335Trp Leu Tyr Phe
Ile Pro Leu Ile Ile Ile Gly Ser Phe Phe Met Leu 340
345 350Asn Leu Val Leu Gly Val Leu Ser Gly Glu Phe
Ala Lys Glu Arg Glu 355 360 365Arg
Val Glu Asn Arg Arg Ala Phe Leu Lys Leu Arg Arg Gln Gln Gln 370
375 380Ile Glu Arg Glu Leu Asn Gly Tyr Met Glu
Trp Ile Ser Lys Ala Glu385 390 395
400Glu Val Ile Leu Ala Glu Asp Glu Thr Asp Gly Glu Gln Arg His
Pro 405 410 415Phe Asp Gly
Ala Leu Arg Arg Thr Thr Ile Lys Lys Ser Lys Thr Asp 420
425 430Leu Leu Asn Pro Glu Glu Ala Glu Asp Gln
Leu Ala Asp Ile Ala Ser 435 440
445Val Gly Ser Pro Phe Ala Arg Ala Ser Ile Lys Ser Ala Lys Leu Glu 450
455 460Asn Ser Thr Phe Phe His Lys Lys
Glu Arg Arg Met Arg Phe Tyr Ile465 470
475 480Arg Arg Met Val Lys Thr Gln Ala Phe Tyr Trp Thr
Val Leu Ser Leu 485 490
495Val Ala Leu Asn Thr Leu Cys Val Ala Ile Val His Tyr Asn Gln Pro
500 505 510Glu Trp Leu Ser Asp Phe
Leu Tyr Tyr Ala Glu Phe Ile Phe Leu Gly 515 520
525Leu Phe Met Ser Glu Met Phe Ile Lys Met Tyr Gly Leu Gly
Thr Arg 530 535 540Pro Tyr Phe His Ser
Ser Phe Asn Cys Phe Asp Cys Gly Val Ile Ile545 550
555 560Gly Ser Ile Phe Glu Val Ile Trp Ala Val
Ile Lys Pro Gly Thr Ser 565 570
575Phe Gly Ile Ser Val Leu Arg Ala Leu Arg Leu Leu Arg Ile Phe Lys
580 585 590Val Thr Lys Tyr Trp
Ala Ser Leu Arg Asn Leu Val Val Ser Leu Leu 595
600 605Asn Ser Met Lys Ser Ile Ile Ser Leu Leu Phe Leu
Leu Phe Leu Phe 610 615 620Ile Val Val
Phe Ala Leu Leu Gly Met Gln Leu Phe Gly Gly Gln Phe625
630 635 640Asn Phe Asp Glu Gly Thr Pro
Pro Thr Asn Phe Asp Thr Phe Pro Ala 645
650 655Ala Ile Met Thr Val Phe Gln Ile Leu Thr Gly Glu
Asp Trp Asn Glu 660 665 670Val
Met Tyr Asp Gly Ile Lys Ser Gln Gly Gly Val Gln Gly Gly Met 675
680 685Val Phe Ser Ile Tyr Phe Ile Val Leu
Thr Leu Phe Gly Asn Tyr Thr 690 695
700Leu Leu Asn Val Phe Leu Ala Ile Ala Val Asp Asn Leu Ala Asn Ala705
710 715 720Gln Glu Leu Thr
Lys Val Glu Ala Asp Glu Gln Glu Glu Glu Glu Ala 725
730 735Ala Asn Gln Lys Leu Ala Leu Gln Lys Ala
Lys Glu Val Ala Glu Val 740 745
750Ser Pro Leu Ser Ala Ala Asn Met Ser Ile Ala Val Lys Glu Gln Gln
755 760 765Lys Asn Gln Lys Pro Ala Lys
Ser Val Trp Glu Gln Arg Thr Ser Glu 770 775
780Met Arg Lys Gln Asn Leu Leu Ala Ser Arg Glu Ala Leu Tyr Asn
Glu785 790 795 800Met Asp
Pro Asp Glu Arg Trp Lys Ala Ala Tyr Thr Arg His Leu Arg
805 810 815Pro Asp Met Lys Thr His Leu
Asp Arg Pro Leu Val Val Asp Pro Gln 820 825
830Glu Asn Arg Asn Asn Asn Thr Asn Lys Ser Arg Ala Ala Glu
Pro Thr 835 840 845Val Asp Gln Arg
Leu Gly Gln Gln Arg Ala Glu Asp Phe Leu Arg Lys 850
855 860Gln Ala Arg Tyr His Asp Arg Ala Arg Asp Pro Ser
Gly Ser Ala Gly865 870 875
880Leu Asp Ala Arg Arg Pro Trp Ala Gly Ser Gln Glu Ala Glu Leu Ser
885 890 895Arg Glu Gly Pro Tyr
Gly Arg Glu Ser Asp His His Ala Arg Glu Gly 900
905 910Ser Leu Glu Gln Pro Gly Phe Trp Glu Gly Glu Ala
Glu Arg Gly Lys 915 920 925Ala Gly
Asp Pro His Arg Arg His Val His Arg Gln Gly Gly Ser Arg 930
935 940Glu Ser Arg Ser Gly Ser Pro Arg Thr Gly Ala
Asp Gly Glu His Arg945 950 955
960Arg His Arg Ala His Arg Arg Pro Gly Glu Glu Gly Pro Glu Asp Lys
965 970 975Ala Glu Arg Arg
Ala Arg His Arg Glu Gly Ser Arg Pro Ala Arg Gly 980
985 990Gly Glu Gly Glu Gly Glu Gly Pro Asp Gly Gly
Glu Arg Arg Arg Arg 995 1000
1005His Arg His Gly Ala Pro Ala Thr Tyr Glu Gly Asp Ala Arg Arg
1010 1015 1020Glu Asp Lys Glu Arg Arg
His Arg Arg Arg Lys Glu Asn Gln Gly 1025 1030
1035Ser Gly Val Pro Val Ser Gly Pro Asn Leu Ser Thr Thr Arg
Pro 1040 1045 1050Ile Gln Gln Asp Leu
Gly Arg Gln Asp Pro Pro Leu Ala Glu Asp 1055 1060
1065Ile Asp Asn Met Lys Asn Asn Lys Leu Ala Thr Ala Glu
Ser Ala 1070 1075 1080Ala Pro His Gly
Ser Leu Gly His Ala Gly Leu Pro Gln Ser Pro 1085
1090 1095Ala Lys Met Gly Asn Ser Thr Asp Pro Gly Pro
Met Leu Ala Ile 1100 1105 1110Pro Ala
Met Ala Thr Asn Pro Gln Asn Ala Ala Ser Arg Arg Thr 1115
1120 1125Pro Asn Asn Pro Gly Asn Pro Ser Asn Pro
Gly Pro Pro Lys Thr 1130 1135 1140Pro
Glu Asn Ser Leu Ile Val Thr Asn Pro Ser Gly Thr Gln Thr 1145
1150 1155Asn Ser Ala Lys Thr Ala Arg Lys Pro
Asp His Thr Thr Val Asp 1160 1165
1170Ile Pro Pro Ala Cys Pro Pro Pro Leu Asn His Thr Val Val Gln
1175 1180 1185Val Asn Lys Asn Ala Asn
Pro Asp Pro Leu Pro Lys Lys Glu Glu 1190 1195
1200Glu Lys Lys Glu Glu Glu Glu Asp Asp Arg Gly Glu Asp Gly
Pro 1205 1210 1215Lys Pro Met Pro Pro
Tyr Ser Ser Met Phe Ile Leu Ser Thr Thr 1220 1225
1230Asn Pro Leu Arg Arg Leu Cys His Tyr Ile Leu Asn Leu
Arg Tyr 1235 1240 1245Phe Glu Met Cys
Ile Leu Met Val Ile Ala Met Ser Ser Ile Ala 1250
1255 1260Leu Ala Ala Glu Asp Pro Val Gln Pro Asn Ala
Pro Arg Asn Asn 1265 1270 1275Val Leu
Arg Tyr Phe Asp Tyr Val Phe Thr Gly Val Phe Thr Phe 1280
1285 1290Glu Met Val Ile Lys Met Ile Asp Leu Gly
Leu Val Leu His Gln 1295 1300 1305Gly
Ala Tyr Phe Arg Asp Leu Trp Asn Ile Leu Asp Phe Ile Val 1310
1315 1320Val Ser Gly Ala Leu Val Ala Phe Ala
Phe Thr Gly Asn Ser Lys 1325 1330
1335Gly Lys Asp Ile Asn Thr Ile Lys Ser Leu Arg Val Leu Arg Val
1340 1345 1350Leu Arg Pro Leu Lys Thr
Ile Lys Arg Leu Pro Lys Leu Lys Ala 1355 1360
1365Val Phe Asp Cys Val Val Asn Ser Leu Lys Asn Val Phe Asn
Ile 1370 1375 1380Leu Ile Val Tyr Met
Leu Phe Met Phe Ile Phe Ala Val Val Ala 1385 1390
1395Val Gln Leu Phe Lys Gly Lys Phe Phe His Cys Thr Asp
Glu Ser 1400 1405 1410Lys Glu Phe Glu
Lys Asp Cys Arg Gly Lys Tyr Leu Leu Tyr Glu 1415
1420 1425Lys Asn Glu Val Lys Ala Arg Asp Arg Glu Trp
Lys Lys Tyr Glu 1430 1435 1440Phe His
Tyr Asp Asn Val Leu Trp Ala Leu Leu Thr Leu Phe Thr 1445
1450 1455Val Ser Thr Gly Glu Gly Trp Pro Gln Val
Leu Lys His Ser Val 1460 1465 1470Asp
Ala Thr Phe Glu Asn Gln Gly Pro Ser Pro Gly Tyr Arg Met 1475
1480 1485Glu Met Ser Ile Phe Tyr Val Val Tyr
Phe Val Val Phe Pro Phe 1490 1495
1500Phe Phe Val Asn Ile Phe Val Ala Leu Ile Ile Ile Thr Phe Gln
1505 1510 1515Glu Gln Gly Asp Lys Met
Met Glu Glu Tyr Ser Leu Glu Lys Asn 1520 1525
1530Glu Arg Ala Cys Ile Asp Phe Ala Ile Ser Ala Lys Pro Leu
Thr 1535 1540 1545Arg His Met Pro Gln
Asn Lys Gln Ser Phe Gln Tyr Arg Met Trp 1550 1555
1560Gln Phe Val Val Ser Pro Pro Phe Glu Tyr Thr Ile Met
Ala Met 1565 1570 1575Ile Ala Leu Asn
Thr Ile Val Leu Met Met Lys Phe Tyr Gly Ala 1580
1585 1590Ser Val Ala Tyr Glu Asn Ala Leu Arg Val Phe
Asn Ile Val Phe 1595 1600 1605Thr Ser
Leu Phe Ser Leu Glu Cys Val Leu Lys Val Met Ala Phe 1610
1615 1620Gly Ile Leu Asn Tyr Phe Arg Asp Ala Trp
Asn Ile Phe Asp Phe 1625 1630 1635Val
Thr Val Leu Gly Ser Ile Thr Asp Ile Leu Val Thr Glu Phe 1640
1645 1650Gly Asn Pro Asn Asn Phe Ile Asn Leu
Ser Phe Leu Arg Leu Phe 1655 1660
1665Arg Ala Ala Arg Leu Ile Lys Leu Leu Arg Gln Gly Tyr Thr Ile
1670 1675 1680Arg Ile Leu Leu Trp Thr
Phe Val Gln Ser Phe Lys Ala Leu Pro 1685 1690
1695Tyr Val Cys Leu Leu Ile Ala Met Leu Phe Phe Ile Tyr Ala
Ile 1700 1705 1710Ile Gly Met Gln Val
Phe Gly Asn Ile Gly Ile Asp Val Glu Asp 1715 1720
1725Glu Asp Ser Asp Glu Asp Glu Phe Gln Ile Thr Glu His
Asn Asn 1730 1735 1740Phe Arg Thr Phe
Phe Gln Ala Leu Met Leu Leu Phe Arg Ser Ala 1745
1750 1755Thr Gly Glu Ala Trp His Asn Ile Met Leu Ser
Cys Leu Ser Gly 1760 1765 1770Lys Pro
Cys Asp Lys Asn Ser Gly Ile Leu Thr Arg Glu Cys Gly 1775
1780 1785Asn Glu Phe Ala Tyr Phe Tyr Phe Val Ser
Phe Ile Phe Leu Cys 1790 1795 1800Ser
Phe Leu Met Leu Asn Leu Phe Val Ala Val Ile Met Asp Asn 1805
1810 1815Phe Glu Tyr Leu Thr Arg Asp Ser Ser
Ile Leu Gly Pro His His 1820 1825
1830Leu Asp Glu Tyr Val Arg Val Trp Ala Glu Tyr Asp Pro Ala Ala
1835 1840 1845Trp Gly Arg Met Pro Tyr
Leu Asp Met Tyr Gln Met Leu Arg His 1850 1855
1860Met Ser Pro Pro Leu Gly Leu Gly Lys Lys Cys Pro Ala Arg
Val 1865 1870 1875Ala Tyr Lys Arg Leu
Leu Arg Met Asp Leu Pro Val Ala Asp Asp 1880 1885
1890Asn Thr Val His Phe Asn Ser Thr Leu Met Ala Leu Ile
Arg Thr 1895 1900 1905Ala Leu Asp Ile
Lys Ile Ala Lys Gly Gly Ala Asp Lys Gln Gln 1910
1915 1920Met Asp Ala Glu Leu Arg Lys Glu Met Met Ala
Ile Trp Pro Asn 1925 1930 1935Leu Ser
Gln Lys Thr Leu Asp Leu Leu Val Thr Pro His Lys Ser 1940
1945 1950Thr Asp Leu Thr Val Gly Lys Ile Tyr Ala
Ala Met Met Ile Met 1955 1960 1965Glu
Tyr Tyr Arg Gln Ser Lys Ala Lys Lys Leu Gln Ala Met Arg 1970
1975 1980Glu Glu Gln Asp Arg Thr Pro Leu Met
Phe Gln Arg Met Glu Pro 1985 1990
1995Pro Ser Pro Thr Gln Glu Gly Gly Pro Gly Gln Asn Ala Leu Pro
2000 2005 2010Ser Thr Gln Leu Asp Pro
Gly Gly Ala Leu Met Ala His Glu Ser 2015 2020
2025Gly Leu Lys Glu Ser Pro Ser Trp Val Thr Gln Arg Ala Gln
Glu 2030 2035 2040Met Phe Gln Lys Thr
Gly Thr Trp Ser Pro Glu Gln Gly Pro Pro 2045 2050
2055Thr Asp Met Pro Asn Ser Gln Pro Asn Ser Gln Ser Val
Glu Met 2060 2065 2070Arg Glu Met Gly
Arg Asp Gly Tyr Ser Asp Ser Glu His Tyr Leu 2075
2080 2085Pro Met Glu Gly Gln Gly Arg Ala Ala Ser Met
Pro Arg Leu Pro 2090 2095 2100Ala Glu
Asn Gln Arg Arg Arg Gly Arg Pro Arg Gly Asn Asn Leu 2105
2110 2115Ser Thr Ile Ser Asp Thr Ser Pro Met Lys
Arg Ser Ala Ser Val 2120 2125 2130Leu
Gly Pro Lys Ala Arg Arg Leu Asp Asp Tyr Ser Leu Glu Arg 2135
2140 2145Val Pro Pro Glu Glu Asn Gln Arg His
His Gln Arg Arg Arg Asp 2150 2155
2160Arg Ser His Arg Ala Ser Glu Arg Ser Leu Gly Arg Tyr Thr Asp
2165 2170 2175Val Asp Thr Gly Leu Gly
Thr Asp Leu Ser Met Thr Thr Gln Ser 2180 2185
2190Gly Asp Leu Pro Ser Lys Glu Arg Asp Gln Glu Arg Gly Arg
Pro 2195 2200 2205Lys Asp Arg Lys His
Arg Gln His His His His His His His His 2210 2215
2220His His Pro Pro Pro Pro Asp Lys Asp Arg Tyr Ala Gln
Glu Arg 2225 2230 2235Pro Asp His Gly
Arg Ala Arg Ala Arg Asp Gln Arg Trp Ser Arg 2240
2245 2250Ser Pro Ser Glu Gly Arg Glu His Met Ala His
Arg Gln Gly Ser 2255 2260 2265Ser Ser
Val Ser Gly Ser Pro Ala Pro Ser Thr Ser Gly Thr Ser 2270
2275 2280Thr Pro Arg Arg Gly Arg Arg Gln Leu Pro
Gln Thr Pro Ser Thr 2285 2290 2295Pro
Arg Pro His Val Ser Tyr Ser Pro Val Ile Arg Lys Ala Gly 2300
2305 2310Gly Ser Gly Pro Pro Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln 2315 2320
2325Gln Gln Gln Ala Val Ala Arg Pro Gly Arg Ala Ala Thr Ser Gly
2330 2335 2340Pro Arg Arg Tyr Pro Gly
Pro Thr Ala Glu Pro Leu Ala Gly Asp 2345 2350
2355Arg Pro Pro Thr Gly Gly His Ser Ser Gly Arg Ser Pro Arg
Met 2360 2365 2370Glu Arg Arg Val Pro
Gly Pro Ala Arg Ser Glu Ser Pro Arg Ala 2375 2380
2385Cys Arg His Gly Gly Ala Arg Trp Pro Ala Ser Gly Pro
His Val 2390 2395 2400Ser Glu Gly Pro
Pro Gly Pro Arg His His Gly Tyr Tyr Arg Gly 2405
2410 2415Ser Asp Tyr Asp Glu Ala Asp Gly Pro Gly Ser
Gly Gly Gly Glu 2420 2425 2430Glu Ala
Met Ala Gly Ala Tyr Asp Ala Pro Pro Pro Val Arg His 2435
2440 2445Ala Ser Ser Gly Ala Thr Gly Arg Ser Pro
Arg Thr Pro Arg Ala 2450 2455 2460Ser
Gly Pro Ala Cys Ala Ser Pro Ser Arg His Gly Arg Arg Leu 2465
2470 2475Pro Asn Gly Tyr Tyr Pro Ala His Gly
Leu Ala Arg Pro Arg Gly 2480 2485
2490Pro Gly Ser Arg Lys Gly Leu His Glu Pro Tyr Ser Glu Ser Asp
2495 2500 2505Asp Asp Trp Cys
25101492368PRTRattus norvegicus 149Met Ala Arg Phe Gly Asp Glu Met Pro
Gly Arg Tyr Gly Ala Gly Gly1 5 10
15Gly Gly Ser Gly Pro Ala Ala Gly Val Val Val Gly Ala Ala Gly
Gly 20 25 30Arg Gly Ala Gly
Gly Ser Arg Gln Gly Gly Gln Pro Gly Ala Gln Arg 35
40 45Met Tyr Lys Gln Ser Met Ala Gln Arg Ala Arg Thr
Met Ala Leu Tyr 50 55 60Asn Pro Ile
Pro Val Arg Gln Asn Cys Leu Thr Val Asn Arg Ser Leu65 70
75 80Phe Leu Phe Ser Glu Asp Asn Val
Val Arg Lys Tyr Ala Lys Lys Ile 85 90
95Thr Glu Trp Pro Pro Phe Glu Tyr Met Ile Leu Ala Thr Ile
Ile Ala 100 105 110Asn Cys Ile
Val Leu Ala Leu Glu Gln His Leu Pro Asp Asp Asp Lys 115
120 125Thr Pro Met Ser Glu Arg Leu Asp Asp Thr Glu
Pro Tyr Phe Ile Gly 130 135 140Ile Phe
Cys Phe Glu Ala Gly Ile Lys Ile Val Ala Leu Gly Phe Ala145
150 155 160Phe His Lys Gly Ser Tyr Leu
Arg Asn Gly Trp Asn Val Met Asp Phe 165
170 175Val Val Val Leu Thr Gly Ile Leu Ala Thr Val Gly
Thr Glu Phe Asp 180 185 190Leu
Arg Thr Leu Arg Ala Val Arg Val Leu Arg Pro Leu Lys Leu Val 195
200 205Ser Gly Ile Pro Ser Leu Gln Val Val
Leu Lys Ser Ile Met Lys Ala 210 215
220Met Ile Pro Leu Leu Gln Ile Gly Leu Leu Leu Phe Phe Ala Ile Leu225
230 235 240Ile Phe Ala Ile
Ile Gly Leu Glu Phe Tyr Lys Gly Lys Phe His Thr 245
250 255Thr Cys Phe Glu Glu Gly Thr Asp Asp Ile
Gln Gly Glu Ser Pro Ala 260 265
270Pro Cys Gly Thr Glu Glu Pro Ala Arg Thr Cys Pro Asn Gly Thr Lys
275 280 285Cys Gln Pro Tyr Trp Glu Gly
Pro Asn Asn Gly Ile Thr Gln Phe Asp 290 295
300Asn Ile Leu Phe Ala Val Leu Thr Val Phe Gln Cys Ile Thr Met
Glu305 310 315 320Gly Trp
Thr Asp Leu Leu Tyr Asn Ser Asn Asp Ala Ser Gly Asn Thr
325 330 335Trp Asn Trp Leu Tyr Phe Ile
Pro Leu Ile Ile Ile Gly Ser Phe Phe 340 345
350Met Leu Asn Leu Val Leu Gly Val Leu Ser Gly Glu Phe Ala
Lys Glu 355 360 365Arg Glu Arg Val
Glu Asn Arg Arg Ala Phe Leu Lys Leu Arg Arg Gln 370
375 380Gln Gln Ile Glu Arg Glu Leu Asn Gly Tyr Met Glu
Trp Ile Ser Lys385 390 395
400Ala Glu Glu Val Ile Leu Ala Glu Asp Glu Thr Asp Val Glu Gln Arg
405 410 415His Pro Phe Asp Gly
Ala Leu Arg Arg Ala Thr Leu Lys Lys Ser Lys 420
425 430Thr Asp Leu Leu Asn Pro Glu Glu Ala Glu Asp Gln
Leu Ala Asp Ile 435 440 445Ala Ser
Val Gly Ser Pro Phe Ala Arg Ala Ser Ile Lys Ser Ala Lys 450
455 460Leu Glu Asn Ser Thr Phe Phe His Lys Lys Glu
Arg Arg Met Arg Phe465 470 475
480Tyr Ile Arg Arg Met Val Lys Thr Gln Ala Phe Tyr Trp Thr Val Leu
485 490 495Ser Leu Val Ala
Leu Asn Thr Leu Trp Leu Ala Ile Val His Tyr Asn 500
505 510Gln Pro Glu Trp Leu Ser Asp Phe Leu Tyr Tyr
Ala Glu Phe Ile Phe 515 520 525Leu
Gly Leu Phe Met Ser Glu Met Phe Ile Lys Met Tyr Gly Leu Gly 530
535 540Thr Arg Pro Tyr Phe His Ser Ser Phe Asn
Cys Phe Asp Cys Gly Val545 550 555
560Ile Ile Gly Ser Ile Phe Glu Val Ile Trp Ala Val Ile Lys Pro
Gly 565 570 575Thr Ser Phe
Gly Ile Ser Val Leu Arg Ala Leu Arg Leu Leu Arg Ile 580
585 590Phe Lys Val Thr Lys Tyr Trp Ala Ser Leu
Arg Asn Leu Val Val Ser 595 600
605Leu Leu Asn Ser Met Lys Ser Ile Ile Ser Leu Leu Phe Leu Leu Phe 610
615 620Leu Phe Ile Val Val Phe Ala Leu
Leu Gly Met Gln Leu Phe Gly Gly625 630
635 640Gln Phe Asn Phe Asp Glu Gly Thr Pro Pro Thr Asn
Phe Asp Thr Phe 645 650
655Pro Ala Ala Ile Met Thr Val Phe Gln Ile Leu Thr Gly Glu Asp Trp
660 665 670Asn Glu Val Met Tyr Asp
Glu Ile Lys Ser Gln Gly Gly Val Gln Gly 675 680
685Gly Met Val Phe Ser Ile Tyr Phe Ile Val Leu Thr Leu Phe
Gly Asn 690 695 700Tyr Thr Leu Leu Asn
Val Phe Leu Ala Ile Ala Val Asp Asn Leu Ala705 710
715 720Asn Ala Gln Glu Leu Thr Lys Asp Glu Gln
Glu Glu Glu Glu Ala Ala 725 730
735Asn Gln Lys Leu Ala Leu Gln Lys Ala Lys Glu Val Ala Glu Val Ser
740 745 750Pro Leu Ser Ala Ala
Asn Met Ser Ile Ala Val Lys Glu Gln Gln Lys 755
760 765Asn Gln Lys Pro Ala Lys Ser Val Trp Glu Gln Arg
Thr Ser Glu Met 770 775 780Arg Lys Gln
Asn Leu Leu Ala Ser Arg Glu Ala Leu Tyr Gly Asp Ala785
790 795 800Ala Glu Arg Trp Pro Thr Thr
Tyr Ala Arg Pro Leu Arg Pro Asp Val 805
810 815Lys Thr His Leu Asp Arg Pro Leu Val Val Asp Pro
Gln Glu Asn Arg 820 825 830Asn
Asn Asn Thr Asn Lys Ser Arg Ala Pro Glu Ala Leu Arg Gln Thr 835
840 845Ala Arg Pro Arg Glu Ser Ala Arg Asp
Pro Asp Ala Arg Arg Ala Trp 850 855
860Pro Ser Ser Pro Glu Arg Ala Pro Gly Arg Glu Gly Pro Tyr Gly Arg865
870 875 880Glu Ser Glu Pro
Gln Gln Arg Glu His Ala Pro Pro Arg Glu His Val 885
890 895Pro Trp Asp Ala Asp Pro Glu Arg Ala Lys
Ala Gly Asp Ala Pro Arg 900 905
910Arg His Thr His Arg Pro Val Ala Glu Gly Glu Pro Arg Arg His Arg
915 920 925Ala Arg Arg Arg Pro Gly Asp
Glu Pro Asp Asp Arg Pro Glu Arg Arg 930 935
940Pro Arg Pro Arg Asp Ala Thr Arg Pro Ala Arg Ala Ala Asp Gly
Glu945 950 955 960Gly Asp
Asp Gly Glu Arg Lys Arg Arg His Arg His Gly Pro Pro Ala
965 970 975His Asp Asp Arg Glu Arg Arg
His Arg Arg Arg Lys Glu Ser Gln Gly 980 985
990Ser Gly Val Pro Met Ser Gly Pro Asn Leu Ser Thr Thr Arg
Pro Ile 995 1000 1005Gln Gln Asp
Leu Gly Arg Gln Asp Leu Pro Leu Ala Glu Asp Leu 1010
1015 1020Asp Asn Met Lys Asn Asn Lys Leu Ala Thr Gly
Glu Pro Ala Ser 1025 1030 1035Pro His
Asp Ser Leu Gly His Ser Gly Leu Pro Pro Ser Pro Ala 1040
1045 1050Lys Ile Gly Asn Ser Thr Asn Pro Gly Pro
Ala Leu Ala Thr Asn 1055 1060 1065Pro
Gln Asn Ala Ala Ser Arg Arg Thr Pro Asn Asn Pro Gly Asn 1070
1075 1080Pro Ser Asn Pro Gly Pro Pro Lys Thr
Pro Glu Asn Ser Leu Ile 1085 1090
1095Val Thr Asn Pro Ser Ser Thr Gln Pro Asn Ser Ala Lys Thr Ala
1100 1105 1110Arg Lys Pro Glu His Met
Ala Val Glu Ile Pro Pro Ala Cys Pro 1115 1120
1125Pro Leu Asn His Thr Val Val Gln Val Asn Lys Asn Ala Asn
Pro 1130 1135 1140Asp Pro Leu Pro Lys
Lys Glu Glu Glu Lys Lys Glu Glu Glu Glu 1145 1150
1155Ala Asp Pro Gly Glu Asp Gly Pro Lys Pro Met Pro Pro
Tyr Ser 1160 1165 1170Ser Met Phe Ile
Leu Ser Thr Thr Asn Pro Leu Arg Arg Leu Cys 1175
1180 1185His Tyr Ile Leu Asn Leu Arg Tyr Phe Glu Met
Cys Ile Leu Met 1190 1195 1200Val Ile
Ala Met Ser Ser Ile Ala Leu Ala Ala Glu Asp Pro Val 1205
1210 1215Gln Pro Asn Ala Pro Arg Asn Asn Val Leu
Arg Tyr Phe Asp Tyr 1220 1225 1230Val
Phe Thr Gly Val Phe Thr Phe Glu Met Val Ile Lys Met Ile 1235
1240 1245Asp Leu Gly Leu Val Leu His Gln Gly
Ala Tyr Phe Arg Asp Leu 1250 1255
1260Trp Asn Ile Leu Asp Phe Ile Val Val Ser Gly Ala Leu Val Ala
1265 1270 1275Phe Ala Phe Thr Gly Asn
Ser Lys Gly Lys Asp Ile Asn Thr Ile 1280 1285
1290Lys Ser Leu Arg Val Leu Arg Val Leu Arg Pro Leu Lys Thr
Ile 1295 1300 1305Lys Arg Leu Pro Lys
Leu Lys Ala Val Phe Asp Cys Val Val Asn 1310 1315
1320Ser Leu Lys Asn Val Phe Asn Ile Leu Ile Val Tyr Met
Leu Phe 1325 1330 1335Met Phe Ile Phe
Ala Val Val Ala Val Gln Leu Phe Lys Gly Lys 1340
1345 1350Phe Phe His Cys Thr Asp Glu Ser Lys Glu Phe
Glu Arg Asp Cys 1355 1360 1365Arg Gly
Lys Tyr Leu Leu Tyr Glu Lys Asn Glu Val Lys Ala Arg 1370
1375 1380Asp Arg Glu Trp Lys Lys Tyr Asp Phe His
Tyr Asp Asn Val Leu 1385 1390 1395Trp
Ala Leu Leu Thr Leu Phe Thr Val Ser Thr Gly Glu Gly Trp 1400
1405 1410Pro Gln Val Leu Lys His Ser Val Asp
Ala Thr Phe Glu Asn Gln 1415 1420
1425Gly Pro Ser Pro Gly Tyr Arg Met Glu Met Ser Ile Phe Tyr Val
1430 1435 1440Val Tyr Phe Val Val Phe
Pro Phe Phe Phe Val Asn Ile Phe Val 1445 1450
1455Ala Leu Ile Ile Ile Thr Phe Gln Glu Gln Gly Asp Lys Met
Met 1460 1465 1470Glu Glu Tyr Ser Leu
Glu Lys Asn Glu Arg Ala Cys Ile Asp Phe 1475 1480
1485Ala Ile Ser Ala Lys Pro Leu Thr Arg His Met Pro Gln
Asn Lys 1490 1495 1500Gln Ser Phe Gln
Tyr Arg Met Trp Gln Phe Val Val Ser Pro Pro 1505
1510 1515Phe Glu Tyr Thr Ile Met Ala Met Ile Ala Leu
Asn Thr Ile Val 1520 1525 1530Leu Met
Met Lys Phe Tyr Gly Ala Ser Val Ala Tyr Glu Asn Ala 1535
1540 1545Leu Arg Val Phe Asn Ile Val Phe Thr Ser
Leu Phe Ser Leu Glu 1550 1555 1560Cys
Val Leu Lys Val Met Ala Phe Gly Ile Leu Asn Tyr Phe Arg 1565
1570 1575Asp Ala Trp Asn Ile Phe Asp Phe Val
Thr Val Leu Gly Ser Ile 1580 1585
1590Thr Asp Ile Leu Val Thr Glu Phe Gly Asn Asn Phe Ile Asn Leu
1595 1600 1605Ser Phe Leu Arg Leu Phe
Arg Ala Ala Arg Leu Ile Lys Leu Leu 1610 1615
1620Arg Gln Gly Tyr Thr Ile Arg Ile Leu Leu Trp Thr Phe Val
Gln 1625 1630 1635Ser Phe Lys Ala Leu
Pro Tyr Val Cys Leu Leu Ile Ala Met Leu 1640 1645
1650Phe Phe Ile Tyr Ala Ile Ile Gly Met Gln Val Phe Gly
Asn Ile 1655 1660 1665Gly Ile Asp Gly
Glu Asp Glu Asp Ser Asp Glu Asp Glu Phe Gln 1670
1675 1680Ile Thr Glu His Asn Asn Phe Arg Thr Phe Phe
Gln Ala Leu Met 1685 1690 1695Leu Leu
Phe Arg Ser Ala Thr Gly Glu Ala Trp His Asn Ile Met 1700
1705 1710Leu Ser Cys Leu Ser Gly Lys Pro Cys Asp
Lys Asn Ser Gly Ile 1715 1720 1725Gln
Lys Pro Glu Cys Gly Asn Glu Phe Ala Tyr Phe Tyr Phe Val 1730
1735 1740Ser Phe Ile Phe Leu Cys Ser Phe Leu
Met Leu Asn Leu Phe Val 1745 1750
1755Ala Val Ile Met Asp Asn Phe Glu Tyr Leu Thr Arg Asp Ser Ser
1760 1765 1770Ile Leu Gly Pro His His
Leu Asp Glu Tyr Val Arg Val Trp Ala 1775 1780
1785Glu Tyr Asp Pro Ala Ala Cys Gly Arg Ile His Tyr Lys Asp
Met 1790 1795 1800Tyr Ser Leu Leu Arg
Val Ile Ser Pro Pro Leu Gly Leu Gly Lys 1805 1810
1815Lys Cys Pro His Arg Val Ala Cys Lys Arg Leu Leu Arg
Met Asp 1820 1825 1830Leu Pro Val Ala
Asp Asp Asn Thr Val His Phe Asn Ser Thr Leu 1835
1840 1845Met Ala Leu Ile Arg Thr Ala Leu Asp Ile Lys
Ile Ala Lys Gly 1850 1855 1860Gly Ala
Asp Lys Gln Gln Met Asp Ala Glu Leu Arg Lys Glu Met 1865
1870 1875Met Ala Ile Trp Pro Asn Leu Ser Gln Lys
Thr Leu Asp Leu Leu 1880 1885 1890Val
Thr Pro His Lys Ser Thr Asp Leu Thr Val Gly Lys Ile Tyr 1895
1900 1905Ala Ala Met Met Ile Met Glu Tyr Tyr
Arg Gln Ser Lys Ala Lys 1910 1915
1920Lys Leu Gln Ala Met Arg Glu Glu Gln Asn Arg Thr Pro Leu Met
1925 1930 1935Phe Gln Arg Met Glu Pro
Pro Ser Pro Thr Gln Glu Gly Gly Pro 1940 1945
1950Ser Gln Asn Ala Leu Pro Ser Thr Gln Leu Asp Pro Gly Gly
Gly 1955 1960 1965Leu Met Ala Gln Glu
Ser Ser Met Lys Glu Ser Pro Ser Trp Val 1970 1975
1980Thr Gln Arg Ala Gln Glu Met Phe Gln Lys Thr Gly Thr
Trp Ser 1985 1990 1995Pro Glu Arg Gly
Pro Pro Ile Asp Met Pro Asn Ser Gln Pro Asn 2000
2005 2010Ser Gln Ser Val Glu Met Arg Glu Met Gly Thr
Asp Gly Tyr Ser 2015 2020 2025Asp Ser
Glu His Tyr Leu Pro Met Glu Gly Gln Thr Arg Ala Ala 2030
2035 2040Ser Met Pro Arg Leu Pro Ala Glu Asn Gln
Arg Arg Arg Gly Arg 2045 2050 2055Pro
Arg Gly Asn Asn Leu Ser Thr Ile Ser Asp Thr Ser Pro Met 2060
2065 2070Lys Arg Ser Ala Ser Val Leu Gly Pro
Lys Ala Arg Arg Leu Asp 2075 2080
2085Asp Tyr Ser Leu Glu Arg Val Pro Pro Glu Glu Asn Gln Arg Tyr
2090 2095 2100His Gln Arg Arg Arg Asp
Arg Gly His Arg Thr Ser Glu Arg Ser 2105 2110
2115Leu Gly Arg Tyr Thr Asp Val Asp Thr Gly Leu Gly Thr Asp
Leu 2120 2125 2130Ser Met Thr Thr Gln
Ser Gly Asp Leu Pro Ser Lys Asp Arg Asp 2135 2140
2145Gln Asp Arg Gly Arg Pro Lys Asp Arg Lys His Arg Pro
His His 2150 2155 2160His His His His
His His His His Pro Pro Ala Pro Asp Arg Glu 2165
2170 2175Arg Tyr Ala Gln Glu Arg Pro Asp Thr Gly Arg
Ala Arg Ala Arg 2180 2185 2190Glu Gln
Arg Trp Ser Arg Ser Pro Ser Glu Gly Arg Glu His Ala 2195
2200 2205Thr His Arg Gln Gly Ser Ser Ser Val Ser
Gly Ser Pro Ala Pro 2210 2215 2220Ser
Thr Ser Gly Thr Ser Thr Pro Arg Arg Gly Arg Arg Gln Leu 2225
2230 2235Pro Gln Thr Pro Cys Thr Pro Arg Pro
Leu Val Ser Tyr Ser Pro 2240 2245
2250Ala Pro Arg Arg Pro Ala Ala Arg Arg Met Ala Gly Pro Pro Ala
2255 2260 2265Pro Pro Gly Gly Ser Pro
Arg Gly Cys Arg Arg Ala Pro Arg Trp 2270 2275
2280Pro Ala His Ala Pro Glu Gly Pro Arg Pro Arg Gly Ala Asp
Tyr 2285 2290 2295Thr Glu Pro Asp Ser
Pro Arg Glu Pro Pro Gly Gly Ala His Glu 2300 2305
2310Pro Ala Pro Arg Ser Pro Arg Thr Pro Arg Ala Ala Gly
Cys Ala 2315 2320 2325Ser Pro Arg His
Gly Arg Arg Leu Pro Asn Gly Tyr Tyr Ala Gly 2330
2335 2340His Gly Ala Pro Arg Pro Arg Thr Ala Arg Arg
Gly Ala His Asp 2345 2350 2355Ala Tyr
Ser Glu Ser Glu Asp Asp Trp Cys 2360
23651506030DNAHomo sapiens 150atggagcaaa cagtgcttgt accaccagga cctgacagct
tcaacttctt caccagagaa 60tctcttgcgg ctattgaaag acgcattgca gaagaaaagg
caaagaatcc caaaccagac 120aaaaaagatg acgacgaaaa tggcccaaag ccaaatagtg
acttggaagc tggaaagaac 180cttccattta tttatggaga cattcctcca gagatggtgt
cagagcccct ggaggacctg 240gacccctact atatcaataa gaaaactttt atagtattga
ataaagggaa ggccatcttc 300cggttcagtg ccacctctgc cctgtacatt ttaactccct
tcaatcctct taggaaaata 360gctattaaga ttttggtaca ttcattattc agcatgctaa
ttatgtgcac tattttgaca 420aactgtgtgt ttatgacaat gagtaaccct cctgattgga
caaagaatgt agaatacacc 480ttcacaggaa tatatacttt tgaatcactt ataaaaatta
ttgcaagggg attctgttta 540gaagatttta ctttccttcg ggatccatgg aactggctcg
atttcactgt cattacattt 600gcgtacgtca cagagtttgt ggacctgggc aatgtctcgg
cattgagaac attcagagtt 660ctccgagcat tgaagacgat ttcagtcatt ccaggcctga
aaaccattgt gggagccctg 720atccagtctg tgaagaagct ctcagatgta atgatcctga
ctgtgttctg tctgagcgta 780tttgctctaa ttgggctgca gctgttcatg ggcaacctga
ggaataaatg tatacaatgg 840cctcccacca atgcttcctt ggaggaacat agtatagaaa
agaatataac tgtgaattat 900aatggtacac ttataaatga aactgtcttt gagtttgact
ggaagtcata tattcaagat 960tcaagatatc attatttcct ggagggtttt ttagatgcac
tactatgtgg aaatagctct 1020gatgcaggcc aatgtccaga gggatatatg tgtgtgaaag
ctggtagaaa tcccaattat 1080ggctacacaa gctttgatac cttcagttgg gcttttttgt
ccttgtttcg actaatgact 1140caggacttct gggaaaatct ttatcaactg acattacgtg
ctgctgggaa aacgtacatg 1200atattttttg tattggtcat tttcttgggc tcattctacc
taataaattt gatcctggct 1260gtggtggcca tggcctacga ggaacagaat caggccacct
tggaagaagc agaacagaaa 1320gaggccgaat ttcagcagat gattgaacag cttaaaaagc
aacaggaggc agctcagcag 1380gcagcaacgg caactgcctc agaacattcc agagagccca
gtgcagcagg caggctctca 1440gacagctcat ctgaagcctc taagttgagt tccaagagtg
ctaaggaaag aagaaatcgg 1500aggaagaaaa gaaaacagaa agagcagtct ggtggggaag
agaaagatga ggatgaattc 1560caaaaatctg aatctgagga cagcatcagg aggaaaggtt
ttcgcttctc cattgaaggg 1620aaccgattga catatgaaaa gaggtactcc tccccacacc
agtctttgtt gagcatccgt 1680ggctccctat tttcaccaag gcgaaatagc agaacaagcc
ttttcagctt tagagggcga 1740gcaaaggatg tgggatctga gaacgacttc gcagatgatg
agcacagcac ctttgaggat 1800aacgagagcc gtagagattc cttgtttgtg ccccgacgac
acggagagag acgcaacagc 1860aacctgagtc agaccagtag gtcatcccgg atgctggcag
tgtttccagc gaatgggaag 1920atgcacagca ctgtggattg caatggtgtg gtttccttgg
ttggtggacc ttcagttcct 1980acatcgcctg ttggacagct tctgccagag gtgataatag
ataagccagc tactgatgac 2040aatggaacaa ccactgaaac tgaaatgaga aagagaaggt
caagttcttt ccacgtttcc 2100atggactttc tagaagatcc ttcccaaagg caacgagcaa
tgagtatagc cagcattcta 2160acaaatacag tagaagaact tgaagaatcc aggcagaaat
gcccaccctg ttggtataaa 2220ttttccaaca tattcttaat ctgggactgt tctccatatt
ggttaaaagt gaaacatgtt 2280gtcaacctgg ttgtgatgga cccatttgtt gacctggcca
tcaccatctg tattgtctta 2340aatactcttt tcatggccat ggagcactat ccaatgacgg
accatttcaa taatgtgctt 2400acagtaggaa acttggtttt cactgggatc tttacagcag
aaatgtttct gaaaattatt 2460gccatggatc cttactatta tttccaagaa ggctggaata
tctttgacgg ttttattgtg 2520acgcttagcc tggtagaact tggactcgcc aatgtggaag
gattatctgt tctccgttca 2580tttcgattgc tgcgagtttt caagttggca aaatcttggc
caacgttaaa tatgctaata 2640aagatcatcg gcaattccgt gggggctctg ggaaatttaa
ccctcgtctt ggccatcatc 2700gtcttcattt ttgccgtggt cggcatgcag ctctttggta
aaagctacaa agattgtgtc 2760tgcaagatcg ccagtgattg tcaactccca cgctggcaca
tgaatgactt cttccactcc 2820ttcctgattg tgttccgcgt gctgtgtggg gagtggatag
agaccatgtg ggactgtatg 2880gaggttgctg gtcaagccat gtgccttact gtcttcatga
tggtcatggt gattggaaac 2940ctagtggtcc tgaatctctt tctggccttg cttctgagct
catttagtgc agacaacctt 3000gcagccactg atgatgataa tgaaatgaat aatctccaaa
ttgctgtgga taggatgcac 3060aaaggagtag cttatgtgaa aagaaaaata tatgaattta
ttcaacagtc cttcattagg 3120aaacaaaaga ttttagatga aattaaacca cttgatgatc
taaacaacaa gaaagacagt 3180tgtatgtcca atcatacaac agaaattggg aaagatcttg
actatcttaa agatgtaaat 3240ggaactacaa gtggtatagg aactggcagc agtgttgaaa
aatacattat tgatgaaagt 3300gattacatgt cattcataaa caaccccagt cttactgtga
ctgtaccaat tgctgtagga 3360gaatctgact ttgaaaattt aaacacggaa gactttagta
gtgaatcgga tctggaagaa 3420agcaaagaga aactgaatga aagcagtagc tcatcagaag
gtagcactgt ggacatcggc 3480gcacctgtag aagaacagcc cgtagtggaa cctgaagaaa
ctcttgaacc agaagcttgt 3540ttcactgaag gctgtgtaca aagattcaag tgttgtcaaa
tcaatgtgga agaaggcaga 3600ggaaaacaat ggtggaacct gagaaggacg tgtttccgaa
tagttgaaca taactggttt 3660gagaccttca ttgttttcat gattctcctt agtagtggtg
ctctggcatt tgaagatata 3720tatattgatc agcgaaagac gattaagacg atgttggaat
atgctgacaa ggttttcact 3780tacattttca ttctggaaat gcttctaaaa tgggtggcat
atggctatca aacatatttc 3840accaatgcct ggtgttggct ggacttctta attgttgatg
tttcattggt cagtttaaca 3900gcaaatgcct tgggttactc agaacttgga gccatcaaat
ctctcaggac actaagagct 3960ctgagacctc taagagcctt atctcgattt gaagggatga
gggtggttgt gaatgccctt 4020ttaggagcaa ttccatccat catgaatgtg cttctggttt
gtcttatatt ctggctaatt 4080ttcagcatca tgggcgtaaa tttgtttgct ggcaaattct
accactgtat taacaccaca 4140actggtgaca ggtttgacat cgaagacgtg aataatcata
ctgattgcct aaaactaata 4200gaaagaaatg agactgctcg atggaaaaat gtgaaagtaa
actttgatca tgtaggattt 4260gggtatctct ctttgcttca agttgccaca ttcaaaggat
ggatggatat aatgtatgca 4320gcagttgatt ccagaaatgt ggaactccag cctaagtatg
aagaaagtct gtacatgtat 4380ctttactttg ttattttcat catctttggg tccttcttca
ccttgaacct gtttattggt 4440gtcatcatag ataatttcaa ccagcagaaa aagaagtttg
gaggtcaaga catctttatg 4500acagaagaac agaagaaata ctataatgca atgaaaaaat
taggatcgaa aaaaccgcaa 4560aagcctatac ctcgaccagg aaacaaattt caaggaatgg
tctttgactt cgtaaccaga 4620caagtttttg acataagcat catgattctc atctgtctta
acatggtcac aatgatggtg 4680gaaacagatg accagagtga atatgtgact accattttgt
cacgcatcaa tctggtgttc 4740attgtgctat ttactggaga gtgtgtactg aaactcatct
ctctacgcca ttattatttt 4800accattggat ggaatatttt tgattttgtg gttgtcattc
tctccattgt aggtatgttt 4860cttgccgagc tgatagaaaa gtatttcgtg tcccctaccc
tgttccgagt gatccgtctt 4920gctaggattg gccgaatcct acgtctgatc aaaggagcaa
aggggatccg cacgctgctc 4980tttgctttga tgatgtccct tcctgcgttg tttaacatcg
gcctcctact cttcctagtc 5040atgttcatct acgccatctt tgggatgtcc aactttgcct
atgttaagag ggaagttggg 5100atcgatgaca tgttcaactt tgagaccttt ggcaacagca
tgatctgcct attccaaatt 5160acaacctctg ctggctggga tggattgcta gcacccattc
tcaacagtaa gccacccgac 5220tgtgacccta ataaagttaa ccctggaagc tcagttaagg
gagactgtgg gaacccatct 5280gttggaattt tcttttttgt cagttacatc atcatatcct
tcctggttgt ggtgaacatg 5340tacatcgcgg tcatcctgga gaacttcagt gttgctactg
aagaaagtgc agagcctctg 5400agtgaggatg actttgagat gttctatgag gtttgggaga
agtttgatcc cgatgcaact 5460cagttcatgg aatttgaaaa attatctcag tttgcagctg
cgcttgaacc gcctctcaat 5520ctgccacaac caaacaaact ccagctcatt gccatggatt
tgcccatggt gagtggtgac 5580cggatccact gtcttgatat cttatttgct tttacaaagc
gggttctagg agagagtgga 5640gagatggatg ctctacgaat acagatggaa gagcgattca
tggcttccaa tccttccaag 5700gtctcctatc agccaatcac tactacttta aaacgaaaac
aagaggaagt atctgctgtc 5760attattcagc gtgcttacag acgccacctt ttaaagcgaa
ctgtaaaaca agcttccttt 5820acgtacaata aaaacaaaat caaaggtggg gctaatcttc
ttataaaaga agacatgata 5880attgacagaa taaatgaaaa ctctattaca gaaaaaactg
atctgaccat gtccactgca 5940gcttgtccac cttcctatga ccgggtgaca aagccaattg
tggaaaaaca tgagcaagaa 6000ggcaaagatg aaaaagccaa agggaaataa
60301511563DNAHomo sapiens 151atgtcctcct cctcctacgc
caagaacggg accgcggacg ggccgcactc ccccacctcg 60caggtggccc gaggcaccac
aacccggagg agcaggttga aaagatccga tggcagcacc 120acttcgacca gcttcatcct
cagacagggt tcagcggatt cctacacaag caggccgtct 180gactccgatg tctctttgga
agaggaccgg gaagcaattc gacaggagag agaacagcaa 240gcagctatcc agcttgagag
agcaaagtcc aaacctgtag catttgccgt gaagacaaat 300gtgagctact gcggcgccct
ggacgaggat gtgcctgttc caagcacagc tatctccttt 360gatgctaaag actttctaca
tattaaagag aaatataaca atgattggtg gataggaagg 420ctggtgaaag agggctgtga
aattggcttc attccaagtc cactcagatt ggagaacata 480cggatccagc aagaacaaaa
aagaggacgt tttcacggag ggaaatcaag tggaaattct 540tcttcaagtc ttggagaaat
ggtatctggg acattccgag caactcccac atcaacagca 600aaacagaagc aaaaagtgac
ggagcacatt cctccttacg atgttgtacc gtcaatgcgt 660ccggtggtgt tagtggggcc
gtcactgaaa ggttacgagg taacagacat gatgcagaaa 720gccctctttg attccctgaa
gcacaggttt gatgggagga tttcaataac gagagtgaca 780gctgacattt ctcttgctaa
gaggtctgtc ctaaataatc ccagcaagag agcaataatt 840gaacgttcga acacccggtc
cagcttagcg gaagtacaaa gtgaaattga aagaatcttt 900gagttggcaa gatctttgca
actggttgtt cttgatgcag acaccatcaa tcacccagca 960caacttataa agacttcctt
agcaccaatt attgttcatg taaaagtctc atctccaaag 1020gttttacagc ggttgattaa
atctagagga aagtcacaaa gtaaacactt gaatgttcaa 1080ctggtggcag ctgataaact
tgcacaatgc cccccagaaa tgtttgatgt tatattggat 1140gaaaatcagc ttgaggatgc
atgtgaacat ctaggggagt acctggaggc gtactggcgt 1200gccacccaca caaccagtag
cacacccatg accccgctgc tgggaaggaa tttgggctcc 1260acggcactct caccatatcc
cacagcaatt tctgggttac agagtcagcg aatgaggcac 1320agcaaccact ccacagagaa
ctctccaatt gaaagacgaa gtctaatgac ctctgatgaa 1380aattatcaca atgaaagggc
tcggaagagt aggaaccgct tgtcttccag ttctcagcat 1440agccgagatc attaccctct
tgtggaagaa gattaccctg actcatacca ggacacttac 1500aaaccccata ggaaccgagg
atcacctggg ggatatagcc atgactcccg acataggctt 1560tga
15631523321DNAOryctolagus
cuniculus 152atggctgcgg gccgcccgct ggcctggacg ctgacacttt ggcaggcgtg
gctgatcctg 60atcgggccct cgtcggagga gccgttccct tcagccgtca ctatcaagtc
atgggtggat 120aagatgcaag aagacctggt cacactggca aaaacagcaa gtggagtcca
tcagcttgtt 180gatatttatg agaaatatca agatttgtat actgtggaac caaataatgc
acgtcagctg 240gtggaaattg cagccagaga cattgagaag cttctcagca acagatctaa
agccctggtg 300cgcctggctt tggaagcaga gaaagttcaa gcagcccacc aatggaggga
agattttgca 360agcaatgaag ttgtctacta taacgcgaag gatgatcttg atcctgaaaa
aaatgacagt 420gaaccaggca gccagaggat caaacctgtt ttcattgacg atgctaactt
tagaagacaa 480gtatcctatc agcacgcagc tgtccatatc cccactgaca tctatgaagg
atcgacaatc 540gtgttaaacg aactcaactg gacaagtgcc ttagatgacg ttttcaaaaa
aaatcgagag 600gaagaccctt cactgttgtg gcaggtgttt ggcagtgcca ctggcctggc
ccggtattac 660ccagcttctc catgggttga taatagccga accccaaaca agattgatct
ttatgatgta 720cgcagaagac catggtacat ccaaggtgct gcatccccta aagatatgct
tattctggtg 780gatgtgagtg gaagcgttag tggactgaca ctcaaactca tccggacatc
cgtctccgaa 840atgttggaaa ccctctcaga tgatgatttt gtgaacgtgg cttcatttaa
cagcaatgct 900caggatgtaa gctgctttca gcaccttgtc caagcaaatg taagaaataa
gaaagtgttg 960aaagatgcag tgaataatat cacagcaaaa ggaatcacag attataagaa
gggctttagt 1020tttgcttttg agcagctgct taattataat gtatccagag ccaactgcaa
taagattatc 1080atgttgttca cggacggagg agaagagaga gcccaggaga tatttgccaa
atacaataaa 1140gacaagaaag tacgtgtatt cacattctca gttggccaac ataattacga
cagaggacct 1200attcagtgga tggcttgcga aaataaaggt tattattatg aaattccatc
cattggagcc 1260ataagaatta atactcagga atacctagat gttctgggaa gaccgatggt
tttagcagga 1320gacaaagcta agcaagtcca atggacaaat gtgtacctgg atgcactgga
actgggactt 1380gtcattactg gaactcttcc ggtcttcaac ataactggcc aatttgaaaa
taagacaaac 1440ttaaagaacc agctgattct tggagtgatg ggagttgatg tgtctttgga
agatattaaa 1500agactgacac cacgttttac actctgcccc aatggctact attttgcaat
tgatcctaat 1560ggttatgtgt tattacatcc aaatcttcag ccaaagccta ttggtgtagg
tataccaaca 1620attaatttga gaaaaaggag acccaatgtt cagaacccca aatctcagga
gccagtgaca 1680ttggatttcc tcgatgcaga gttggagaat gacattaaag tggagattcg
aaataaaatg 1740atcgatggag aaagtggaga aaaaacattc agaactctgg ttaaatctca
agatgagaga 1800tatattgaca aaggaaacag gacatacacg tggactcctg tcaacggcac
agattatagc 1860agtttggcct tggtattacc aacctacagt ttttactata taaaagccaa
aatagaagag 1920acaataactc aggccagata ttcagaaaca ctgaaaccgg ataattttga
agaatctggc 1980tacacattcc tagcaccaag agattactgc agtgacctta aaccttcaga
taataacact 2040gaatttcttt taaatttcaa tgagtttatt gatagaaaaa ctccaaacaa
cccatcctgt 2100aatacagact tgattaatag agtcttgctg gatgcaggct ttacaaatga
acttgttcaa 2160aattactgga gtaagcagaa gaatatcaag ggagtgaaag cacggtttgt
tgtgactgat 2220ggtgggatta ccagagttta tcccaaagag gctggagaaa attggcagga
aaacccagag 2280acatatgaag acagcttcta taaaaggagc ctcgataatg ataactacgt
tttcactgct 2340ccctacttta acaaaagtgg acctggggcc tatgagtcag gcattatggt
aagcaaagct 2400gtagaaatat atatccaagg aaaacttctt aaacctgcag ttgttggaat
taaaattgat 2460gtaaattctt ggatagagaa tttcaccaaa acttcaatca gggatccgtg
tgctggtcca 2520gtttgtgact gcaaacgaaa cagtgatgta atggattgtg tgattctaga
tgacggtggg 2580tttcttttga tggccaacca tgatgattat accaatcaga ttggaagatt
ctttggagag 2640attgatccaa gcttgatgag acacctggtc aatatatcag tttatgcctt
taacaaatct 2700tatgattatc agtcggtgtg tgaacctggt gctgcgccaa agcagggagc
agggcaccgc 2760tcggcttatg tgccatcaat agcagacata ctgcagattg gatggtgggc
cactgctgct 2820gcctggtcta ttcttcagca gtttctgttg agtttgactt ttccacggct
ccttgaggca 2880gctgatatgg aggatgacga cttcactgcc tccatgtcaa agcagagctg
catcactgag 2940caaacccagt atttcttcga taatgacagc aaatcgttca gtggggtatt
agactgtggg 3000aattgttcca gaatctttca tgtagaaaag ctcatgaaca ccaatttaat
attcataatg 3060gtagagagca aggggacatg tccctgtgac acacggctgc tcatacaagc
agagcaaact 3120tctgatggac cagatccttg tgatatggtt aagcaaccca gatatcgaaa
agggccagat 3180gtctgctttg acaacaatgt cctggaggat tatactgact gcggtggggt
ctctggatta 3240aatccttccc tgtggtccat catcgggata cagtttgtac tgctttggct
ggtttctggc 3300agcagacact gcctgttatg a
3321
User Contributions:
Comment about this patent or add new information about this topic: