Abstract:
The invention relates to methods of and kits for detecting polymorphisms in a nucleic acid encoding a human histamine H 2  receptor.

Description:
This application is a continuation of U.S. Ser. No. 08/875,540, filed Jan. 5, 1998, U.S. Pat. No. 6,015,888, which claims priority from International Patent Application No. PCT/EP96/00397, filed Jan. 30, 1996, which claims priority from United Kingdom Patent Application No. 9503866.7, filed Jan. 30, 1995. 
    
    
     TECHNICAL FIELD 
     This invention relates to the detection of variations in human H 2  receptors, and more particularly to the development of new compounds useful in the sequencing and identification of a human histamine H 2  receptor and their use in the diagnosis and treatment of certain human disorders, for example, brain disorders. The invention also relates to new compounds and a method for detecting an allelic polymorphic variation within the human population for the gene encoding the histamine H 2  receptor and their use in the diagnosis and treatment of human disorders. 
     BACKGROUND 
     The human H 2  receptor was first identified by Black et al Nature (1972), 236, 385-390. This was followed by the demonstration of the receptor in the mammalian brain by Baudry et al (1975) Nature 253, 362-363, and Haas and Bucher (1975) Nature 255, 634-635. Gantz et al (1991) Biochem. Biophys. Res. Comm. 178, 3, 1386-1392 have recently identified the sequence of a human H2 receptor cDNA from gastric parietal cells by using the polymerase chain reaction (PCR) and degenerated oligonucleotide primers whose sequence was obtained from the canine H 2  receptor previously cloned by this group, Gantz et al (1991) Proc. Nat. Acad. Sci. USA 88, 429-433. This sequence was characterised as an intronless gene encoding a typical seven transmembrane domain aminergic receptor protein. 
     The receptor is coupled to heterotrimeric GTPases (G proteins), but differs from other monoamine receptors in this G protein coupled superfamily in several respects. The human gastric H 2  receptor is shorter than most other. receptors in this class (359 amino acids) and lacks the two serine residues in the fifth transmembrane region (TM5). There exists instead an aspartate and a threonine residue, so far unique in this region. These two residues may be important for binding with the nitrogen atoms of the imidazole ring of histamine as suggested by Birdsall (1991) Trends in Pharmacological Sci. Jan, 12, 9-10. 
     Histamine is a natural constituent of many organs and tissues including the gastrointestinal tract, the immune system and the brain, Green et al (1987) Agents and Actions 22, 1-15. It is a central neurotransmitter in the brain and is formed in the posterior hypothalamus from exogenous histidine by histidine decarboxylase (HDC). It is subsequently metabolised by histamine methyltransferase (HMT), Prell et al (1986) Ann. Rev. Neurosci. 9, 209-254. The cell bodies and neuronal pathways for histamine have been mapped in the human brain using immunocytochemistry by Panula et al (1990) Neuroscience 34, 127-132. Its cells project from the tuberomamillary nucleus of the posterior hypothalamus to almost every region of the brain. There are three known histamine receptors; H 1 , H 2  and H 3 , the latter functioning as an autoreceptor. The H 2  receptor specifically has been localised in the human brain by Traiffort et al (1992) J. of Neurochem. 59, 1, 290-299. using receptor autoradiography. 
     Histamine is known to have significant effects in the central nervous system (CNS). It has been implicated in the CNS mediated mechanisms of arousal ever since the sedating effect of H 1  receptor antagonists (eg,. chlorpheniramine, chloropromazine) had been noticed clinically. The use of H 2  receptor antagonists in the human brain however, has shown, that these compounds, unlike those acting on the H 1  receptor, do not produce any effect on psychomotor functioning, or a subjective feeling of sedation or arousal in healthy subjects, White et al (1988) Psychopharmacology 95, 1-14. Some H 2  receptor antagonists (eg. cimetidine) are known to cause confusion in elderly or severely medically ill patients, perhaps in part due to a co-existing anti-cholinergic effect. H 1  and H 2  receptor antagonists in large doses have been reported to cause hallucinations, Csillag et al (1973) Med. J. Aug. 1, 653-654, Argawal (1978) J. Am. Med. Assoc. 240, 214. Animal studies have shown that histamine applied directly to the hippocampus, where there is the highest level of activity of the H 2  receptor, will induce psychomotor withdrawal and decreased exploratory behaviour. The above evidence has led to the conclusion that H 1  receptor systems are excitatory in the terms of arousal and motivated behaviour whilst H 2  receptor systems are inhibitory in this respect, Alvarez and Banzan (1985) Physiol. and Beh. 34, 661-664 and (1986) Physiol. and Beh. 37, 39-45, White et al (1988) supra. 
     The H 2  receptor is a site of action of various compounds used in the treatment of psychiatric disorders eg. amitriptyline and mianserin, Traiffort et al (1992) supra. Kaminsky et al (1990) The Lancet 335, 1351-1352 and (1991) Schizophrenia Bull. 4, 318-319 have reported the successful response of patients with chronic, predominantly negative type schizophrenia, to the highly specific H 2  receptor antagonist famotidine. For example in one patient there was a substantial amelioration of the deficit symptoms of schizophrenia (eg apathy, social withdrawal, and blunted affect) while on famotidine, relapse in these symptoms on withdrawal, and improvement on re-institution of this drug, Kaminski, U.S. Pat. Nos. 5,070,101 and 5,177,081. Prell et al (1992) Abstract, part 1, 199.6 Soc. for Neurosci. Annual Meeting, Anaheim Cal. have shown substantially raised levels of N-tele-methyl histamine, a metabolite of histamine in the cerebrospinal fluid of patients with schizophrenia which correlates with those patients with the occurrence of negative symptoms of this disorder assessed using the Psychiatric Symptoms Assessment Scale. These levels were not significantly different between patients free from medication and those on neuroleptic therapy. It is therefore postulated that there is an increase in histaminergic activity in patients with chronic schizophrenia. 
     The disclosures of all the above mentioned publications are incorporated herein by reference for all purposes. 
     Additionally, histamine, acting via its receptors, including the H 2  receptor, is believed to be critically involved in a number of diseases of organs other than the brain; these include peptic ulceration, allergic reactions, including asthma, immune-mediated disorders, and possibly some tumours. 
     The histamine H 2  receptor is one of many receptors in the body. Compounds used to treat many diseases work by activating a receptor or inhibiting the action if its natural ligand. Variations in receptors amongst the population are known to be caused by allelic variation and this variation, can alter the response of a disease to a drug amongst patients. An example of this would be the response to clozapine, used to treat, schizophrenia associated with allelic variation in the 5-HT 2A  receptor demonstrated by Arranz el al (1995) Lancet, 346(8970), 291-282. 
     SUMMARY OF THE INVENTION 
     The present invention is concerned in one aspect with improvements in the diagnosis and/or treatment of human neurological and psychiatric disorders, and more particularly in the diagnosis and treatment of schizophrenia. In another aspect, the invention is concerned with improvements in the diagnosis and/or treatment of diseases of other systems or organs of the human body. 
     As a first step to the present invention, the Applicants devised a new oligonucleotide probe to the human H 2  receptor mRNA in accordance with the published cDNA sequence available for the gastric parietal cell. Surprisingly, studies using this probe with in-situ hybridization histochemistry on human post-mortem brain tissue produced evidence of a mismatch in the nucleotide sequence for the brain H 2  receptor and the sequence for the gastric parietal cell H 2  receptor. This discovery was made by recording melt-curve estimations for the optimum hybridization incubation temperature using the method of Davis et al (1986) “Basic Methods in Molecular Biology” page 77 Elsevier Science Publishing Co. It was found that the sequence mismatch is of the order of 10%. 
     It was apparent, therefore, that there is a hitherto unrecognised allele or subtype of the human histamine H 2  receptor gene, which may be specific to the brain. 
     In a first aspect, the invention provides a sequence for a novel allele of a human histamine H 2  receptor gene comprising up to six single base substitutions compared with the cDNA sequence published by Gantz et al (1991) Biochem Biophys Res Comm 178, 3, 1386-1392 as follows: 
     
       
         
               
               
               
             
           
               
                   
               
               
                 site of change 
                 base change 
                 amino acid alteration 
               
               
                   
               
             
             
               
                 398 
                 T - C 
                 Val - Gly 
               
               
                 525 
                 A - T 
                 Lys - Asn 
               
               
                 620 
                 A - G 
                 Lys - Asp 
               
               
                 649 
                 A - G 
                 Asn - Asp 
               
               
                 692 
                 A - G 
                 Lys - Arg 
               
               
                 802 
                 G - A 
                 Val - Met 
               
               
                   
               
             
          
         
       
     
     In another aspect, the invention provides a nucleotide sequence coding for a region of a human histamine H 2  receptor, comprising one or more of the following base substitutions compared with the published sequence in Gantz et al (1991) supra, and from which the positional notation is taken: 
     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 site of change 
                 base 
               
               
                   
                   
               
             
             
               
                   
                 398 
                 C 
               
               
                   
                 525 
                 T 
               
               
                   
                 620 
                 G 
               
               
                   
                 649 
                 G 
               
               
                   
                 692 
                 G 
               
               
                   
                 802 
                 A 
               
               
                   
                   
               
             
          
         
       
     
     The nucleotide sequence of the invention can, for example, comprise the following sequence (as also listed in SEQ ID NO:1): 
     
       
         
               
               
             
           
               
                 5′ 
                   
               
               
                 CAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTC 
               
               
                   
               
               
                 CTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAAT 
               
               
                   
               
               
                 CATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATG 
               
               
                   
               
               
                 GGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCAT 
               
               
                   
               
               
                 CTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAG 
               
               
                   
               
               
                 GCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGG 
               
               
                   
               
               
                 CCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAG 
               
               
                   
               
               
                 AGGGGATGATGCCATCAATGAGATGTTA 3′ 
               
             
          
         
       
     
     As a specific exemplification, the nucleotide sequence of the invention can comprise the following sequence (as also listed in SEQ ID NO:2): 
     
       
         
               
               
             
           
               
                 5′ 
                   
               
               
                 CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCAT 
               
               
                   
               
               
                 CACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTC 
               
               
                   
               
               
                 TGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCG 
               
               
                   
               
               
                 TGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGC 
               
               
                   
               
               
                 CATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTAC 
               
               
                   
               
               
                 ACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCA 
               
               
                   
               
               
                 GCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCAC 
               
               
                   
               
               
                 CCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTG 
               
               
                   
               
               
                 TCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCA 
               
               
                   
               
               
                 ATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGA 
               
               
                   
               
               
                 TGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGC 
               
               
                   
               
               
                 ATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGA 
               
               
                   
               
               
                 AGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGG 
               
               
                   
               
               
                 GGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTG 
               
               
                   
               
               
                 AGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCT 
               
               
                   
               
               
                 ATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCG 
               
               
                   
               
               
                 CACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAA 
               
               
                   
               
               
                 ACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCA 
               
               
                   
               
               
                 GGCAACAGGAAGAGMACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG 
               
               
                   
               
               
                 3′ 
               
             
          
         
       
     
     In another aspect of the invention, a series of new oligonucleotide primers have been developed for the identification of sequences in a sample comprising a human histamine H 2  receptor DNA, cDNA or RNA originating from a tissue sample or body fluid. 
     In this aspect, the invention provides new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H 2  receptor, having nucleotide sequences selected from: 
     1) 5′ CCAATGGCACAGCCTCTT 3′ (as listed in SEQ ID NO: 3) 
     2) 5′ CGTGACTTCTGTCCCACT 3′ (as listed in SEQ ID NO: 4) 
     3) 5′ CCAGGCAACAGGAAGAGA 3′ (as listed in SEQ ID NO: 5) 
     4) 5′ TCTCTTCCTGTTGCCTGG 3′ (as listed in SEQ ID NO: 6) 
     5) 5′ GCAGCAGAAGAGCTGTTG 3′ (as listed in SEQ ID NO: 7) 
     6) 5′ TCCAGGTCAATGAAGTGT 3′ (as listed in SEQ ID NO: 8) 
     7) 5′ ACACTTCATTGACCTGGA 3′ (as listed in SEQ ID NO: 9) 
     8) 5′ CCAAGAGGATCAATCACA 3′ (as listed in SEQ ID NO: 10) 
     9) 5′ TGTGATTGATCCTCTTGG 3′ (as listed in SEQ ID NO: 11) 
     and a diagnostic kit comprising one or more of the new oligonucleotides. 
     The direction and base start numbers for the novel oligonucleotide primers are as follows: 
     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Primer 
                 Base Start No. 
               
               
                   
                   
               
             
             
               
                   
                 1) Upstream 
                 8 
               
               
                   
                 2) Downstream 
                 1036 and 1095 
               
               
                   
                 3) Upstream 
                 995 
               
               
                   
                 4) Downstream 
                 1012 (no. 3) in reverse) 
               
               
                   
                 5) Downstream 
                 898 and 1171 
               
               
                   
                 6) Upstream 
                 527 
               
               
                   
                 7) Downstream 
                 544 (no. 6) in reverse) 
               
               
                   
                 8) Upstream 
                 638 
               
               
                   
                 9) Downstream 
                 655 (no. 8) in reverse) 
               
               
                   
                   
               
             
          
         
       
     
     Information on the human histamine H 2  receptor was obtained from the MRC Daresbury database accessing “Genem 61” File no. M64799—Human histamine H 2  receptor gene. 
     The above mentioned substitutions alter and in some instances introduce or remove new sites for cleavage by specific restriction endonucleases as follows: 
     
       
         
               
               
             
           
               
                   
               
               
                 base change site 
                 alters restriction map of: 
               
               
                   
               
             
             
               
                 398 
                 AluI, AvaI, BspWI, BsrI, CviJI 
               
               
                 525 
               
               
                 620 
                 Eco57 
               
               
                 649 
                 ClaI, Sau3A, TaqI 
               
               
                 692 
               
               
                 802 
                 MnlI 
               
               
                   
               
             
          
         
       
     
     The invention further provides a diagnostic kit comprising one or more of the new oligonucleotide primers and preferably, one or more of the above mentioned endonucleases, optionally with one or more buffers. 
     A kit may be used to establish genotype or base variations. This information may be used in predicting an individuals disease susceptibility, disease course, prognosis and/or response to treatment as would be understood by those skilled in the art from the disclosure contained therein. The treatment response or efficacy which may be predicted may include drug treatment such as for example, use of the H2 receptor antagonist like famotidine or other forms of treatment such as social or psychological intervention. 
     Eucaryotic expression vectors comprising a DNA sequence coding for a protein and/or a peptide according to the invention are new materials and are also included in the invention. Host cells, for example, cloned human cell lines, such as NTera 2 c.dl, can be transformed using the new expression vectors and are also included in the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is a photograph of an agarose gel containing isolated nucleic acids of the invention. 
    
    
     DETAILED DESCRIPTION 
     Expression vectors and host cells transformed thereby, in accordance with the invention, can be prepared, for example, as detailed below, and the encoded protein studies, by one or more of the following exemplary methods: 
     1. Total RNA is extracted from homogenised human tissue, eg. brain, by the acid guanidine thiocyanate method (Chomczynski &amp; Saach (1987), Anal. Biochem. 161, 156-159). Messenger RNA (mRNA) is purified from this by hybridisation of oligo(d)T to the polyadenylated tails present on the majority of mRNA&#39;s, for example, using the Promega PolyAttract ® system. Reverse transcription of the mRNA using specific reverse transcriptase enzyme, eg. Superscipt II, Gibco BRL, is followed by PCR amplification of the resultant product using specific oligonucleotide primers, for example, those previously described. The resulting amplified cDNA is ligated into an expression vector, eg. pGEMEX®-1 vector available from Promega. Competent cells, eg. bacterial strain JM109(DE3), also available from Promega, are transformed using this vector, effective transforms selected and cultured. Expression of the encoded protein is then induced with a suitable promotor, eg. IPTG, and the expressed protein purified from the cell culture using standard biochemical procedures, eg. cell lysis and polyacrylamide gel electrophoresis. 
     2. An alternative method for examining the functional protein encoded by the cDNA described above, is to induce transcription of the cloned cDNA, as above, and to purify the specific mRNA from the cell culture as described. The purified mRNA is introduced into competent cells, eg. frog oocytes or Chinese hamster ovary cells, and the function of the encoded protein studied by standard pharmacological and physiological techniques, eg. microelectrode recording and receptor binding techniques. 
     3. As 1 above, but introducing the cDNA into a coupled transcription-translation system, eg. TNT, Promega with subsequent purification and analysis of the encoded protein as described. 
     The invention is illustrated by the following Examples: 
     EXAMPLE 1 
     This example describes the identification and sequencing of an allelic human H 2  receptor gene using certain novel oligonucleotide primers according to the invention. 
     A polymerase chain reaction (PCR) product is prepared from human DNA. 
     DNA was extracted from human brain tissue by first pulverizing approximately 1 g of tissue in liquid nitrogen then adding to 10 ml lysis buffer (0.32M sucrose, 10 mM Tris, 5 mM magnesium chloride 1% Triton X-100 pH8.0). This solution was centrifuged (9,000 rpm 15 mins) to pellet the tissue, the lysis buffer was drawn off and the pellet resuspended in 4.5 ml 75 mM sodium chloride, 24 mM EDTA. This solution was then incubated for 3 hours with 250 μl 10% SDS and 2 mg proteinase K at 56° C. This aqueous phase was then extracted twice with 5 ml of phenol:chloroform:isoamyl alcohol (25:24:1). Then sodium acetate to 0.3M pH7.5 and 2 volumes of ethanol (at −20° C.) were added to the aqueous phase and the DNA hooked out into TE buffer. The concentration of the DNA was determined by measuring the optical density of the sample, at a wavelength of 260 nm. 
     The DNA was then amplified by the polymerase chain reaction using the oligonucleotide primers 1) and 2) (as hereinbefore described) for 36 cycles. The timing for each cycle was as follows; 1 min at 94° C., 1.5 min at 56° C. and 2 mins at 72° C., this was then followed by a 10 min extension at 72° C. (Amplitaq DNA polymerase Perkin-Elmer Cetus). This reaction produced a DNA fragment of 1047 base pair when analyzed by gel electrophoresis. 
     Following PCR amplification of the DNA, the PCR products were immediately ligated and cloned into the TA cloning system (InvitroGen). The transformed cells were plated onto Luria-Bertani plates containing 50 μl/ml amplicillin and 1.6 mg X-Gal. Plates were then incubated overnight at 37° C., then moved to 4° C. for 4 hours to allow for colour development. Positive (white colonies) were then analyzed by growing a 5 ml culture overnight at 37° C. extracting the plasmids (Qiaspin minipreps (Qiagen)) and performing an EcoRI digest to ensure the correct size product was contained in the plasmid. The plasmid used to clone the PCR product is the pCR™II Vector, which is transformed into One Shot™ INVaF′ Competent cells. 
     Both strands of the cloned PCR product were sequenced using the dideoxynucleotide chain-terminated method, carried out with Seguenase version 2.0 (Amersham/USB). Partial sequencing of short stretches of the cloned DNA utilised all the oligonucleotide primers 1) to 9) hereinbefore described. The cloned PCR product was shown to be identical to the gastric cDNA of Gantz et al except for the previously mentioned six single base changes. 
     Results and Discussions 
     The sequence derived from the above described method is listed below and in SEQ ID NO: 2. 
     
       
         
               
               
             
           
               
                 5′ 
                   
               
               
                   
               
               
                 CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCAT 
               
               
                   
               
               
                 CACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTC 
               
               
                   
               
               
                 TGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCG 
               
               
                   
               
               
                 TGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGC 
               
               
                   
               
               
                 CATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTAC 
               
               
                   
               
               
                 ACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCA 
               
               
                   
               
               
                 GCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCAC 
               
               
                   
               
               
                 CCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTG 
               
               
                   
               
               
                 TCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCA 
               
               
                   
               
               
                 ATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGA 
               
               
                   
               
               
                 TGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGC 
               
               
                   
               
               
                 ATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGA 
               
               
                   
               
               
                 AGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGG 
               
               
                   
               
               
                 GGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTG 
               
               
                   
               
               
                 AGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCT 
               
               
                   
               
               
                 ATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGACACTTCCG 
               
               
                   
               
               
                 CACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAA 
               
               
                   
               
               
                 ACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCA 
               
               
                   
               
               
                 GGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG 
               
               
                   
               
               
                 3′ 
               
             
          
         
       
     
     EXAMPLE 2 
     This example describes the confirmation of the presence of the base changes in a larger population. This is made possible by an assay based upon PCR amplification of a 909 base pair fragment of the H 2  receptor gene from human DNA, followed by cleavage utilizing specific restriction endonucleases. It is apparent to those skilled in the art that single base changes could be detected using other techniques known to those in the art which include single stranded confirmational polymorphisms (sscp), chemical cleavage, PCR thermoligase reactions, etc. 
     Samples of blood are collected from human volunteers into EDTA coated tube, 1 ml of this blood is heated to 100° C. for 15 minutes then spun in a microcentrifuge at 13,000×g for 15 minutes. This supernatant is collected, and the cell debris is discarded. Then 0.5-3 μl of this supernatant is utilised as template DNA for a PCR reaction to amplify a portion of the receptor gene between bases 8 and 915. The conditions for this PCR reaction are 3 mM MgCl 2  (Gibco BRL),1×PCR buffer (Gibco BRL) 1 mM of each dATP, dGTP, dGTP and dTTP (Promega) 10 pmoles of each of oligonucleotide primers 1) and 5) (hereinbefore described) and 1 unit Taq DNA polymerase (Gibco BRL), in a total volume adjusted to 50 μl by sterile DNAse free water. This mix is then subjected to the following conditions; 96° C. 5 minutes, then 35 cycles of 96° C. for 1 minute, 56° C. for 1 minute, 72° C. for 1 minute and 20 seconds. 
     10 μl of the resultant products are then analysed on a 1% agarose gel to ensure that the above reaction is correctly amplifying the target DNA fragment. Then 11.5 μl of the PCR mix is added to 2 units Taql restriction endonuclease (Fermentas) and 1.5 μl of 10× buffer and incubated at 65° C. for 3-24 hours. The products of this reaction are then analysed on a 2.5% agarose gel. If the original sequence described by Gantz (nominated H 2 A) has been amplified, then bands of 574 and 335 base pairs are seen which indicates that the individual is an A/A homozygote. If the sequence described in Example 1 (nominated H 2 B) has been amplified, then following the TaqI cleavage of the PCR product, bands of 335, 306 and 268 base pairs can be seen, indicating that individual is a B/B homozygote. If bands of 574, 335, 306 and 268 base pairs can be seen, then that individual is an A/B heterozygote. 
     Typical results are illustrated in FIG. 1, which shows a 2.5% TBE Agarose gel stained with ethidium bromide, showing TaqI digestion patterns of a 909 base pair PCR fragment, from 4 separate individuals. 
     Lanes A+G−100 base pair DNA marker (Gibco BRL) 
     Lanes B+F−Band pattern indicative of an A/B heterozygote 
     Lanes C+E−Band pattern indicative of an B/B homozygote 
     Lane D−Blank 
     Arrows indicated the sizes of the DNA fragments in lanes B to F. 
     Primers: Oswell DNA Services) 
     1) upstream: 5′ CCAATGGCACAGCCTCTT 3′ (as in SEQ ID NO:1) 
     2) upstream: 5′ CCAGGCAACAGGAAGAGA 3′ (as in SEQ ID NO:5) 
     5) downstream: 5′ GCAGCAGAAGAGCTGTTG 3′ (as in SEQ ID NO:7) 
     EXAMPLE 3 
     A method as described in example 2 is applied to a series of DNA samples extracted from schizophrenic individuals, their first degree relatives, and normal controls. There is observed a statistically significant difference of P less than 0.01 the pattern seen in the genotype of these individuals, as described in the table below: 
     
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 H 2  Genotype 
               
             
          
           
               
                   
                 Diagnosis 
                 A/A 
                 A/B 
                 B/B 
               
               
                   
                   
               
             
          
           
               
                   
                 Controls 
                 12.1% 
                 48.5% 
                 39.4% 
               
               
                   
                 Schizophrenia 
                 9.8% 
                 26.8% 
                 63.4% 
               
               
                   
                 1st degree 
                 6.1% 
                 12.1% 
                 81.8% 
               
               
                   
                 relatives 
               
               
                   
                   
               
             
          
         
       
     
     Discussion 
     The variable sequence is explained by a polymorphic allelic variation within the human population for the gene encoding the H 2  receptor protein. This allelic polymorphism may lead to substantial variation in the effect of activation of the encoded receptor by histamine, either in the efficacy of histamine binding, the duration of activation, or the intracellular effects of such activation. It is envisaged that such variation resulting from allelic polymorphism may underline susceptibility to specific disorders, both affecting the brain and/or involving other systems or organs. In summary, this variation in the human H 2  receptor gene and its products, including, for example, mRNA and proteins, could be used as a method of establishing individual risk to a particular psychiatric or neurological or other illness eg. schizophrenia. 
     Alternative embodiments of the invention can be envisaged by those skilled in the art from the information contained herein. All such alternative embodiments are intended to lie within the scope of this application. 
     The reader&#39;s attention is directed to all papers and documents which are filed concurrently with this specification and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. 
     All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps or any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. 
     Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. 
     The invention is not restricted to the details of the foregoing embodiments. This invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. 
     
       
         
           
             15 
           
           
             1 
             413 
             DNA 
             Homo sapiens 
             
               CDS 
               (3)...(413) 
             
           
            1
ca gct cgg gtc gcc atc tct ctg gtc tta att tgg gtc atc tcc att        47
   Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser Ile
     1               5                  10                  15
acc ctg tcc ttt ctg tct atc cac ctg ggg tgg aac agc agg aac gag       95
Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn Glu
                 20                  25                  30
acc agc aag ggc aat cat acc acc tct aag tgc aat gtc cag gtc aat      143
Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val Asn
             35                  40                  45
gaa gtg tac ggg ctg gtg gat ggg ctg gtc acc ttc tac ctc ccg cta      191
Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu
         50                  55                  60
ctg atc atg tgc atc acc tac tac cgc atc ttc agg gtc gcc cgg gat      239
Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg Asp
     65                  70                  75
cag gcc aag agg atc gat cac att agc tcc tgg aag gca gcc acc atc      287
Gln Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr Ile
 80                  85                  90                  95
agg gag cac aga gcc aca gtg aca ctg gcc gcc gtc atg ggg gcc ttc      335
Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala Phe
                100                 105                 110
atc atc tgc tgg ttt ccc tac ttc acc gcg ttt gtg tac cgt ggg ctg      383
Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu
            115                 120                 125
aga ggg gat gat gcc atc aat gag atg tta                              413
Arg Gly Asp Asp Ala Ile Asn Glu Met Leu
        130                 135
 
           
             2 
             1046 
             DNA 
             Homo sapiens 
             
               CDS 
               (3)...(1046) 
             
           
            2
cc aat ggc aca gcc tct tcc ttt tgc ctg gac tct acc gca tgc aag        47
   Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala Cys Lys
     1               5                  10                  15
atc acc atc acc gtg gtc ctt gcg gtc ctc atc ctc atc acc gtt gct       95
Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr Val Ala
                 20                  25                  30
ggc aat gtg gtc gtc tgt ctg gcc gtg ggc ttg aac cgc cgg ctc cgc      143
Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg Leu Arg
             35                  40                  45
aac ctg acc aat tgt ttc atc gtg tcc ttg gct atc act gac ctg ctc      191
Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp Leu Leu
         50                  55                  60
ctc ggc ctc ctg gtg ctg ccc ttc tct gcc atc tac cag ctg tcc tgc      239
Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu Ser Cys
     65                  70                  75
aag tgg agc ttt ggc aag gtc ttc tgc aat atc tac acc agc ctg gat      287
Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser Leu Asp
 80                  85                  90                  95
gtg atg ctc tgc aca gcc tcc att ctt aac ctc ttc atg atc agc ctc      335
Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile Ser Leu
                100                 105                 110
gac cgg tac tgc gct gtc atg gac cca ctg cgg tac cct gtg ctg gtc      383
Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val Leu Val
            115                 120                 125
acc cca gct cgg gtc gcc atc tct ctg gtc tta att tgg gtc atc tcc      431
Thr Pro Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser
        130                 135                 140
att acc ctg tcc ttt ctg tct atc cac ctg ggg tgg aac agc agg aac      479
Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn
    145                 150                 155
gag acc agc aag ggc aat cat acc acc tct aag tgc aat gtc cag gtc      527
Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val
160                 165                 170                 175
aat gaa gtg tac ggg ctg gtg gat ggg ctg gtc acc ttc tac ctc ccg      575
Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro
                180                 185                 190
cta ctg atc atg tgc atc acc tac tac cgc atc ttc agg gtc gcc cgg      623
Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg
            195                 200                 205
gat cag gcc aag agg atc gat cac att agc tcc tgg aag gca gcc acc      671
Asp Gln Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr
        210                 215                 220
atc agg gag cac aga gcc aca gtg aca ctg gcc gcc gtc atg ggg gcc      719
Ile Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala
    225                 230                 235
ttc atc atc tgc tgg ttt ccc tac ttc acc gcg ttt gtg tac cgt ggg      767
Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly
240                 245                 250                 255
ctg aga ggg gat gat gcc atc aat gag atg tta gaa gcc atc gtt ctg      815
Leu Arg Gly Asp Asp Ala Ile Asn Glu Met Leu Glu Ala Ile Val Leu
                260                 265                 270
tgg ctg ggc tat gcc aac tca gcc ctg aac ccc atc ctg tat gct gcg      863
Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr Ala Ala
            275                 280                 285
ctg aac aga gac ttc cgc acc ggg tac caa cag ctc ttc tgc tgc agg      911
Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys Cys Arg
        290                 295                 300
ctg gcc aac cgc aac tcc cac aaa act tct ctg agg tcc aac gcc tct      959
Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn Ala Ser
    305                 310                 315
cag ctg tcc agg acc caa agc cga gaa ccc agg caa cag gaa gag aaa     1007
Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu Glu Lys
320                 325                 330                 335
ccc ctg aag ctc cag gtg tgg agt ggg aca gaa gtc acg                 1046
Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr
                340                 345
 
           
             3 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            3
ccaatggcac agcctctt                                                   18
 
           
             4 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            4
cgtgacttct gtcccact                                                   18
 
           
             5 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            5
ccaggcaaca ggaagaga                                                   18
 
           
             6 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            6
tctcttcctg ttgcctgg                                                   18
 
           
             7 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            7
gcagcagaag agctgttg                                                   18
 
           
             8 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            8
tccaggtcaa tgaagtgt                                                   18
 
           
             9 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            9
acacttcatt gacctgga                                                   18
 
           
             10 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            10
ccaagaggat caatcaca                                                   18
 
           
             11 
             18 
             DNA 
             Artificial sequence 
             
               Synthetic Oligonucleotide 
             
           
            11
tgtgattgat cctcttgg                                                   18
 
           
             12 
             137 
             PRT 
             Homo sapiens 
           
            12
Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser Ile Thr
  1               5                  10                  15
Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn Glu Thr
             20                  25                  30
Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val Asn Glu
         35                  40                  45
Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu Leu
     50                  55                  60
Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg Asp Gln
 65                  70                  75                  80
Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr Ile Arg
                 85                  90                  95
Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala Phe Ile
            100                 105                 110
Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu Arg
        115                 120                 125
Gly Asp Asp Ala Ile Asn Glu Met Leu
    130                 135
 
           
             13 
             348 
             PRT 
             Homo sapiens 
           
            13
Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala Cys Lys Ile
  1               5                  10                  15
Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr Val Ala Gly
             20                  25                  30
Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg Leu Arg Asn
         35                  40                  45
Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp Leu Leu Leu
     50                  55                  60
Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu Ser Cys Lys
 65                  70                  75                  80
Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser Leu Asp Val
                 85                  90                  95
Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile Ser Leu Asp
            100                 105                 110
Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val Leu Val Thr
        115                 120                 125
Pro Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser Ile
    130                 135                 140
Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn Glu
145                 150                 155                 160
Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val Asn
                165                 170                 175
Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu
            180                 185                 190
Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg Asp
        195                 200                 205
Gln Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr Ile
    210                 215                 220
Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala Phe
225                 230                 235                 240
Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu
                245                 250                 255
Arg Gly Asp Asp Ala Ile Asn Glu Met Leu Glu Ala Ile Val Leu Trp
            260                 265                 270
Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr Ala Ala Leu
        275                 280                 285
Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys Cys Arg Leu
    290                 295                 300
Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn Ala Ser Gln
305                 310                 315                 320
Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu Glu Lys Pro
                325                 330                 335
Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr
            340                 345
 
           
             14 
             1080 
             DNA 
             Homo sapiens 
             
               CDS 
               (1)...(1077) 
             
           
            14
atg gca ccc aat ggc aca gcc tct tcc ttt tgc ctg gac tct acc gca       48
Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala
  1               5                  10                  15
tgc aag atc acc atc acc gtg gtc ctt gcg gtc ctc atc ctc atc acc       96
Cys Lys Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr
             20                  25                  30
gtt gct ggc aat gtg gtc gtc tgt ctg gcc gtg ggc ttg aac cgc cgg      144
Val Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg
         35                  40                  45
ctc cgc aac ctg acc aat tgt ttc atc gtg tcc ttg gct atc act gac      192
Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp
     50                  55                  60
ctg ctc ctc ggc ctc ctg gtg ctg ccc ttc tct gcc atc tac cag ctg      240
Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu
 65                  70                  75                  80
tcc tgc aag tgg agc ttt ggc aag gtc ttc tgc aat atc tac acc agc      288
Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser
                 85                  90                  95
ctg gat gtg atg ctc tgc aca gcc tcc att ctt aac ctc ttc atg atc      336
Leu Asp Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile
            100                 105                 110
agc ctc gac cgg tac tgc gct gtc atg gac cca ctg cgg tac cct gtg      384
Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val
        115                 120                 125
ctg gtc acc cca gtt cgg gtc gcc atc tct ctg gtc ata att tgg gtc      432
Leu Val Thr Pro Val Arg Val Ala Ile Ser Leu Val Ile Ile Trp Val
    130                 135                 140
atc tcc att acc ctg tcc ttt ctg tct atc cac ctg ggg tgg aac agc      480
Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser
145                 150                 155                 160
agg aac gag acc agc aag ggc aat cat acc acc tct aag tgc aaa gtc      528
Arg Asn Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Lys Val
                165                 170                 175
cag gtc aat gaa gtg tac ggg ctg gtg gat ggg ctg gtc acc ttc tac      576
Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr
            180                 185                 190
ctc ccg cta ctg atc atg tgc atc acc tac tac cgc atc ttc aag gtc      624
Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Lys Val
        195                 200                 205
gcc cgg gat cag gcc aag agg atc aat cac att agc tcc tgg aag gca      672
Ala Arg Asp Gln Ala Lys Arg Ile Asn His Ile Ser Ser Trp Lys Ala
    210                 215                 220
gcc acc atc agg gag cac aaa gcc aca gtg aca ctg gcc gcc gtc atg      720
Ala Thr Ile Arg Glu His Lys Ala Thr Val Thr Leu Ala Ala Val Met
225                 230                 235                 240
ggg gcc ttc atc atc tgc tgg ttt ccc tac ttc acc gcg ttt gtg tac      768
Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr
                245                 250                 255
cgt ggg ctg aga ggg gat gat gcc atc aat gag gtg tta gaa gcc atc      816
Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Glu Val Leu Glu Ala Ile
            260                 265                 270
gtt ctg tgg ctg ggc tat gcc aac tca gcc ctg aac ccc atc ctg tat      864
Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr
        275                 280                 285
gct gcg ctg aac aga gac ttc cgc acc ggg tac caa cag ctc ttc tgc      912
Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys
    290                 295                 300
tgc agg ctg gcc aac cgc aac tcc cac aaa act tct ctg agg tcc aac      960
Cys Arg Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn
305                 310                 315                 320
gcc tct cag ctg tcc agg acc caa agc cga gaa ccc agg caa cag gaa     1008
Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu
                325                 330                 335
gag aaa ccc ctg aag ctc cag gtg tgg agt ggg aca gaa gtc acg gcc     1056
Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr Ala
            340                 345                 350
ccc cag gga gcc aca gac agg taa                                     1080
Pro Gln Gly Ala Thr Asp Arg
        355
 
           
             15 
             359 
             PRT 
             Homo sapiens 
           
            15
Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala
  1               5                  10                  15
Cys Lys Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr
             20                  25                  30
Val Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg
         35                  40                  45
Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp
     50                  55                  60
Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu
 65                  70                  75                  80
Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser
                 85                  90                  95
Leu Asp Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile
            100                 105                 110
Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val
        115                 120                 125
Leu Val Thr Pro Val Arg Val Ala Ile Ser Leu Val Ile Ile Trp Val
    130                 135                 140
Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser
145                 150                 155                 160
Arg Asn Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Lys Val
                165                 170                 175
Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr
            180                 185                 190
Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Lys Val
        195                 200                 205
Ala Arg Asp Gln Ala Lys Arg Ile Asn His Ile Ser Ser Trp Lys Ala
    210                 215                 220
Ala Thr Ile Arg Glu His Lys Ala Thr Val Thr Leu Ala Ala Val Met
225                 230                 235                 240
Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr
                245                 250                 255
Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Glu Val Leu Glu Ala Ile
            260                 265                 270
Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr
        275                 280                 285
Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys
    290                 295                 300
Cys Arg Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn
305                 310                 315                 320
Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu
                325                 330                 335
Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr Ala
            340                 345                 350
Pro Gln Gly Ala Thr Asp Arg
        355