Abstract:
The invention relates to allelic variants of human histamine H 2  receptor genes and primer sequences useful in detecting such genes.

Description:
This is a 371 of International Patent Application No. PCT/EP96/00397, with an international filing date of Jan. 30, 1996, now pending, which claims priority from United Kingdom Patent Application No. 9503866.7 with a filing date of Jan. 30, 1995. 
    
    
     TECHNICAL FIELD 
     This invention relates to the detection of variations in human histamine 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 ART 
     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 H 2  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. Aust. 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. 5070101 and 5177081. Prell et al (1992) Abstract, part 1, 199.6 Soc. for Neurosci. Annual Meeting, Anaheim Calif. 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), 281-282. 
     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) &#34;Basic Methods in Molecular Biology&#34; 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 - Gly525          A - T     Lys - Asn620          A - G     Lys - Asp649          A - G     Asn - Asp692          A - G     Lys - Arg802          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&#39;CAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAGAGGGGATGATGCCATCAATGAGATGTTA 3&#39; 
     As a specific exemplification, the nucleotide sequence of the invention can comprise the following sequence (as also listed in SEQ ID NO: 2):5&#39;CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCATCACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTCTGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCGTGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGCCATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTACACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCAGCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCACCCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCTATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCGCACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAAACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCAGGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG3&#39; 
     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&#39; CCAATGGCACAGCCTCTT 3&#39; (as listed in SEQ ID NO: 3) 
     2) 5&#39; CGTGACTTCTGTCCCACT 3&#39; (as listed in SEQ ID NO: 4) 
     3) 5&#39; CCAGGCAACAGGAAGAGA 3&#39; (as listed in SEQ ID NO: 5) 
     4) 5&#39; TCTCTTCCTGTTGCCTGG 3&#39; (as listed in SEQ ID NO: 6) 
     5) 5&#39; GCAGCAGAAGAGCTGTTG 3&#39; (as listed in SEQ ID NO: 7) 
     6) 5&#39; TCCAGGTCAATGAAGTGT 3&#39; (as listed in SEQ ID NO: 8) 
     7) 5&#39; ACACTTCATTGACCTGGA 3&#39; (as listed in SEQ ID NO: 9) 
     8) 5&#39; CCAAGAGGATCAATCACA 3&#39; (as listed in SEQ ID NO: 10) 
     9) 5&#39; TGTGATTGATCCTCTTGG 3&#39; (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      82)      Downstream    1036 and 10953)      Upstream      9954)      Downstream    1012 (no. 3) in reverse)5)      Downstream    898 and 11716)      Upstream      5277)      Downstream    544 (no. 6) in reverse)8)      Upstream      6389)      Downstream    655 (no. 8) in reverse)______________________________________ 
    
     Information on the human histamine H 2  receptor was obtained from the MRC Daresbury database accessing &#34;Genem 61&#34; 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, CviJI525620          Eco57649          ClaI, Sau3A, TaqI692802          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 herein. The treatment response or efficacy which may be predicted may include drug treatment such as for example, use of H 2  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.d1, 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 studied, 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™ INVαF&#39; Competent cells. 
     Both strands of the cloned PCR product were sequenced using the dideoxynucleotide chain-terminated method, carried out with Sequenase 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&#39;CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCATCACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTCTGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCGTGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGCCATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTACACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCAGCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCACCCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCATCTTCAGGGTCGCCCGGGATCACGCCAAGAGGATCGATCACATTAGCTCCTGGAAGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCTATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCGCACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAAACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCAGGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG3&#39; 
     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 utilising specific restriction endonucleases. It will 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 13,000Xg 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),1X 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 Taq1 restriction endonuclease (Fermentas) and 1.5 μl of 10X 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 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 TαqI 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&#39; CCAATGGCACAGCCTCTT 3&#39; (as in SEQ ID NO:1) 
     2) upstream: 5&#39; CCAGGCAACAGGAAGAGA 3&#39; (as in SEQ ID NO:5) 
     5) downstream: 5&#39; GCAGCAGAAGAGCTGTTG 3&#39; (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.sub.2  GenotypeDiagnosis   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. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- &lt;160&gt; NUMBER OF SEQ ID NOS: 15- &lt;210&gt; SEQ ID NO 1&lt;211&gt; LENGTH: 413&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Homo sapiens&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: CDS&lt;222&gt; LOCATION: (3)...(413)- &lt;400&gt; SEQUENCE: 1- ca gct cgg gtc gcc atc tct ctg gtc tta att - # tgg gtc atc tcc att  47#Ile Trp Val Ile Ser Ileer Leu Val Leu#  15- acc ctg tcc ttt ctg tct atc cac ctg ggg tg - #g aac agc agg aac gag  95Thr Leu Ser Phe Leu Ser Ile His Leu Gly Tr - #p Asn Ser Arg Asn Glu#                 30- acc agc aag ggc aat cat acc acc tct aag tg - #c aat gtc cag gtc aat 143Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cy - #s Asn Val Gln Val Asn#             45- gaa gtg tac ggg ctg gtg gat ggg ctg gtc ac - #c ttc tac ctc ccg cta 191Glu Val Tyr Gly Leu Val Asp Gly Leu Val Th - #r Phe Tyr Leu Pro Leu#         60- ctg atc atg tgc atc acc tac tac cgc atc tt - #c agg gtc gcc cgg gat 239Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Ph - #e Arg Val Ala Arg Asp#     75- cag gcc aag agg atc gat cac att agc tcc tg - #g aag gca gcc acc atc 287Gln Ala Lys Arg Ile Asp His Ile Ser Ser Tr - #p Lys Ala Ala Thr Ile# 95- agg gag cac aga gcc aca gtg aca ctg gcc gc - #c gtc atg ggg gcc ttc 335Arg Glu His Arg Ala Thr Val Thr Leu Ala Al - #a Val Met Gly Ala Phe#               110- atc atc tgc tgg ttt ccc tac ttc acc gcg tt - #t gtg tac cgt ggg ctg 383Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Ph - #e Val Tyr Arg Gly Leu#           125#          413     cc atc aat gag atg ttaArg Gly Asp Asp Ala Ile Asn Glu Met Leu#       135- &lt;210&gt; SEQ ID NO 2&lt;211&gt; LENGTH: 1046&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Homo sapiens&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: CDS&lt;222&gt; LOCATION: (3)...(1046)- &lt;400&gt; SEQUENCE: 2- cc aat ggc aca gcc tct tcc ttt tgc ctg gac - # tct acc gca tgc aag  47#Asp Ser Thr Ala Cys Lyser Phe Cys Leu#  15- atc acc atc acc gtg gtc ctt gcg gtc ctc at - #c ctc atc acc gtt gct  95Ile Thr Ile Thr Val Val Leu Ala Val Leu Il - #e Leu Ile Thr Val Ala#                 30- ggc aat gtg gtc gtc tgt ctg gcc gtg ggc tt - #g aac cgc cgg ctc cgc 143Gly Asn Val Val Val Cys Leu Ala Val Gly Le - #u Asn Arg Arg Leu Arg#             45- aac ctg acc aat tgt ttc atc gtg tcc ttg gc - #t atc act gac ctg ctc 191Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Al - #a Ile Thr Asp Leu Leu#         60- ctc ggc ctc ctg gtg ctg ccc ttc tct gcc at - #c tac cag ctg tcc tgc 239Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Il - #e Tyr Gln Leu Ser Cys#     75- aag tgg agc ttt ggc aag gtc ttc tgc aat at - #c tac acc agc ctg gat 287Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Il - #e Tyr Thr Ser Leu Asp# 95- gtg atg ctc tgc aca gcc tcc att ctt aac ct - #c ttc atg atc agc ctc 335Val Met Leu Cys Thr Ala Ser Ile Leu Asn Le - #u Phe Met Ile Ser Leu#               110- gac cgg tac tgc gct gtc atg gac cca ctg cg - #g tac cct gtg ctg gtc 383Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Ar - #g Tyr Pro Val Leu Val#           125- acc cca gct cgg gtc gcc atc tct ctg gtc tt - #a att tgg gtc atc tcc 431Thr Pro Ala Arg Val Ala Ile Ser Leu Val Le - #u Ile Trp Val Ile Ser#       140- att acc ctg tcc ttt ctg tct atc cac ctg gg - #g tgg aac agc agg aac 479Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gl - #y Trp Asn Ser Arg Asn#   155- gag acc agc aag ggc aat cat acc acc tct aa - #g tgc aat gtc cag gtc 527Glu Thr Ser Lys Gly Asn His Thr Thr Ser Ly - #s Cys Asn Val Gln Val160                 1 - #65                 1 - #70                 1 -#75- aat gaa gtg tac ggg ctg gtg gat ggg ctg gt - #c acc ttc tac ctc ccg 575Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Va - #l Thr Phe Tyr Leu Pro#               190- cta ctg atc atg tgc atc acc tac tac cgc at - #c ttc agg gtc gcc cgg 623Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Il - #e Phe Arg Val Ala Arg#           205- gat cag gcc aag agg atc gat cac att agc tc - #c tgg aag gca gcc acc 671Asp Gln Ala Lys Arg Ile Asp His Ile Ser Se - #r Trp Lys Ala Ala Thr#       220- atc agg gag cac aga gcc aca gtg aca ctg gc - #c gcc gtc atg ggg gcc 719Ile Arg Glu His Arg Ala Thr Val Thr Leu Al - #a Ala Val Met Gly Ala#   235- ttc atc atc tgc tgg ttt ccc tac ttc acc gc - #g ttt gtg tac cgt ggg 767Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Al - #a Phe Val Tyr Arg Gly240                 2 - #45                 2 - #50                 2 -#55- ctg aga ggg gat gat gcc atc aat gag atg tt - #a gaa gcc atc gtt ctg 815Leu Arg Gly Asp Asp Ala Ile Asn Glu Met Le - #u Glu Ala Ile Val Leu#               270- tgg ctg ggc tat gcc aac tca gcc ctg aac cc - #c atc ctg tat gct gcg 863Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pr - #o Ile Leu Tyr Ala Ala#           285- ctg aac aga gac ttc cgc acc ggg tac caa ca - #g ctc ttc tgc tgc agg 911Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gl - #n Leu Phe Cys Cys Arg#       300- ctg gcc aac cgc aac tcc cac aaa act tct ct - #g agg tcc aac gcc tct 959Leu Ala Asn Arg Asn Ser His Lys Thr Ser Le - #u Arg Ser Asn Ala Ser#   315- cag ctg tcc agg acc caa agc cga gaa ccc ag - #g caa cag gaa gag aaa1007Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Ar - #g Gln Gln Glu Glu Lys320                 3 - #25                 3 - #30                 3 -#35#   1046g aag ctc cag gtg tgg agt ggg aca ga - #a gtc acgPro Leu Lys Leu Gln Val Trp Ser Gly Thr Gl - #u Val Thr#               345- &lt;210&gt; SEQ ID NO 3&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 3#  18              tt- &lt;210&gt; SEQ ID NO 4&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 4#  18              ct- &lt;210&gt; SEQ ID NO 5&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 5#  18              ga- &lt;210&gt; SEQ ID NO 6&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 6#  18              gg- &lt;210&gt; SEQ ID NO 7&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 7#  18              tg- &lt;210&gt; SEQ ID NO 8&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 8#  18              gt- &lt;210&gt; SEQ ID NO 9&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 9#  18              ga- &lt;210&gt; SEQ ID NO 10&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 10#  18              ca- &lt;210&gt; SEQ ID NO 11&lt;211&gt; LENGTH: 18&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Artificial sequence&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: Synthetic Oligonucleotide- &lt;400&gt; SEQUENCE: 11#  18              gg- &lt;210&gt; SEQ ID NO 12&lt;211&gt; LENGTH: 137&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Homo sapiens- &lt;400&gt; SEQUENCE: 12- Ala Arg Val Ala Ile Ser Leu Val Leu Ile Tr - #p Val Ile Ser Ile Thr#                 15- Leu Ser Phe Leu Ser Ile His Leu Gly Trp As - #n Ser Arg Asn Glu Thr#             30- Ser Lys Gly Asn His Thr Thr Ser Lys Cys As - #n Val Gln Val Asn Glu#         45- Val Tyr Gly Leu Val Asp Gly Leu Val Thr Ph - #e Tyr Leu Pro Leu Leu#     60- Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Ar - #g Val Ala Arg Asp Gln# 80- Ala Lys Arg Ile Asp His Ile Ser Ser Trp Ly - #s Ala Ala Thr Ile Arg#                 95- Glu His Arg Ala Thr Val Thr Leu Ala Ala Va - #l Met Gly Ala Phe Ile#           110- Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Va - #l Tyr Arg Gly Leu Arg#       125- Gly Asp Asp Ala Ile Asn Glu Met Leu#   135- &lt;210&gt; SEQ ID NO 13&lt;211&gt; LENGTH: 348&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Homo sapiens- &lt;400&gt; SEQUENCE: 13- Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Se - #r Thr Ala Cys Lys Ile#                 15- Thr Ile Thr Val Val Leu Ala Val Leu Ile Le - #u Ile Thr Val Ala Gly#             30- Asn Val Val Val Cys Leu Ala Val Gly Leu As - #n Arg Arg Leu Arg Asn#         45- Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Il - #e Thr Asp Leu Leu Leu#     60- Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Ty - #r Gln Leu Ser Cys Lys# 80- Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Ty - #r Thr Ser Leu Asp Val#                 95- Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Ph - #e Met Ile Ser Leu Asp#           110- Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Ty - #r Pro Val Leu Val Thr#       125- Pro Ala Arg Val Ala Ile Ser Leu Val Leu Il - #e Trp Val Ile Ser Ile#   140- Thr Leu Ser Phe Leu Ser Ile His Leu Gly Tr - #p Asn Ser Arg Asn Glu145                 1 - #50                 1 - #55                 1 -#60- Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cy - #s Asn Val Gln Val Asn#               175- Glu Val Tyr Gly Leu Val Asp Gly Leu Val Th - #r Phe Tyr Leu Pro Leu#           190- Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Ph - #e Arg Val Ala Arg Asp#       205- Gln Ala Lys Arg Ile Asp His Ile Ser Ser Tr - #p Lys Ala Ala Thr Ile#   220- Arg Glu His Arg Ala Thr Val Thr Leu Ala Al - #a Val Met Gly Ala Phe225                 2 - #30                 2 - #35                 2 -#40- Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Ph - #e Val Tyr Arg Gly Leu#               255- Arg Gly Asp Asp Ala Ile Asn Glu Met Leu Gl - #u Ala Ile Val Leu Trp#           270- Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Il - #e Leu Tyr Ala Ala Leu#       285- Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Le - #u Phe Cys Cys Arg Leu#   300- Ala Asn Arg Asn Ser His Lys Thr Ser Leu Ar - #g Ser Asn Ala Ser Gln305                 3 - #10                 3 - #15                 3 -#20- Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gl - #n Gln Glu Glu Lys Pro#               335- Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Va - #l Thr#           345- &lt;210&gt; SEQ ID NO 14&lt;211&gt; LENGTH: 1080&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Homo sapiens&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: CDS&lt;222&gt; LOCATION: (1)...(1077)- &lt;400&gt; SEQUENCE: 14- atg gca ccc aat ggc aca gcc tct tcc ttt tg - #c ctg gac tct acc gca  48Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cy - #s Leu Asp Ser Thr Ala#                 15- tgc aag atc acc atc acc gtg gtc ctt gcg gt - #c ctc atc ctc atc acc  96Cys Lys Ile Thr Ile Thr Val Val Leu Ala Va - #l Leu Ile Leu Ile Thr#             30- gtt gct ggc aat gtg gtc gtc tgt ctg gcc gt - #g ggc ttg aac cgc cgg 144Val Ala Gly Asn Val Val Val Cys Leu Ala Va - #l Gly Leu Asn Arg Arg#         45- ctc cgc aac ctg acc aat tgt ttc atc gtg tc - #c ttg gct atc act gac 192Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Se - #r Leu Ala Ile Thr Asp#     60- ctg ctc ctc ggc ctc ctg gtg ctg ccc ttc tc - #t gcc atc tac cag ctg 240Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Se - #r Ala Ile Tyr Gln Leu# 80- tcc tgc aag tgg agc ttt ggc aag gtc ttc tg - #c aat atc tac acc agc 288Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cy - #s Asn Ile Tyr Thr Ser#                 95- ctg gat gtg atg ctc tgc aca gcc tcc att ct - #t aac ctc ttc atg atc 336Leu Asp Val Met Leu Cys Thr Ala Ser Ile Le - #u Asn Leu Phe Met Ile#           110- agc ctc gac cgg tac tgc gct gtc atg gac cc - #a ctg cgg tac cct gtg 384Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pr - #o Leu Arg Tyr Pro Val#       125- ctg gtc acc cca gtt cgg gtc gcc atc tct ct - #g gtc ata att tgg gtc 432Leu Val Thr Pro Val Arg Val Ala Ile Ser Le - #u Val Ile Ile Trp Val#   140- atc tcc att acc ctg tcc ttt ctg tct atc ca - #c ctg ggg tgg aac agc 480Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile Hi - #s Leu Gly Trp Asn Ser145                 1 - #50                 1 - #55                 1 -#60- agg aac gag acc agc aag ggc aat cat acc ac - #c tct aag tgc aaa gtc 528Arg Asn Glu Thr Ser Lys Gly Asn His Thr Th - #r Ser Lys Cys Lys Val#               175- cag gtc aat gaa gtg tac ggg ctg gtg gat gg - #g ctg gtc acc ttc tac 576Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gl - #y Leu Val Thr Phe Tyr#           190- ctc ccg cta ctg atc atg tgc atc acc tac ta - #c cgc atc ttc aag gtc 624Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Ty - #r Arg Ile Phe Lys Val#       205- gcc cgg gat cag gcc aag agg atc aat cac at - #t agc tcc tgg aag gca 672Ala Arg Asp Gln Ala Lys Arg Ile Asn His Il - #e Ser Ser Trp Lys Ala#   220- gcc acc atc agg gag cac aaa gcc aca gtg ac - #a ctg gcc gcc gtc atg 720Ala Thr Ile Arg Glu His Lys Ala Thr Val Th - #r Leu Ala Ala Val Met225                 2 - #30                 2 - #35                 2 -#40- ggg gcc ttc atc atc tgc tgg ttt ccc tac tt - #c acc gcg ttt gtg tac 768Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Ph - #e Thr Ala Phe Val Tyr#               255- cgt ggg ctg aga ggg gat gat gcc atc aat ga - #g gtg tta gaa gcc atc 816Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Gl - #u Val Leu Glu Ala Ile#           270- gtt ctg tgg ctg ggc tat gcc aac tca gcc ct - #g aac ccc atc ctg tat 864Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Le - #u Asn Pro Ile Leu Tyr#       285- gct gcg ctg aac aga gac ttc cgc acc ggg ta - #c caa cag ctc ttc tgc 912Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Ty - #r Gln Gln Leu Phe Cys#   300- tgc agg ctg gcc aac cgc aac tcc cac aaa ac - #t tct ctg agg tcc aac 960Cys Arg Leu Ala Asn Arg Asn Ser His Lys Th - #r Ser Leu Arg Ser Asn305                 3 - #10                 3 - #15                 3 -#20- gcc tct cag ctg tcc agg acc caa agc cga ga - #a ccc agg caa cag gaa1008Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Gl - #u Pro Arg Gln Gln Glu#               335- gag aaa ccc ctg aag ctc cag gtg tgg agt gg - #g aca gaa gtc acg gcc1056Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gl - #y Thr Glu Val Thr Ala#           350#              1080ca gac agg taaPro Gln Gly Ala Thr Asp Arg   355- &lt;210&gt; SEQ ID NO 15&lt;211&gt; LENGTH: 359&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Homo sapiens- &lt;400&gt; SEQUENCE: 15- Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cy - #s Leu Asp Ser Thr Ala#                 15- Cys Lys Ile Thr Ile Thr Val Val Leu Ala Va - #l Leu Ile Leu Ile Thr#             30- Val Ala Gly Asn Val Val Val Cys Leu Ala Va - #l Gly Leu Asn Arg Arg#         45- Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Se - #r Leu Ala Ile Thr Asp#     60- Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Se - #r Ala Ile Tyr Gln Leu# 80- Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cy - #s Asn Ile Tyr Thr Ser#                 95- Leu Asp Val Met Leu Cys Thr Ala Ser Ile Le - #u Asn Leu Phe Met Ile#           110- Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pr - #o Leu Arg Tyr Pro Val#       125- Leu Val Thr Pro Val Arg Val Ala Ile Ser Le - #u Val Ile Ile Trp Val#   140- Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile Hi - #s Leu Gly Trp Asn Ser145                 1 - #50                 1 - #55                 1 -#60- Arg Asn Glu Thr Ser Lys Gly Asn His Thr Th - #r Ser Lys Cys Lys Val#               175- Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gl - #y Leu Val Thr Phe Tyr#           190- Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Ty - #r Arg Ile Phe Lys Val#       205- Ala Arg Asp Gln Ala Lys Arg Ile Asn His Il - #e Ser Ser Trp Lys Ala#   220- Ala Thr Ile Arg Glu His Lys Ala Thr Val Th - #r Leu Ala Ala Val Met225                 2 - #30                 2 - #35                 2 -#40- Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Ph - #e Thr Ala Phe Val Tyr#               255- Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Gl - #u Val Leu Glu Ala Ile#           270- Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Le - #u Asn Pro Ile Leu Tyr#       285- Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Ty - #r Gln Gln Leu Phe Cys#   300- Cys Arg Leu Ala Asn Arg Asn Ser His Lys Th - #r Ser Leu Arg Ser Asn305                 3 - #10                 3 - #15                 3 -#20- Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Gl - #u Pro Arg Gln Gln Glu#               335- Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gl - #y Thr Glu Val Thr Ala#           350- Pro Gln Gly Ala Thr Asp Arg   355__________________________________________________________________________