20110301071 | Stabilized Liquid Enzyme Composition | December, 2011 | Nielsen et al. |
20100111969 | IL-4 RECEPTOR AND IL-13 AS PROGNOSTIC MARKERS FOR COLON AND PANCREAS TUMORS | May, 2010 | Fricke et al. |
20020119458 | Novel computation with nucleic acid molecules, computer and software for computing | August, 2002 | Suyama et al. |
20070099186 | Methods and means for the treatment of disorders associated with cellular senescence | May, 2007 | D'adda Di et al. |
20010036626 | Screening assay methods and systems using target pooling | November, 2001 | Farinas et al. |
20020123098 | 55063, a novel human NMDA family member and uses thereof | September, 2002 | Curtis |
20070184533 | MICROBIAL TRANSFORMATION METHOD FOR THE PREPARATION OF AN EPOTHILONE | August, 2007 | Li et al. |
20040002151 | Antisense modulation of selenophosphate synthetase 2 expression | January, 2004 | Watt et al. |
20060286564 | Modified Pol III replicases and uses thereof | December, 2006 | Peters |
20130316335 | SAMPLE HANDLING | November, 2013 | Ross et al. |
20050170476 | Method for producing phospholipid | August, 2005 | Sakai et al. |
[0001] The present application is a Continuation-In-Part application (“CIP”) of U.S. Provisional Application Serial No. 60/029,278, filed Oct. 28, 1996. The aforementioned application is explicitly incorporated herein by reference in its entirety and for all purposes.
[0002] The present invention relates to compositions and methods for determining the genotype associated with an increased or decreased susceptibility to manic-depressive illness. The invention also provides a means to determine an individual's increased or decreased risk of developing manic-depressive illness.
[0003] Genome screening efforts by several groups, designed to identify regions linked to bipolar disorder, have revealed evidence for potential susceptibility loci on chromosome 18. Berrettini (1994)
[0004] In addition to bipolar disorder, more than 25 other diseases have been localized to chromosome 18, approximately 80% of which still await the discovery of the underlying defective gene (Overhauser et al.
[0005] In one aspect the present invention is directed to a method for determining a genotype associated with increased susceptibility to manic-depressive illness. The method comprises determining the genotype of an affected individual with at least one polymorphic marker localized within the chromosomal region defined by and including markers D18S843 and D18S869, and determining therefrom the genotype associated with increased susceptibility to manic-depressive disorder.
[0006] In preferred embodiments the polymorphic marker is amplified by primers which selectively hybridize, under stringent conditions, to the same nucleic acid sequences as primers of SEQ ID NO:1 and SEQ ID NO:2 (see Table 1, below, forward and reverse primers to amplify Clone 22). Typically the polymorphic marker is amplified by the polymerase chain reaction.
[0007] In other embodiments the method of further comprises determining the genotype of a tested individual wherein the genotype is determined with at least one polymorphic marker localized within the chromosomal region defined by and including markers D18S843 and D18S869. The genotype of the tested individual is compared to the genotype associated with increased susceptibility to manic-depressive illness and the increased or decreased risk of the tested individual developing manic-depressive illness is
TABLE 1 PCR PRIMER SEQUENCES Name Primers SEQ ID NO: Name Primers SEQ ID NO: Clone 22 F-TACAAAAGAGGACAAAGCAC 30 D18S73 F-TGCCACTGCAACAATGC 31 R-GGTGCCTGTATATAAGTTGA 32 R-CCCAGCAATCAACCTTTAAG 33 Clone 24 F-CTACAGAATAGAATACATGGCG 34 D18S869 F-TGTTTATTTGTTTGACTCAATGG 35 R-GAGCTCTGAACTGTATTCAGA 36 R-GAGTGAATGCTGTACAAACAGC 37 Clone 29 F-TCTCAGCTTACTCAACCT 38 D18S996 F-GATGGAAAGCCATTTTATTTTTC 39 R-GATGAGGTGGAACAATCAC 40 R-TCGTACTATGAAATTTTTAAGCCTT 41 GNAL F-GGTCTGTACAGTGTAATAAACC 42 FB14A10 F-CCTTCCCCTCTATTCTCAAA 43 R-CTACTGCAAAATGTGTCCTGTC 44 R-GAGCGAGACTGTCTCAAAAA 45 Clone 37 F-CACATTAGCCAGTCTGATAAAG 46 GC32001 F-GAGTTGTGGGGGGGAATAGT 47 R-AAGTTACACACAGTAGCTGA 48 R-ATACGGAGGTTGAACTAGGAAGG 49 AFMa058yg5 F-TAGATGCTATATTAGGCTGGGTCTC 50 GP4B15 F-CGGTTCTGGATTTATCAGTA 51 R-GAACTTACAGCACTGGCTCTCC 52 R-AGGGTTGCAATGAGCTGAG 53 AFMa152wg9 F-AAGAACAAAAGGTCACCTGTCA 54 IB-1114 F-GCCACACACAAATTTTTCTC 55 R-TGTCTCACCTCTGCTCACTCAT 56 R-ACAGGGTGTAAGAGGAGAGG 57 CHLC.GGA16G02 F-ATGGAAGGAXAAACAGAGGG 58 NIB-1802 F-CTGATCACATTTCATACAGC 59 R-GAACTCTTCAAGAGGGGAGC 60 R-TGTATGTGGGCTTAACTGTT 61 D18S1114 F-ATCAGTATAATGATGGATGAATCAC 62 SGC-31363 F-CTACTGGGAGGTAGGTAATCTCAG 63 R-TGAGGCAAGAGGGTCAC 64 R-GCAAAACCAACCACATCAAA 65 D18S1116 F-TCTGCCACTTTTTATGGG 66 SGC34207 F-GATCCTGTTCTTTCAGCAGG 67 R-CAATGTTTTAACTTCTAGGACAAAT 68 R-TTTAACCAGCTGGAGTGAAGG 69 D18S1150 F-GGCACAGGAAACGTGAAT 70 WI-11680 F-ACAGATACTTTTCCACGCAACA 71 R-CACAAGGATGCCAGCC 72 R-AAAAAGATGTACGGTCTGGCC 73 D18S1153 F-ATGGAGGCTCTGAGACCCTT 74 WI-13171 F-TTTTATTTGGACAAGAGAACTTGTG 75 R-CTTGCCTGATGCCTGAAAT 76 R-ATGATCAGCTCTGAGGTGCA 77 D18S1158 F-GCATCTATGCAGTGCCAAAT 78 WI-18080 F-TGGCATAAAGTTTGCAAATATCA 79 R-TCATTAGCAACAAGGATCTCC 80 R-ATACACCAAAGGAGAAGGATTAACA 81 D18S1228 F-AGACAGTTGAAAAGGACACAAATG 82 D18S1066 F-TGCTGTTGCCTCTCAGCATCTC 83 R-TGGTGATGGGACTTTTCAAA 84 R-CACCTTTCAAGTGCTTGGCAGTC 85 D18S378 F-AGCCTGGGTGACAGAGCAA 86 D18S1215 F-GTTTGCTGCATCTCCCAATT 87 R-ACAGGGAAAGCTGGGGGAT 88 R-GTGCCCACATTGTTGTGAAG 89 D18S40 F-CAAGATAGATGCATTTTCCAGT 90 D18S1299 F-TTTAAGCCTCAAGGGACCCT 91 R-CATCCAAAGGGTGAATGTGT 92 R-AGATTGAGGACCAGGTGGTG 93 D18S464 F-GCCAGACTTTGTGCCATTTC 94 D18S1226 F-CTCTTAAGTTGAGTGAAGTGGAAGC 95 R-TTTCCTGAATCTCTTGTGGTTTG 96 R-CGCAAAAGTCAGGAAAGAGG 97 D18S482 F-ATGAGTGAATGCCAACTTCG 98 SHGC-32282 F-TTACGCATTTTGTATCAGACTTACA 99 R-CCTGGCTGACAGAGTGAGT 100 R-GGTGGAGTATCAGAAGTGATTTTAG 101 D18S53 F-GGTCACCTACAACTTTGGATG 102 D18S1315 F-TGGACTTCTACCCCCATCTG 103 R-TGCATGTAAATATCAGAGTCTGTT 104 R-TTTGAAACCTGGACACTTTGG 105 D18S71 F-ACCCGCTCAAAAGCCT 106 D18S843 F-GTCCTCATCTGTAAAACGGG 107 R-TTAATGGATTATCAAGAGTGGTTCT 108 R-CCACTAACTAGTTTGTGACTTTGG 109
[0008] determined therefrom. Generally, the polymorphic marker of the tested individual is amplified by primers which selectively hybridize, under stringent conditions, to the same nucleic acid sequences as primers of SEQ ID NO:11 and SEQ ID NO:2.
[0009] In another aspect, the present invention is directed to a nucleic acid composition comprising oligonucleotide primers which selectively hybridize, under stringent conditions, to the same nucleic acid sequence as primers of SEQ ID NO:1 and SEQ ID NO:2. In an additional aspect the present invention is directed to a nucleic acid of less than 10 kB and comprising a polymorphic marker amplified by oligonucleotide primers of SEQ ID NO:1 and SEQ ID NO:2.
[0010] In yet another aspect, the present invention is directed to a method for determining an increased susceptibility to manic-depressive illness in an individual, comprising determining the genotype of the individual with oligonucleotide primers. The oligonucleotide primers amplify a polymorphic site as primers of SEQ ID NO:1 and SEQ ID NO:2. This polymorphic marker can be found in at least two forms, designated as “allele 1” of clone 22 (SEQ ID NO:14) or “allele 2” of clone 22 (SEQ ID NO:15). The presence of allele 2 of the polymorphic marker indicates an increased susceptibility to manic-depressive illness.
[0011] The invention further provides for a isolated nucleic acid encoding an IMP.18p myo-inositol monophosphatase, the protein defined as having a calculated molecular weight of between about 22 to 34 ka, and where the protein's activity includes hydrolysis of myo-inositol 1-phosphate to generate inositol and inorganic phosphate; and where the protein specifically binds to an antibody raised against an IMP.18p myo-inositol monophosphatase protein, or immunogenic fragment thereof, consisting of SEQ ID NO:17; or, having at least 60% amino acid sequence identity to an IMP.18p myo-inositol monophosphatase protein consisting of SEQ ID NO:17, as measured using a sequence comparison algorithm. In one embodiment, the nucleic acid encodes a IMP.18p myo-inositol monophosphatase having a calculated molecular weight of about 28 to 29 kDa. In other embodiments, the isolated nucleic acid; encodes a protein which has at least 80% amino acid sequence identity to the IMP.18p myo-inositol monophosphatase protein of SEQ ID NO:17. as measured using a sequence comparison algorithm; encodes a protein having the sequence set forth in SEQ ID NO:17; specifically hybridizes to SEQ ID NO:16 under stringent conditions; or, encodes an IMP.18p myo-inositol monophosphatase protein which specifically binds to an antibody directed against a protein having a sequence as set forth in SEQ ID NO:17.
[0012] In further embodiments, the invention also provides for a polynucleotide or fragment thereof comprising a purified antisense nucleotide capable of hybridizing to and having a nucleic acid sequence complementary to at least a portion of an IMP.18p myo-inositol monophosphatase polynucleotide. The invention also provides for an expression vector comprising a nucleic acid encoding an IMP.18p myo-inositol monophosphatase or its antisense sequence. Further embodiments provide for a cell comprising an exogenous nucleic acid sequence encoding an IMP.18p myo-inositol monophosphatase protein. Another embodiment provides for an organism into which an exogenous nucleic acid sequence which specifically hybridizes under stringent conditions to SEQ ID NO:16 or which comprises a nucleic acid encoding an IMP.18p myo-inositol monophosphatase or fragment thereof, has been introduced, and the organism expresses the exogenous nucleic acid as an IMP.18p myo-inositol monophosphatase protein, or fragment thereof. In one embodiment, the organism's exogenous nucleic acid sequence is translated into an IMP.18p myo-inositol monophosphatase protein which is expressed externally from the organism.
[0013] The invention also provides for an isolated IMP.18p myo-inositol monophosphatase protein having a calculated molecular weight of about 22 to 34 kDa; where the protein's activity includes hydrolysis of myo-inositol 1-phosphate to generate inositol and inorganic phosphate; and specifically binds to an antibody raised against a myo-inositol monophosphatase protein, or immunogenic fragment thereof, consisting of SEQ ID NO:17, or has at least 60% amino acid sequence identity to a myo-inositol monophosphatase protein consisting of SEQ ID NO:17, as measured using a sequence comparison algorithm. In one embodiment, the isolated IMP.18p myo-inositol monophosphatase protein can also be found in humans. In further embodiments, the isolated IMP.18p myo-inositol monophosphatase protein has a calculated molecular weight of about 28 to 29 kDa; or, has a sequence as set forth in SEQ ID NO:17.
[0014] The invention further provides for an isolated antibody which is specifically immunoreactive under immunologically reactive conditions to an IMP.18p myo-inositol monophosphatase protein having the sequence as set forth in SEQ ID NO:17. In another embodiment, the isolated antibody is specifically immunoreactive under immunologically reactive conditions to an IMP.18p myo-inositol monophosphatase protein encoded by a IMP.18p myo-inositol monophosphatase nucleic acid of the invention.
[0015] Also provided for in the invention is a pharmaceutical composition comprising an acceptable carrier and an IMP.18p myo-inositol monophosphatase protein; an anti-IMP.18p myo-inositol monophosphatase antibody or binding fragment thereof; or a polynucleotide encoding an IMP.18p myo-inositol monophosphatase protein.
[0016] The invention also provides for a method for quantifying the amount of a myo-inositol monophosphatase in a mammal, comprising: obtaining a cell or tissue sample from the mammal; and, determining the amount of an IMP.18p myo-inositol monophosphatase gene product in the cell or tissue.
[0017] Another embodiment provides for a method for detecting the presence of a polynucleotide sequence encoding at least a portion of an IMP.18p myo-inositol monophosphatase in a biological sample, comprising the steps of providing a biological sample suspected of containing a IMP.18p myo-inositol monophosphatase-encoding nucleic acid and a probe capable of hybridizing to at least a portion of an IMP.18p myo-inositol monophosphatase nucleotide sequence, or a fragment thereof, from a biological sample; then combining the nucleic acid-containing biological sample with the probe under conditions such that a hybridization complex is formed between the nucleic acid and the probe; and detecting the hybridization complex. In one embodiment the nucleic acid in the biological sample is ribonucleic acid. In another embodiment, the detected hybridization complex correlates with expression of an IMP.18p myo-inositol monophosphatase in the biological sample.
[0018] The invention also provides for a method of determining whether a test compound is a modulator of an IMP.18p myo-inositol monophosphatase activity, the method comprising the steps of: providing a composition comprising an IMP.18p myo-inositol monophosphatase protein; contacting the monophosphatase with the test compound; and measuring the activity of the monophosphatase, wherein a change in monophosphatase activity in the presence of the test compound is an indicator of whether the test compound modulates monophosphatase activity. In one embodiment, the composition comprises monophosphatase is encoded a an IMP.18p myo-inositol monophosphatase polypeptide of the invention. In further embodiments, the composition comprises a cell or an organism.
[0019]
[0020]
[0021]
[0022]
[0023]
[0024]
[0025]
[0026]
[0027] In the present invention, a region of chromosome 18 has been identified that is tightly linked to a locus associated with susceptibility to manic-depressive illness, including affective disorders. Linkage disequilibrium between a particular form of a marker in the population and the presence of the manic-depressive illness provides a means to determine the increased susceptibility of an individual to manic-depressive illness. Accordingly, the methods and compositions of the present invention provide a means to alert clinicians to a genetic predisposition towards developing manic-depressive illness. The methods of the invention are useful in genetic counseling of individuals from families affected with manic-depressive illness, and aid in the differential diagnosis of manic-depressive illness from other psychiatric pathologies.
[0028] A susceptibility region for bipolar disorder has been found on the pericentromeric portion of chromosome 18 (Berrettini (1994)
[0029] This novel, full-length cDNA, designated IMP.18p (alternatively designated IMPA2), was isolated and sequenced (SEQ ID NO:16, see
[0030] The invention also provides for novel anti-IMP.18p reagents in the form of anti-IMP.18p antibodies and IMP.18p-encoding nucleic acids to identify polymorphic variants of IMP.18p within the scope of the claimed invention. Use these novel reagents in various antibody-based and nucleic acid-based assays to clearly describe the identification and isolation of such polymorphic variants are described below.
[0031] To provide a more precise location of this gene, mapping with a panel of radiation hybrids (RH) was conducted. Multipoint RH analysis placed the gene between GNAL and D18S71 within the 18p11.2 region (see
[0032] Lithium is the most commonly prescribed medication and effective treatment for manic depression/bipolar disorder. Its therapeutic action is in part mediated through the inhibition of IMP, an enzyme which has a crucial role in the phosphatidylinositol signaling pathway (reviewed in Atack (1996) “Inositol monophosphatase, the putative therapeutic target for lithium.”
[0033] As IMP is a molecular target for the therapeutic effects of lithium, inhibitors of IMP can be lithium-mimetics. Thus, the novel IMP.18p of the invention, which is distantly related to inositol monophosphatase enzymes, can be used to not only to identify inhibitors specific for IMP.18p, but also as a novel means to identify and isolate new inhibitors of IMPs as alternatives to lithium.
[0034] In disease states associated with increased levels of IMP activity, such as bipolar disease, the enzymatic activity and levels of IMP.18p is altered in specific brain areas. Thus, the IMP.18p nucleic acid sequence of the invention provides for novel means to measure levels of IMP and diagnose the corresponding disease state.
[0035] Because of the location and function of IMP.18p, it qualifies as a novel target for diagnosis, therapeutics and molecular scanning, i.e., identification of mutations, polymorphisms and further members of this new myo-inositol monophosphatase enzyme family.
[0036] Definitions
[0037] Units, prefixes, and symbols can be denoted in their SI accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[0038] As used herein, “manic-depressive illness” and bipolar disorder, including bipolar I (BPI) and bipolar II (BPII), refer to the same phenotype and can be used interchangeably. Manic depressive disorder includes reference to schizoaffective disorder, or recurrent Major Depressive Illness (i.e., recurrent unipolar illness). See, “Research Diagnostic Criteria,” Spitzer et al.
[0039] As used herein, “marker” includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A “polymorphic marker” includes reference to a marker which can appear in multiple forms, i.e., these different forms sometimes referred to as “alleles” (alleles are defined as different variations of a gene or marker). Different forms of the marker can be used to follow their transmission from parent to child and throughout generations (when they are present in a homologous pair, allow transmission of each of the chromosomes in that pair to be followed).
[0040] A genotype may be defined by use of a single or a plurality of markers.
[0041] As used herein, “chromosomal region” includes reference to a length of chromosome which may be measured by reference to the linear segment of DNA which it comprises. The chromosomal region can be defined by reference to two unique DNA sequences, i.e. markers.
[0042] As used herein, “genotype associated with increased susceptibility to manic-depressive illness” includes reference to a genotype which has a higher probability of occurrence in a manic-depressive illness affected individual than in members of the general United States population who are past the age of onset but unaffected by manic-depressive illness.
[0043] As used herein, “increased” means greater than that of the U.S. population average. Thus, an increased susceptibility to manic-depressive illness includes reference to a greater risk of developing manic-depressive illness than the average risk for the U.S. population.
[0044] As used herein, “decreased” means less than that of the U.S. population average. Thus, a decreased susceptibility to manic-depressive illness includes reference to a lesser risk of developing manic-depressive illness than the average risk for the U.S. population.
[0045] As used herein, “determining” the “risk of the tested individual developing familial manic-depressive illness” means ascertaining the probability of the tested individual developing manic-depressive illness after the individual reaches the age of onset. The determination of risk may be a quantitatively assessed or may be assessed qualitatively as higher, lower, or equivalent to the average risk to the U.S. population.
[0046] As used herein, “tested individual” includes reference to a human whose genotype is being determined. The tested individual may be pre- or post-partum.
[0047] As used herein, “localized within the chromosomal region defined by and including” with respect to particular markers includes reference to a contiguous length of a chromosome delimited by and including the stated markers.
[0048] As used herein, “manic-depressive illness genotype” includes reference to a genotype determined with at least one polymorphic marker within the chromosomal region defined by markers linked to the locus associated with susceptibility to manic-depressive illness. Preferably, the genotype is deter-mined using polymorphic markers within 5 centimorgans of the polymorphic marker defined by SEQ ID NO:1 and SEQ ID NO:2. In a preferred embodiment, the chromosomal region is defined (flanked) by and includes chromosomal markers D18S843 and D18S869. In a particularly preferred embodiment, the genotype is determined using the marker amplified by oligonucleotide primers of SEQ ID NO:1 and SEQ ID NO:2 (Table 1).
[0049] As used herein, “isolated.” “purified” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment. Purity and homogeneity are typically determined using analytical chemistry techniques, e.g., sequence analysis, gel electrophoresis or high performance liquid chromatography (HPLC). A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated IMP.18p or clone 22 nucleic acid is separated from open reading frames which flank the IMP.18p or clone 22 gene and encode proteins other than IMP.18p or clone 22. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
[0050] As used herein, “nucleic acid,” “polynucleotide,” or “nucleic acid sequence” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al.,
[0051] As used herein, “encoding” with respect to a specified nucleic acid, includes reference to nucleic acids which comprise the information for translation into the specified protein. The information is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium
[0052] As used herein, “having amino acid (or nucleic acid) sequence identity as measured using a sequence comparison algorithm” means optimal alignment of sequences for comparison using any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method of termed “PILEUP” (see below); by the local homology algorithm of Smith & Waterman,
[0053] One example, PILEUP, creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle,
[0054] Another example of algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul et al.
[0055] The BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul,
[0056] A “comparison window”, as used herein, includes reference to a segment of about 10 to 20 residues in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981)
[0057] By “selectively hybridizing to,” “specifically hybridizing to” or “selective hybridization” is meant hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree than its hybridization to non-target nucleic acid sequences. Specifically, as used herein, a specific or selective hybridization reaction (which is, by definition, under stringent hybridization conditions) will be at least about 10 times greater than the background signal or noise. Generally, selectively hybridizing primer sequences yield an amplicon composition which can comprise at least 90% of the target amplicon. Selectively hybridizing sequences can have at least about 80% sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other. “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window (10-20 nucleotides), wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
[0058] As used herein, “stringent conditions” includes reference to conditions under which a nucleic acid sequence, such as a probe, will preferentially hybridize to its target sequence and/or hybridize to its target sequence to the substantial exclusion of non-target sequences. As defined herein, a specific or selective hybridization reaction under stringent hybridization conditions will be at least about 5 to 10 times greater than the background signal or noise. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 2×SSC at 50° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. “Stringent hybridization conditions” or “stringent conditions” in the context of nucleic acid hybridization assay formats are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993)
[0059] As used herein, “antibody composition” includes reference to at least one antibody. In turn, “antibody” includes reference to an immunoglobulin molecule obtained by in vitro or in vivo generation of the humoral response, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and recombinant single chain Fv fragments (scFv). The term “antibody” also includes antigen binding forms of antibodies (e.g., Fab′, F(ab′)
[0060] As used herein, “specifically reactive” includes reference to the preferential association of a ligand, in whole or part, with a particular target molecule (i.e., “binding partner” or “binding moiety”) relative to compositions lacking that target molecule. As defined herein, a specific or selective binding reaction will be at least about 10 times greater than the background signal or noise. It is, of course, recognized that a certain degree of non-specific interaction may occur between a ligand and a non-target molecule. Nevertheless, specific binding, may be distinguished as mediated through specific recognition of the target molecule. Typically specific binding results in a much stronger association between the ligand and the target molecule than between the ligand and non-target molecule. Specific binding by an antibody to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. The affinity constant of the antibody binding site for its cognate monovalent antigen is at least between 10
[0061] A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein and its polymorphic variants, as discussed in detail below. Solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunorcactive with a protein (see, e.g., Harlow & Lane,
[0062] An “immunogen” or “immunogenic fragment” refers to a compound or composition comprising a carbohydrate, peptide, polypeptide or protein which is “immunogenic,” i.e., capable of eliciting, augmenting or boosting a cellular and/or humoral immune response, either alone or in combination or linked or fused to another substance. An immunogenic composition can be a peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, or preferably, the a fragment 15 amino acids in length and more preferably a fragment 20 amino acids in length or greater. The immunogen (immunogenic fragment) can comprise a “carrier” polypeptide and a hapten (e.g., a carrier polypeptide fused or linked (chemically or otherwise) to a peptide/protein fragment against which the desired antibody will specifically recognize). The immunogen can be recombinantly expressed in an immunization vector, which can be simply naked DNA comprising the immunogen's coding sequence operably linked to a promoter. The immunogen (immunogenic fragment) includes antigenic determinants, or epitopes (described below), to which antibodies or TCRs bind, which are typically 3 to 10 amino acids in length. An “immunological carrier” is an composition which, when linked, joined, chemically coupled or fused to a second composition (e.g., protein, peptide, polysaccharide or the like) boosts or augments the cellular or humoral response to the composition. Any physiologic mechanism can be involved in this augmentation or boosting of the immune response. An immunogenic carrier is typically a polypeptide linked or fused to a second composition of interest—the immunogenic fragment—comprising a protein, peptide or polysaccharide, where the carrier stimulates a cellular (T cell mediated) immune response that boosts or augments the humoral (B cell mediated, antibody-generating) immune response to the composition of interest. These second compositions can be “haptens,” which are typically defined as compounds of low molecular weight that are not immunogenic by themselves, but that, when coupled to carrier molecules, can elicit antibodies directed to epitopes on the hapten. Alternatively, an immunogenic fragment can be linked to a carrier simply to facilitate manipulation of the peptide in the generation of the immune response (see, for example, Rondard (1997)
[0063] The term “immunologically reactive conditions” refers to any environment in which antibodies can bind to antigens, such as the IMP.18p of the invention or immunogenic fragments thereof. These conditions can be physiologic conditions similar to those seen in vivo, or, in vitro conditions compatible with antibody-antigen binding, such as in an immunological binding assay.
[0064] As used herein, “polypeptide”, “peptide” and “protein” are used interchangeably and include reference to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The amino acids and analogs referred to herein are described by shorthand designations as follows:
Amino Acid Nomenclature Name 3-letter 1 letter Alanine Ala A Arginine Mg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Homoserine Hse — Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Methionine sulfoxide Met (O) — Methionine Met (S-Me) — methylsulfonium Norleucine Nle — Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0065] Those of ordinary skill will readily understand that proteins of the present invention embrace minor variants of the isoforms of clone 22 SEQ ID NO:3 and SEQ ID NO:4; and, IMP.18p proteins. Accordingly, the present invention embraces conservatively modified variants of the clone 22 and IMP.18p proteins and substantially similar variants of clone 22 and IMP.18p proteins. The following six groups each contain amino acids that are conservative substitutions for one another:
[0066] 1) Alanine (A), Serine (S), Threonine (T);
[0067] 2) Aspartic acid (D), Glutamic acid (E);
[0068] 3) Asparagine (N), Glutamine (O);
[0069] 4) Arginine (R), Lysine (K);
[0070] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
[0071] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0072] See also, Creighton (1984) Proteins W. H. Freeman and Company.
[0073] One of ordinary skill will recognize that individual substitutions, deletions or additions to a protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
[0074] As used herein, “calculated molecular weight” of a polypeptide or peptide is the molecular weight based on the polypeptide's or peptide's deduced amino acid sequence—the deduced translation product—as encoded by the corresponding nucleic acid. In contrast, the “apparent” molecular weight is measured, empirical value. The apparent molecular weight of a protein can be determined by many different methods, all known to one of skill in the art. Some methods of determination include: SDS gel electrophoresis, native gel electrophoresis, molecular exclusion chromatography, zonal centrifugation, mass spectroscopy. Disparity between results of different techniques can be due to factors inherent in the technique. For example, native gel electrophoresis, molecular exclusion chromatography and zonal centrifugation depend on the size of the protein. The proteins that are cysteine rich can form many disulfide bonds, both intra- and intermolecular. SDS gel electrophoresis depends on the binding of SDS to amino acids present in the protein. Some amino acids bind SDS more tightly than others, therefore, proteins will migrate differently depending on their amino acid composition. Mass spectroscopy and calculated molecular weight from the sequence in part depend upon the frequency that particular amino acids are present in the protein and the molecular weight of the particular amino acid. If a protein is glycosylated, mass spectroscopy results will reflect the glycosvlation but a calculated molecular weight may not.
[0075] As used herein, “recombinant” includes reference to a protein produced using cells that do not have in their native form an endogenous copy of the DNA able to express the protein. The cells produce the recombinant protein because they have been genetically altered by the introduction of the appropriate isolated nucleic acid sequence. The term also includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
[0076] As used herein, “encoding” with respect to a specified nucleic acid, includes reference to nucleic acids which comprise the information for translation into the specified protein. The information is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium
[0077] As used herein, “immunologically cross-reactive” or “immunologically reactive” includes reference to an antigen which is specifically reactive with an antibody which was generated using the same (“immunologically reactive”) or different (“immunologically cross-reactive”) antigen.
[0078] As used herein, “isoform” includes reference to a family of functionally related proteins that differ in their amino acid sequences but are derived from the same nuclear transcript.
[0079] The term “modulator” refers to any synthetic or natural compound or composition that can change in any way activity of protein of the invention, including IMP.18p or clone 22 proteins. An modulator can be an agonist or an antagonist. A modulator can be, but is not limited to, any organic and inorganic compound; including, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids and the like.
[0080] Method of Determining Increased Susceptibility to Manic-Depressive Illness
[0081] The present invention is directed to a method for determining a genotype associated with increased susceptibility to manic-depressive illness. The method comprises determining the genotype of a human individual diagnosed as manic-depressive. Methods of genotyping are well known to those of ordinary skill in the art. The genotype is determined using at least one polymorphic marker from within the region of chromosome 18 localized by and including the markers D18S843 and D18S869, see
[0082] Primers for polymorphic markers within this region of chromosome 18, including the markers D18S843 and D18S869, are publicly available on the internet. See, for example, The Genome Database at URL: http://gdbwww.gdb.org/; National Center for Biotechnology Information at URL: http://www.ncbi.nim.nih.gov/SCIENCE96/(cited in
[0083] In preferred embodiments, genotyping within the interval of chromosome 18 localized by markers D18S843 and D18S869 (see TABLE 3 Infant brain derived cDNA clones mapping to chromosome 18. Clone Our Insert dhEST Insert GenBank Accession Number Gene EST Cytogenetic Number Size (kB) Size (kb) 5° 3° Homology* Homology* Bin 1 2.4 1.7 R51685 R51596 HK63KDAP NA M 2 1.4 NA R61592 R61536 unknown EST64032 M 3 1.4 2.1 T77800 R38384 HS63KDAP NA M 4 1.4 1.6 R56762 R56915 unknown unknown M 5 1.2 1.4 H08437 H08745 unknown unknown S 6 1.5 1.5 R54360 R54361 unknown unknown S 7 1.4 1.9 T78290 R37939 MBP NA S 8 1.3 2.4 R20367 R43753 unknown unknown M 9 1.2 1.2 R18592 R41672 unknown EST197262 S 10 1.3 1.4 R18875 R37298 HS63KDAP NA M 11 1.5 1.5 R34535 R49065 PTPRM NA A 12 1.9 2.0 H17695 H17080 MBP NA S 13 1.7 1.9 R52596 R52541 unknown unknown L 14 1.8 2.0 R13520 R20642 unknown unknown A 15 1.4 NA R16321 R41398 unknown EST228925 M 16 1.7 NA H08970 H09539 unknown unknown S 17 1.6 2.1 R17799 R43004 unknown EST64032 M 18 1.5 1.0 R22831 R46021 MBF NA S 19 2.0 2.8 R14016 R39139 unknown unknown M 20 1.1 1.3 R11914 R39106 unknown EST197262 S 21 1.5 2.0 R19053 R44040 unknown EST228925 M 22 1.1 1.2 R19445 R44696 unknown unknown C 23 1.1 1.2 T80229 R38716 unknown D135928E S 24 1.2 1.5 R35001 R49388 unknown EST91427 A 25 1.3 2.1 R17655 R43373 unknown unknown M 26 1.0 1.2 R20441 R44144 unknown EST197262 S 27 1.3 1.4 R19332 R44600 unknown EST91427 A 28 1.8 NA H08354 H08355 unknown EST91427 A 29 1.8 1.7 none R39845 unknown unknown B 30 1.3 1.4 R52394 R52395 unknown EST30984 M 31 1.1 1.3 H17749 H17636 GNAL NA B 32 1.2 1.2 H06013 H05964 unknown EST91427 A 33 2.7 1.3 T74001 T87210 unknown unknown M 34 1.9 1.3 T80579 R38876 unknown unknown A 35 1.4 NA R60481 R60245 unknown EST91427 A 36 1.2 NA R59504 R59505 unknown unknown K 37 1.9 2.1 R20248 R43704 unknown unknown B 38 1.1 1.1 H08492 H08770 unknown unknown M 39 1.6 1.7 H11689 H11600 unknown unknown G 40 1.7 1.3 R19498 R43846 HUMKIAAN NA N 41 1.6 2.0 H17610 H17501 unknown unknown S 42 1.8 5.3 R17567 R42907 unknown EST91427 A 43 1.5 1.5 R20380 R43767 unknown unknown S 44 1.6 1.6 H17267 H17268 unknown EST91427 A 45 1.4 1.4 T80517 R38994 PTPRM NA A 46 1.6 1.4 R20075 none MBP NA S 47 1.2 1.3 T66113 T65029 unknown unknown S 48 1.3 1.3 R15279 none unknown EST91427 A
[0084] As will be recognized by those of skill, the complementary sequences of these primers may likewise be employed for amplifying or selectively hybridizing and detecting their target marker. Additional target regions may be identified by walking from known chromosome markers as described above. Techniques for chromosome walking are well known in the art as described in Sambrook et al.,
[0085] New markers may result from physical mapping of the interval defined by (flanked by) markers D18S843 and D18S869, see
[0086] Markers from within the region localized by and including markers D18S843 and D18S869 are linked to a locus associated with susceptibility to manic-depressive illness (bipolar disorder). Linkage disequilibrium between a polymorphism from this region and the appearance of manic-depressive illness provides a means of associating the appearance of that polymorphism in an individual with an increased susceptibility to manic-depressive illness. Consequently, a polymorphism exhibiting linkage disequilibrium with the appearance of manic-depressive illness can be used as a standard against which an increased susceptibility to manic-depressive illness can be determined for an individual whose disease status is unknown.
[0087] In the present method, a statistically significant correlation between the presence of a particular polymorphism with the presence of manic-depressive illness in an individual allows for the determination of the genotype(s) associated with increased or decreased susceptibility to familial manic-depressive illness. In a preferred embodiment, the transmission disequilibrium test (TDT) is employed to determine a genotype associated with increased susceptibility to manic-depressive illness. See, Spieiman et al.,
[0088] The genotype of the tested individual can be conveniently determined with at least one polymorphic marker localized within the chromosomal region defined (flanked) by and including markers D18S43 and D18S869 (
[0089] Methods of amplifying sequences are well known to those of ordinary skill in the art. Amplification systems include the polymerase chain reaction (PCR) system, strand displacement amplification (SDA), see, e.g.,
[0090] The PCR process is well-known in the art and is thus not described in detail herein. For a review of PCR methods and protocols, see, e.g., Innis, et al, eds.
[0091] In a preferred embodiment of the PCR process, strand separation is achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase. Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically DATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering system. Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis. The methods of the present invention may be performed on a wide variety of human cells including somatic cell hybrids, purified nuclei, chromosomal preparations or nucleic acid sequences comprising a marker to a chromosomal region of the present invention. The cells may be somatic or germline and from any time in gestation including fertilized embryo or preimplantation blastocysts. Preferably, somatic cells are employed to avoid the possibility of meiotic recombination events between a marker and locus associated with susceptibility to manic-depressive illness and to more readily allow determination of the genotype for a homologous chromosome pair.
[0092] The methods of the present invention may conveniently be practiced with markers which differ as to sequence or length, such as RFLPs (restriction fragment length polymorphisms) and microsatellite markers such as STRPs (short tandem repeat polymorphisms) or VNTRs (variable number tandem repeats). Generally, the sizes will be determined by standard gel electrophoresis techniques as described in Sambrook et al.,
[0093] Amplification of markers is generally performed with labeled nucleotide bases that provide a means for identifying the amplified product following the procedure. Alternatively, labeled nucleic acid primers can be employed as probes.
[0094] Probes can be used to selectively hybridize and detect and isolate a nucleic acid sequence (e.g., a cDNA or gene) of interest. For example, labeled probes can be used to detect RFLP markers which differ in size after digestion with one or more restriction enzymes which have been separated, as by electrophoresis.
[0095] Where the nucleic acid encoding a clone 22 or IMP.18p protein is to be used as a nucleic acid probe, it is often desirable to label the nucleic acid with detectable labels. The labels may be incorporated by any of a number of means well known to those of skill in the art. The label can be simultaneously incorporated during the amplification procedure in the preparation of the nucleic acids. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In another preferred embodiment, transcription amplification using a labeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.
[0096] Alternatively, a label may be added directly to an original nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the amplification is completed. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example nick translation or end-labeling (e.g., with a labeled RNA) by phosphorylation of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).
[0097] Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate. Probes may be labeled with visual labels such as photoluminescents, Texas red, rhodamine and its derivatives, red leuco dye and 3,3′,5,5°-tetramethylbenzidine (TMB), fluorescein and its derivatives, dansyl, umbelliferone and the like. Enzymes such as horse radish peroxidase, alkaline phosphatase, or equivalents can be used, especially in ELISAs. Magnetic beads, fluorescent dyes (e g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g.,
[0098] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
[0099] Those of skill will recognize that polymorphic markers within the region localized within and including D18S843 and D18S869 can be identified by variations at the protein level when the polymorphism occurs within a coding region. The present invention includes the use of polymorphisms which manifest themselves at both the nucleic acid and protein sequence levels. Accordingly, means of distinguishing polymorphisms include, but are not limited to, differences arising from antigenicity, substrate specificity, or activity of encoded proteins.
[0100] Isolation of nucleic acids from biological samples for use in the present invention may be carried out by a variety of means well known in the art. For example, see those described in Rothbart et al. 1989, in
[0101] In another aspect, the present invention provides a method for determining an increased susceptibility to manic-depressive illness in an individual. Due to linkage disequilibrium the presence of allele 2 (SEQ ID NO:15) of the clone 22 polymorphism appears more frequently amongst individuals in the U.S. population who have increased susceptibility to manic-depressive illness than individuals who lack this allele. Consequently, the presence of allele of clone 22 is itself determinative of an increased susceptibility to manic-depressive illness. The tested individual may be a member of any racial or ethnic group, including, for example, individuals of European, African, or Asian descent. In preferred embodiments, the tested individual is of European descent. The method comprises determining the genotype of the individual using the polymorphic marker of clone 22. The polymorphic marker of clone 22 can be amplified with oligonucleotide primers which amplify the same polymorphic marker as primers of SEQ ID NO:1 and SEQ ID NO:2. Use of such primers on a target comprising allele 1 yields the nucleic acid having the sequence shown in SEQ ID NO:14. The allele 1 polymorphism consists of 10 trinucleotide (GCT) repeats. Use these same primers with a target nucleic acid of allele 2 yields the nucleic acid having the sequence shown in SEQ ID NO:15. The allele 2 polymorphism consists of 9 trinucleotide (GCT) repeats. Thus, primers of the present invention will amplify the region of the trinucleotide repeat polymorphism of clone 22. Those of skill will recognize that the priming of a target sequence is performed under stringent conditions such that the primers selectively hybridize to their target sequence. Preferably, the primers employed to amplify the polymorphism of clone 22 comprise the sequence of SEQ ID NO:1 and SEQ ID NO:2. The primers of SEQ ID NO:1 and SEQ ID NO:2 may comprise additional sequences to aid in such processes as purification, labeling, or subcloning. The use of additional 5′ terminal sequences (i.e. tails) or 5′ labels is well known to the skilled artisan.
[0102] Nucleic Acid and Protein Compositions
[0103] The invention provides for novel nucleic acids, and proteins encoded therefrom, derived from a specific area of human chromosome 18. Genetic variations in this chromosomal region have been shown to be associated with manic depressive illness, including bipolar disease, making these nucleic acids and proteins useful as diagnostic markers and targets for preventive and therapeutic treatments. Specific embodiments include novel nucleic acids and proteins identified as clone 22 and IMP.18p, both of which are encoded in this chromosome 18 region. These and other sequences within the region localized by and including markers D18S843 and D18S869. being linked to a locus associated with susceptibility to manic-depressive illness, are also used as diagnostic markers in the invention. The invention provides for novel nucleic acid and antibody reagents used to identify and isolate these nucleic acids sequences and proteins, The invention also provides for characterization and isolation of related species of clone 22 and IMP18.p using the novel reagents of the invention.
[0104] For example, one embodiment provides for a method for detecting the presence of, and thereby isolating, a polynucleotide sequence encoding at least a portion of an IMP.18p myo-inositol monophosphatase in a biological sample, comprising the steps of reacting a biological sample suspected of containing an IMP.18p nucleic acid with a probe comprising a nucleotide sequence of an IMP.18p, or a fragment thereof, capable of hybridizing to a myo-inositol monophosphatase-encoding nucleic acid from the biological sample. Embodiments which provide for a means of detecting these novel nucleic acids or proteins thus also provide means to diagnosing a myo-inositol monophosphatase-related conditions in a mammal. These methods comprise obtaining a cell or tissue sample from the mammal; determining the amount of an gene product in the cell or tissue; and comparing the amount of the gene product in the cell or tissue with the amount in a healthy cell or tissue of the same type; wherein a different amount of gene product in the sample from the mammal and the healthy cell or tissue is diagnostic of a myo-inositol monophosphatase-related condition.
[0105] On another embodiment, the invention provides for clone 22 nucleic acid and protein encoded therefrom. The common subsequence of the native (naturally occurring) clone 22 mRNA transcript is shown in DNA form as SEQ ID NO:6. This common sequence is expressed with one of two different 5′ untranslated regions, SEQ ID NO:12 or SEQ ID NO:13. The present invention includes isolated nucleic acids comprising the common sequence, the 5′ untranslated regions of SEQ ID NO:12 and SEQ ID NO:13, and subsequences thereof.
[0106] Two isoforms of clone 22 proteins are provided herein. The present invention includes these isolated proteins and subsequences thereof. One isoform of a clone 22 protein has the amino acid sequence shown in SEQ ID NO:3. The present invention provides isolated nucleic acids comprising a nucleic acid encoding the clone 22 protein of SEQ ID NO:3 and subsequences thereof. The present invention also provides isolated proteins comprising the amino acid sequence shown SEQ ID NO:3 and subsequences thereof.
[0107] The second isoform of the clone 22 protein comprises the amino acid sequence of SEQ ID NO:3 but lacks the amino acid sequence from position 113 to 130 (i.e., EGCLWPSDSAAPRLGASE) (SEQ ID NO:5). The second isoform has the protein sequence shown in SEQ ID NO:4. The present invention includes isolated nucleic acids comprising a nucleic acid encoding the alternatively spliced clone 22 protein of SEQ ID NO:4 and subsequences thereof. The present invention also provides isolated proteins comprising the amino acid sequence shown in SEQ ID NO:4 and subsequences thereof. Thus, the present invention provides nucleic acids (“clone 22 nucleic acids”) and proteins (“clone 22 proteins”) which include both full-length and subsequences of isolated native nucleic acids and proteins of clone 22.
[0108] With the amino acid sequences of the clone 22 and IMP.18p proteins provided herein, one of skill can readily construct a variety of clones containing nucleic acids which encode the same protein but vary in nucleic acid sequence due to the degeneracy of the genetic code. Cloning methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook, et al.,
[0109] In some embodiments the isolated nucleic acids of the present invention comprise the sequence shown in SEQ ID NO:6 from nucleotide 116 to 1033 (i.e., the sequence coding for the protein of SEQ ID NO:3); this nucleic acid is identified herein as SEQ ID NO:7. In other embodiments nucleic acids of the present invention comprise the sequence shown in SEQ ID NO:6 from nucleotide 116 to 1033 but lacking the sequence from nucleotide 452 to 505 corresponding to the region from Glu 113 to Glu 130 (i.e., lacking the region coding for the protein of SEQ ID NO:5); this nucleic acid is identified herein as SEQ ID NO:8.
[0110] A nucleic acid encoding the protein of SEQ ID NO:3 or SEQ ID NO:4 can be amplified from human brain cDNA libraries using primers which selectively hybridize, under stringent conditions, to the same nucleic acid sequence as primers of SEQ ID NO:9 and SEQ ID NO:10. Thus, for example, isolated nucleic acids encoding the isolated proteins of SEQ ID NO:3 or SEQ ID NO:4 can be amplified using oligonucleotide primers which selectively hybridize, under stringent conditions, to the same nucleic acid sequences of SEQ ID NO:7 and SEQ ID NO:8, respectively, as primers of SEQ ID NO:9 and SEQ ID NO:10.
[0111] The IMP.18p nucleic acid sequence (SEQ ID NO:16) and protein sequence information (SEQ ID NO:17) can be used to design PCR primers which can be used to identify related IMP species, such as: SEQ ID NO:18 and SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:21; and, SEQ ID NO:22 and SEQ ID NO:23, can be used to directly amplify IMP species. The SEQ ID NO:18 (forward) and SEQ ID NO:19 (reverse) primer pair amplifies full length IMP.18p cDNA protein coding sequence:
5′-ATG AAG CCG AGC GGC GAG GAG-3′ (SEQ ID NO:18) 5′-CTT CTC ATC ATC CCG CCC ATA G-3′ (SEQ ID NO:19)
[0112] PCR primers such as SEQ ID NO:20 (forward, beginning at residue number 901, see 5′-CTC GAC CTC ATG GCT TGC AGA G-3′ (SEQ ID NO:20) 5′-CTG AGA ACG ATC CGC TTT ATC-3′ (SEQ ID NO:21)
[0113] PCR primers such as SEQ ID NO:22 (forward primer) and SEQ ID NO:23 (reverse) can also be used to directly amplify new IMP species and isoforms or to generate a DNA probe that would include an internal subset of IMP coding sequence. SEQ ID NO:22 and SEQ ID NO:23 primer pair amplifies an internal block of the coding sequence of IMP.18p protein. SEQ ID NO:22 and SEQ ID NO:23 correspond to coding sequence immediately upstream and downstream of motif A and motif B (discussed below), respectively (amino acids number 98 to 111 and 230 to 244, respectively, see 5′-GTG TGT GCT CAC CCC GAC TGT-3′ (SEQ ID NO:22) 5′-CCC GAA GTG TCT ATC ACG ATG-3′ (SEQ ID NO:23)
[0114] The subsequences of the isolated nucleic acids of the present invention are at least N nucleotides in length, where N is any one of the integers selected from the group consisting of from 15 to 900. Typically, the subsequences are at least 20 nucleotides in length, preferably at least 25 nucleotides in length, preferably at least 30 nucleotides in length, and often at least 35.40, or 50 nucleotides in length. The subsequences of the isolated proteins of the present invention are at least N′ amino acids in length, where N′ is any one of the integers from 5 to 300. The amino acid subsequences are derived from contiguous amino acids from the protein sequences of SEQ ID NO:3 or SEQ ID NO:4. The nucleic acid subsequences are derived from contiguous nucleotides from the nucleic acid sequences of SEQ ID NO:7 or SEQ ID NO:8. “Contiguous” with respect to a specified number of amino acid residues or nucleotides, includes reference to a sequence of amino acids or nucleotides, respectively, of the specified number from within the specified reference sequence which has the identical order of amino acids or nucleotides and the same adjacent amino acids or nucleotides as in the reference sequence.
[0115] The present invention also provides isolated mammalian proteins comprising a clone 22 protein subsequence and an IMP.18p subsequence of at least 10 contiguous amino acids, preferably at least 15 contiguous amino acids, more preferably at least 20 contiguous amino acids, and most preferably at least 25, 30, 35, or 40 contiguous amino acids. In the case of clone 22, these amino acid sequences are from SEQ ID NO:3. In the case of IMP.18p, these amino acid sequences are from SEQ ID NO:17. The isolated mammalian proteins are immunologically cross-reactive to an antibody composition that is generated from (e.g., screened, synthesized, or elicited) and specifically reactive to a protein immunogen of SEQ ID NO:3 and SEQ ID NO:17 for clone 22 and IMP.18p, respectively. The mammalian protein may be isolated from any number of mammals including: rat, mice, cattle, dog, pig, guinea pig, or rabbit, and most preferably a primate such as macaques, chimpanzees, or humans.
[0116] The isolated clone 22 and IMP.18p proteins of the present invention can be constructed using standard recombinant or synthetic methods. Solid phase synthesis of isolated proteins of the present invention of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Merrifield,
[0117] Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N′-dicycylohexyl carbodiimide) is known to those of skill.
[0118] Subsequences of nucleic acids can be used as probes to detect or isolate the clone 22 and IMP.18p encoding nucleic acids for further analysis of the polymorphism contained therein for purposes described more fully, supra. Additionally, subsequences can be utilized as primers for amplification of the clone 22 and IMP.18p polymorphisms. The subsequence may be derived from within any portion of the clone 22 isoforms and IMP.18p coding sequence. Probes specific to one or the other isoform of clone 22 can be used to study differential transcription of these isoforms.
[0119] Isolated nucleic acids of the present invention can also be used for recombinant expression of the proteins of the present invention for use as immunogens in the preparation of antibodies. Subsequences can also be used for detecting and/or quantifying clone 22 protein and IMP.18p expression by assaying for the gene transcript (e.g., nuclear RNA, mRNA) using nucleic acids coding for clone 22 and IMP.18p proteins. The assay can be for the presence or absence of the normal gene or gene product, for the presence or absence of an abnormal gene or gene product or quantification of the transcription levels of normal or abnormal clone 22 and IMP.18p gene product. Nucleic acid assays are well known in the art and included in standard molecular biology references such as those incorporated by reference herein.
[0120] For example, amongst the various hybridization formats well known to the skilled artisan is included solution phase, solid phase, mixed phase, or in situ hybridization assays. Briefly, in solution (or liquid) phase hybridizations, both the target nucleic acid and the probe or primer are free to interact in the reaction mixture. In solid phase hybridization assays, probes or primers are typically linked to a solid support where they are available for hybridization with target nucleic in solution. In mixed phase, nucleic acid intermediates in solution hybridize to target nucleic acids in solution as well as to a nucleic acid linked to a solid support. In in situ hybridization, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis. The following articles provide an overview of the various hybridization assay formats: Singer et al.,
[0121] Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. A %s the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0% to 50%.
[0122] The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100 percent; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium as described below. Thus, despite the lack of 100 percent complementarity under reduced conditions of stringency, functional nucleic acids of the present invention having minor base differences from the nucleic acid targets are possible. Therefore, under hybridization conditions of reduced stringency, it may be possible to construct an oligonucleotide having substantial identity to an oligonucleotide complementary to the target sequence while maintaining an acceptable degree of specificity. Substantial identity in the context of nucleic acids means that the two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. to 200 C lower than the thermal melting point (T
[0123] The nucleic acids of the present invention, whether derived from a biological source, artificially constructed or both, can be operably linked to a promoter. Those of ordinary skill will recognize that an isolated duplex clone 22 or IMP.18p nucleic acid operably linked to a promoter in forward orientation can direct transcription of mRNA which can be translated into a clone 22 or IMP.18p protein of the present invention. An isolated duplex clone 22 or IMP.18p nucleic acid operably linked to a promoter in reverse orientation can direct transcription of antisense mRNA. Antisense nucleic acids can be used for probes in assays for normal or abnormal gene product or to quantitate the expression of mRNA coding for the clone 22 or IMP.18p protein in, for example, drug assays. Accordingly, the isolated nucleic acids of the present invention are inclusive of both sense and antisense nucleic acids.
[0124] The isolated nucleic acid compositions of this invention, whether RNA, cDNA, genomic DNA, or a hybrid of the various combinations, are isolated from biological sources or synthesized in vitro. Deoxynucleotides encoding isolated proteins of the present invention can be prepared by any suitable method including, for example, cloning and restriction of appropriate sequences as discussed supra, or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al.
[0125] Once the nucleic acid encoding a protein of the present invention is isolated and cloned, one may express the desired protein in a recombinantly engineered cell such as bacteria, yeast, insect (especially employing baculoviral vectors), and mammalian cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of proteins. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made. In brief, the expression of natural or synthetic nucleic acids encoding the isolated proteins of the invention will typically be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding the protein. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. One of skill would recognize that minor modifications can be made to a clone 22 or IMP.18p protein. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
[0126] Examples of techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel,
[0127] 1. Expression in Prokaryotes
[0128] Bacterial strains which can be used to express the nucleic acid of the invention include
[0129] Examples of regulatory regions suitable for this purpose in
[0130] The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for clone 22 proteins are available using
[0131] When expressing clone 22 or IMP.18p proteins in
[0132] An example of how this can be achieved is based on the his operon of Salmonella. Two steps are involved in this process. First, a segment of the his operon must be deleted in the Salmonella strain selected as the carrier. Second, a plasmid carrying the deleted his region downstream of the gene encoding the clone 22 or IMP.18p protein is transfected into the his Salmonella strain. Integration of both the his sequences and a gene encoding a clone 22 or IMP.18p protein occurs, resulting in recombinant strains which can be selected as his
[0133] Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Bacteria are grown according to standard procedures in the art. Because some proteins can be difficult to isolate with intact biological activity, preferably fresh bacteria cells are used for isolation of protein. Use of cells that are frozen after growth but prior to lysis typically results in negligible yields of active protein.
[0134] Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.
[0135] Proteins expressed in bacteria may form insoluble aggregates (“inclusion bodies”). Several protocols are suitable for purification of inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be homogenized using a Polytron (Brinkman Instruments, Westbury, N.Y.). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et al., supra: Ausubel et al., supra).
[0136] The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer that does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.
[0137] Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties); the proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents which are capable of solubilizing aggregate-forming proteins for example SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of immunologically and/or biologically active protein. After solubilization, the protein can se separated from other bacterial proteins by standard separation techniques.
[0138] Alternatively, it is possible to purify the protein of interest from bacteria periplasm. Where IMP.18p or clone 22, for example, is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art. To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO
[0139] 2. Expression in Eukaryotes
[0140] A variety of eukaryotic expression systems such as yeast, insect cell lines, bird, fish, frog, and mammalian cells, are known to those of skill in the art. As explained briefly below, the isolated proteins of the present invention may be expressed in these eukaryotic systems.
[0141] Yeast expression systems being eukaryotic, provide an attractive alternative to bacterial systems for some applications, for an overview of yeast expression systems, see,
[0142] Yeast strains which can be used to express exogenous nucleic acids include
[0143] Synthesis of heterologous proteins in yeast is well known.
[0144] Two procedures are used in transfecting yeast cells. In one case, yeast cells are first converted into protoplasts using zymolyase, lyticase or glusulase, followed by addition of DNA and polyethylene glycol (PEG). The PEG-treated protoplasts are then regenerated in a 3% agar medium under selective conditions. Details of this procedure are given in the papers by J. D. Beggs, (1978),
[0145] Clone 22 proteins or IMP.18p, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
[0146] The sequences encoding clone 22 or IMP.18p proteins can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, bird, amphibian, or fish origin. Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the CHO cell lines, and various human cells such as COS cell lines, HeLa cells, myeloma cell lines, Jurkat cells. Other animal cells useful for production of IMP18.p and clone 22 proteins are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition. 1992).
[0147] Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al. (1986)
[0148] The DNA sequence encoding the IMP18.p and clone 22 proteins can typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell. Such signal peptides would include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of
[0149] Additional elements of the expression cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
[0150] Appropriate vectors for expressing clone 22 or IMP.18p proteins in insect cells arc usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See Schneider
[0151] As indicated above, the vector, e.g., a plasmid which is used to transfect the host cell, preferably contains DNA sequences to initiate transcription and sequences to control the translation of the protein. These sequences are referred to as expression control sequences.
[0152] As with yeast, when higher animal host cells are employed, polyadenlyation or transcription terminator sequences from known mammalian genes need to be incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, J. et al., (1983),
[0153] Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein Bar virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A
[0154] Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Gene amplification, whether by higher vector copy number or by replication of a gene in a chromosome, can increase yields of recombinant proteins in mammalian and other cells. One in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit coding for a selectable marker. As another example, gene amplification of the gene of interest can be achieved by linking it to a dihydrofolate reductase (Dhfr) gene and administering methotrexate to the transfected cells, this method can increase recombinant protein production many fold (see Monaco (1996)
[0155] Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a bacculovirus vector in insect cells, with for example an IMP.18p encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters. A commonly used insect system utilizes
[0156] The host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art.
[0157] The clone 22 or IMP.18p proteins of the present invention which are produced by recombinant DNA technology may be purified by standard techniques well known to those of skill in the art. Recombinantly produced clone 22 or IMP.18p proteins can be directly expressed or expressed as a fusion protein. The recombinant clone 22 or IMP.18p protein can be purified by a combination of cell lysis (e.g., sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant clone 22 or IMP.18p protein.
[0158] The clone 22 or IMP.18p proteins of this invention, recombinant or synthetic, may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes,
[0159] Antibodies
[0160] The present invention provides antibodies specifically reactive, under immunologically reactive conditions, to an isolated protein of the present invention. Antibodies are raised to a protein of the present invention, including individual, allelic strain, or species variants and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated.
[0161] Many methods of making antibodies are known to persons of skill. The following discussion is presented as a general overview of the techniques available; however, one of skill will recognize that many variations upon the following methods are known.
[0162] A. Antibody Production
[0163] A number of immunogens are used to produce antibodies immunologically reactive with a clone 22 or IMP.18p protein. An isolated recombinant, synthetic, or native clone 2e protein of 5 contiguous amino acids in length or greater from SEQ ID NO:3 or 4 is the preferred immunogens (antigen) for the production of anti-clone 22 polypeptide monoclonal or polyclonal antibodies. An isolated recombinant, synthetic, or native IMP.18p protein of 5 contiguous amino acids in length or greater from SEQ ID NO:17 is the preferred immunogens (antigen) for the production of anti-IMP.18p polypeptide monoclonal or polyclonal antibodies. In one class of preferred embodiments, an immunogenic protein conjugate is also included as an immunogen. Naturally occurring clone 22 or IMP.18p proteins are also used either in pure or impure form.
[0164] The clone 22 or IMP.18p protein is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the clone 22 or IMP.18p protein. Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen (antigen), preferably a purified clone 22 or IMP.18p protein, a clone 22 or IMP.18p protein coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a clone 22 or IMP.18p protein incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the clone 22 or IMP.18p protein of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the clone 22 or IMP.18p protein is performed where desired (see, e.g., Coligan (1991)
[0165] Antibodies, including binding fragments and single chain recombinant versions thereof, against predetermined fragments of clone 22 or IMP.18p protein are raised by immunizing animals, e.g., with conjugates of the fragments with carrier proteins as described above. Typically, the immunogen of interest is a clone 22 or IMP.18p protein of at least about 5 amino acids, more typically the clone 22 or IMP.18p protein is at least 10 amino acids in length, preferably, at least 15 amino acids in length, more preferably at least 25 amino acids in length. In particularly preferred embodiments, the immunogen is derived from the extra- or intra-cytoplasmic region of the clone 22 protein. The peptides are typically coupled to a carrier protein (e.g., as a fusion protein), or are recombinantly expressed in an immunization vector. Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.
[0166] Monoclonal antibodies are prepared from cells secreting the desired antibody. Monoclonals antibodies are screened for binding to a clone 22 or IMP.18p protein from which the immunogen was derived. Specific monoclonal and polyclonal antibodies will usually bind with an affinity constant of at least between 10
[0167] In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g., Stites et al. (eds.)
[0168] Alternative methods of immortalization include transfection with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The clone 22 or IMP.18p proteins and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies.
[0169] Other suitable techniques involve selection of libraries of recombinant antibodies in phase or similar vectors (see, e.g., Huse et al. (1989)
[0170] Frequently, the clone 22 or IMP.18p proteins and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989)
[0171] The antibodies of this invention are also used for affinity chromatography in isolating clone 22 or IMP.18p protein. Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified clone 22 or IMP.18p protein are released.
[0172] The antibodies can be used to screen expression libraries for particular expression products such as normal or abnormal human clone 22 or IMP.18p proteins. Usually the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
[0173] Antibodies raised against a clone 22 or IMP.18p protein can also be used to raise anti-idiotypic antibodies. These are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens.
[0174] B. Human or Humanized (Chimeric) Antibody Production
[0175] The anti-clone 22 or anti-IMP.18p protein antibodies of this invention can also be administered to a mammal (e.g., a human patient) for therapeutic purposes (e.g., as targeting molecules when conjugated or fused to effector molecules such as labels, cytotoxins, enzymes, growth factors, drugs, etc.). Antibodies administered to an organism other than the species in which then, are raised are often immunogenic. Thus, for example, murine antibodies administered to a human often induce an immunologic response against the antibody (e.g., the human anti-mouse antibody (HAMA) response) on multiple administrations. The immunogenic properties of the antibody are reduced by altering portions, or all, of the antibody into characteristically human sequences thereby producing chimeric or human antibodies, respectively.
[0176] i) Humanized (Chimeric) Antibodies
[0177] Humanized (chimeric) antibodies are immunoglobulin molecules comprising a human and non-human portion. More specifically, the antigen combining region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g., murine) and the constant region of the chimeric antibody (which confers biological effector function to the immunoglobulin) is derived from a human source. The humanized chimeric antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule. A large number of methods of generating chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and 4,975,369). Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Pat. No. 5,482,856.
[0178] ii) Human Antibodies
[0179] In another embodiment, this invention provides for fully human anti-clone 22 or anti-IMP.18p protein antibodies. Human antibodies consist entirely of characteristically human polypeptide sequences. The human anti-clone 22 or anti-IMP.18p protein antibodies of this invention can be produced in using a wide variety of methods (see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, for review).
[0180] In preferred embodiments, the human anti-clone 22 or anti-IMP.18p protein antibodies of the present invention are usually produced initially in trioma cells. Genes encoding the antibodies are then cloned and expressed in other cells, particularly, nonhuman mammalian cells. The general approach for producing human antibodies by trioma technology has been described by Ostberg et al. (1983),
[0181] The genes encoding the heavy and light chains of immunoglobulins secreted by trioma cell lines are cloned according to methods, including the polymerase chain reaction, known in the art (see, e.g., Sambrook et al.,
[0182] Clone 22 and IMP.18p Protein Immunoassays
[0183] Embodiments include means of detecting the clone 22 or IMP.18p proteins of the present invention using novel reagents provided for by the invention. In one embodiment, the clone 22 or IMP.18p proteins are detected and/or quantified using the novel antibodies provided for by the invention utilizing any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also
[0184] Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled clone 22 or IMP.18p protein or a labeled anti-clone 22 or anti-IMP.18p protein antibody. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/clone 22 protein complex.
[0185] In some embodiments, the labeling agent is a second clone 22 or IMP.18p protein antibody bearing a label. Alternatively, the second clone 22 or IMP.18p protein antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
[0186] Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al. (1973)
[0187] Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 101C to 40° C.
[0188] While the details of the immunoassays of the present invention may vary with the particular format employed, the method of detecting a clone 22 or IMP.18p protein in a biological sample generally comprises the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to the clone 22 or IMP.18p protein. The antibody is allowed to bind to the clone 22 or IMP.18p protein under immunologically reactive conditions, and the presence of the bound antibody is detected directly or indirectly.
[0189] A. Non-Competitive Assay Formats
[0190] Immunoassays for detecting clone 22 or IMP.18p proteins of the present invention include competitive and noncompetitive formats. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case clone 22 or IMP.18p protein) is directly measured. In one preferred “sandwich” assay, for example, the capture agent (anti-clone 22 or anti-IMP.18p protein antibodies) can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture clone 22 or IMP.18p protein present in the test sample. The clone 22 or IMP.18p protein thus immobilized is then bound by a labeling agent, such as a second human clone 22 or IMP.18p protein antibody bearing a label. Alternatively, the second clone 22 or IMP.18p protein antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
[0191] B. Competitive Assay Formats
[0192] In competitive assays, the amount of analyte (clone 22 or IMP.18p protein) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (clone 22 or IMP.18p protein) displaced (or competed away) from a capture agent (anti clone 22 or IMP.18p protein antibody) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, clone 22 or IMP.18p protein is added to the sample and the sample is then contacted with a capture agent, in this case an antibody that specifically binds clone 22 or IMP.18p protein. The amount of clone 22 or IMP.18p protein bound to the antibody is inversely proportional to the concentration of clone 22 or IMP.18p protein present in the sample.
[0193] In some embodiments, the antibody is immobilized on a solid substrate. The amount of clone 22 or IMP.18p protein bound to the antibody may be determined either by measuring the amount of clone 22 or IMP.18p protein present in a clone 22 or IMP.18p protein/antibody complex, or alternatively by measuring the amount of remaining uncomplexed clone 22 or IMP.18p protein. The amount of clone 22 or IMP.18p protein may be detected by providing a labeled clone 22 or IMP.18p protein molecule.
[0194] A hapten inhibition assay is another preferred competitive assay. In this assay a known analyte, in this case clone 22 or IMP.18p protein is immobilized on a solid substrate. A known amount of anti-clone 22 or anti-IMP.18p protein antibody is added to the sample, and the sample is then contacted with the immobilized clone 22 or IMP.18p protein. In this case, the amount of anti-clone 22 or IMP.18p protein antibody bound to the immobilized clone 22 or IMP.18p protein is inversely proportional to the amount of clone 22 or IMP.18p protein present in the sample. Again the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
[0195] Immunoassays in the competitive binding format are also used for crossreactivity determinations to permit one of skill to determine if a novel protein is a homologue, allele, or polymorphic variant of the IMP.18p polypeptide having the sequence set forth as SEQ ID NO:17, thus falling within the scope of the claimed invention. In this assay, the IMP.18p polypeptide with the sequence set forth as SEQ ID NO:17 is immobilized to a solid support. Putative IMP.18p polymorphic variants are added to the assay to compete with immobilized IMP.18p antigen for binding to a characterized anti-IMP.18p antisera. The ability of the putative IMP.18p polymorphic variants to compete with immobilized IMP.18p antigen for binding to the anti-IMP.18p antisera is compared to the ability of IMP.18p of SEQ ID NO:17, or immunogenic fragments thereof, to compete with immobilized antigen for binding to the antisera. The percent crossreactivity for the above proteins is calculated, using standard calculations.
[0196] To prepare the antisera for use in this competitive binding immunoassay, all IMP cross-reacting antibodies are first removed by immuno-absorption with known IMP polypeptides. Specifically, antisera are immunosorbed with the human IMP (huIMP) defined by McAllister (1992)
[0197] In this competitive binding immunoassay, the IMP.18p protein of SEQ ID NO:17 competes with a second, putative IMP.18p polymorphic variant in an antibody binding reaction. The known and uncharacterized IMP.18p polypeptides are competitively reacted with antisera developed against and specifically reactive with the IMP.18p of SEQ ID NO:17 (antisera immunosorbed to ensure no cross-reactivity with previously characterized IMPs, as described above). The two polypeptides are each assayed at a wide range of concentrations. The amount of each polypeptide required to inhibit 50% of the binding of the anti-IMP.18p (SEQ ID NO:17) antisera to immobilized IMP.18p (SEQ ID NO:17) polypeptide is determined. If the amount of the second (uncharacterized) protein required is less than 10 times the amount of the characterized immunogen (IMP.18p/SEQ ID NO:17) that is required, then the second protein is said to specifically bind to an antibody generated to the characterized (IMP.18p/SEQ ID NO:17) immunogen.
[0198] Immunoassays in the competitive binding format can be used for crossreactivity determinations to permit one of skill to determine if a novel anti-IMP.18p antibody or antisera is sufficiently related to the anti-IMP.18p polypeptide of the invention with the sequence set forth as SEQ ID NO:17 so as to fall under (within the scope of) the claims of this invention. For example, the IMP.18p/SEQ ID NO:17 polypeptide is immobilized to a solid support. Test antibodies are added to the assay to compete with the binding of the known anti-IMP18.p/SEQ ID NO:17 antisera to the immobilized antigen (IMP.18p/SEQ ID NO:17). The ability of the test antisera to compete with the binding of the known antisera to the immobilized IMP.18p is compared. The percent crossreactivity for the above antibodies is calculated, using standard calculations.
[0199] C. Other Assay Formats
[0200] In other embodiments. Western blot (immunoblot) analysis is used to detect and quantify the presence of clone 22 or IMP.18p protein in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind clone 22 or IMP.18p protein. The anti-clone 22 or anti-IMP.18p protein antibodies specifically bind to clone 22 or IMP.18p protein, respectively, on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-clone 22 or anti-IMP.18p protein.
[0201] Assaying for Activity and Modulators of IMP.18p Myo-Inositol Monophosphatase
[0202] The invention also provides for means to assay the activity of the novel IMP18.p myo-inositol monophosphatase enzyme. Using such assays, one embodiment provides for a method of determining whether a test compound is a modulator, such as an inhibitor/antagonist or agonist, of IMP.18p myo-inositol monophosphatase activity. The method involves contacting an active IMP.18p with a putative modulator test compound and measuring the activity of the IMP.18p. A change in the activity of the IMP.18p in the presence of the test compound is an indicator of whether the test compound is an antagonist or agonist/activator of IMP.18p. A variety of myo-inositol monophosphatase activity assays are known in the art which can be adapted by the skilled artisan to be used using the novel IMP.18p in the methods of the invention. Illustrative examples of such assays are set forth below.
[0203] Myo-inositol monophosphatases are major enzymes controlling the inositol intracellular signaling pathway. Numerous diacylglycerol and calcium-mobilizing enzymes are associated with this pathway, including serotonergic, muscarinic, adrenergic, metabotropic, histaminergic, cholecystokinin, tachykinin, bombesin, neurotensin and bradykinin receptors, to name a few examples. Activation of these receptors activates GTP binding proteins, which results in the phospholipase C hydrolysis of inositol-phospholipid. This reaction releases two intracellular messengers: myo-inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 releases intracellular calcium stores, which in turn activates a variety of second signals, triggering numerous physiologic effects, for example, ion channel activation. Levels of IP3 are controlled by sequential dephosphorylation, the last step generating the products inositol and phosphate from the substrate myo-inositol monophosphate (myo-inositol 1-phosphate) by the enzyme myo-inositol monophosphatase. Thus, the activity of myo-inositol monophosphates can be monitored in vitro or in vivo by measuring the loss or accumulation of a substrate or a product, respectively, over time.
[0204] Monitoring the activity and assessment of potential modulators of the novel IMP.18p of the invention can be accomplished in vitro by measuring the accumulation of either myo-inositol monophosphatase product in the form of radiolabeled inositol (e.g.,
[0205] As in Vadnal (1995) supra, the reaction mixture can consist of 0.05 ml of 120 mM Tris-HCl, pH 7.8; 0.05 ml of 18 mM or 3 mM magnesium chloride; 0.05 ml of 4.2 mM D-myo-inositol 1-phosphate, 0.125 ml water alone or with positive controls or putative modulator test compounds or compositions. Known myo-inositol monophosphatase inhibitors (antagonists), such as lithium, carbamazepine and/or valproic acid, in varying amounts can be used as controls. A 0.025 ml solution of myo-inositol monophosphatase (e.g., IMP.18p, or another myo-inositol monophosphatase as a positive control) is added and the reaction mixture is incubated at 37° C. for about 15 minutes to an hour. The reaction is stopped by the addition of 0.05 nl of 20% trichloroacetic acid (TCA). The suspension is centrifuged and 0.10 ml of supernatant is used to estimate the liberated Pi using the malachite green reagent method, as, for example, described by Eisenberg (1987)
[0206] Kinetic activity and assessment of potential modulators of the IMP.18p of the invention can also be accomplished in vivo by measuring accumulation of the substrate myo-inositol monophosphate (myo-inositol 1-phosphate) using, for example, assays described by Atack (1993)
[0207] Using these assays and variations thereof, the kinetics of the IMP.18p enzyme with and without test modulators (e.g., competitive or non-competitive antagonists) can be analyzed using known methods (e.g., Lineweaver-Burke plots, as used, for example by Lee (1996)
[0208] High-Throughput Screening of Candidate IMP.18p Modulators
[0209] Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity (in this case, e.g., an antagonist or agonist of IMP.18p), creating variants of the lead compound, and evaluating the property and activity of those variant compounds. However, the current trend is to shorten the time scale for all aspects of drug discovery. Because of the ability to test large numbers quickly and efficiently, high throughput screening (HTS) methods are replacing conventional lead compound identification methods.
[0210] In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic or diagnostic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays, some of which are described above, to identify those library members (particular chemical species or subclasses) that display the desired characteristic activity (e.g., modulation of the activity of IMP.18p). The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics. See also, van Breemen (1997)
[0211] a. Combinatorial Chemical Libraries
[0212] Recently, attention has focused on the use of combinatorial chemical libraries to assist in the generation of new chemical compound leads. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, one commentator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop et al. (1994) 37(9): 1233-1250).
[0213] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991)
[0214] Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention. Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, Dec. 26, 1991), encoded peptides (PCT Publication WO 93/20242, Oct. 14, 1993), random bio-oligomers (PCT Publication WO 92/00091, Jan. 9, 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993)
[0215] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech. Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.).
[0216] A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton. N.J., Asinex, Moscow, Ru, Tripos, Inc. St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
[0217] b. High Throughput Assays of Chemical Libraries
[0218] Any of the assays for compounds inhibiting the virulence described herein are amenable to high throughput screening. As described above, having identified the nucleic acid associated with virulence, likely drug candidates either inhibit expression of the gene product, or inhibit the activity of the expressed protein. Preferred assays thus detect inhibition of transcription (i.e., inhibition of mRNA production) by the test compound(s), inhibition of protein expression by the test compound(s), or binding to the gene (e.g., gDNA, or cDNA) or gene product (e.g., mRNA or expressed protein) by the test compound(s). Alternatively, the assay can detect inhibition of the characteristic activity of the gene product or inhibition of or binding to a receptor or other transduction molecule that interacts with the gene product.
[0219] High throughput assays for the presence, absence, or quantification of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays are similarly well known. Thus, for example, U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins, U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays), while U.S. Pat. Nos. 5,576,220 and 5,541.061 disclose high throughput methods of screening for ligand/antibody binding.
[0220] In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high thruput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example. Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
[0221] Rational Drug Design
[0222] Potential modulators of enzyme activity can also be investigated utilizing “rational drug design” approaches. This involves an integrated set of methodologies that include structural analysis of target molecules, synthetic chemistries, and advanced computational tools. When used to design modulators, such as antagonists/inhibitors of protein targets, such as IMP.18p polypeptides, the objective of rational drug design is to understand a molecule's three-dimensional shape and chemistry. Rational drug design is aided by X-ray crystallographic data or NMR data, which can now be determined for the IMP.18p polypeptide in accordance with the methods and using the reagents provided by the invention. Calculations on electrostatics, hydrophobicities and solvent accessibility is also helpful. See, for example, Coldren (1997)
[0223] Inhibitory Natural Compounds as Modulators of IMP.18p Activity
[0224] In addition, a large number of potentially useful activity-modifying compounds can be screened in extracts from natural products as a source material. Sources of such extracts can be from a large number of species of fungi, actinomyces, algae, insects, protozoa, plants, and bacteria. Those extracts showing inhibitory activity can then be analyzed to isolate the active molecule. See for example, Turner (1996)
[0225] Inhibitory Oligonucleotides
[0226] One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind mRNA encoding IMP.18p or clone 22 polypeptides or to their corresponding genes. In either case, these oligos prevent or inhibit the production of functional protein.
[0227] Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of IMP.18p or clone 22 mRNA. That is, the oligonucleotide is chemically modified or has enzyme activity which causes such cleavage, such as ribozymes. As noted above, one may screen a pool of many different such oligonucleotides for those with the desired activity.
[0228] Another useful class of inhibitors includes oligonucleotides which bind polypeptides. Double- or single-stranded DNA or single-stranded RNA molecules that bind to specific polypeptides targets are called “aptamers.” The specific oligonucleotide-polypeptide association may be mediated by electrostatic interactions. For example, aptamers specifically bind to anion-binding exosites on thromoin, which physiologically binds to the polyanionic heparin (Bock (1992)
[0229] Antisense Oligonucleotides
[0230] IMP.18p or clone 22 activity can be inhibited by targeting their respective mRNA with antisense oligonucleotides capable of binding the mRNA. In some situations, naturally occurring nucleic acids used as antisense oligonucleotides may need to be relatively long (18 to 40 nucleotides) and present at high concentrations. AR wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997)
[0231] As noted above, combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the IMP.18p of the invention, can be utilized (for general background information Gold (1995)
[0232] Inhibitory Ribozymes
[0233] Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
[0234] In some circumstances, the enzymatic nature of a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide. This potential advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
[0235] The enzymatic ribozyme RNA molecule has complementarity to the target, such as the mRNA encoding IMP.18p. The enzymatic ribozyme RNA molecule is able to cleave RNA and thereby inactivate a target RNA molecule. The complementarity functions to allow sufficient hybridization of the enzymatic ribozyme RNA molecule to the target RNA for cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be employed. The present invention provides ribozymes targeting any portion of the coding region for an IMP.18p or clone 22 gene that cleaves their corresponding mRNA in a manner that will inhibit the translation of the mRNA and thus reduce enzymatic activity. In addition, the invention provides ribozymes targeting the nascent RNA transcript of the IMP.18p or clone 22 gene to reduce activity.
[0236] The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNaseP-like RNA (in association with an RNA guide sequence ). Examples of such hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989)
[0237] Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
[0238] The following examples are offered to illustrate, but not to limit the claimed invention.
[0239] A human chromosome 18-specific cosmid library, LL18NC02, was provided by the Human Genome Center at the Lawrence Livermore Laboratory. The source of the chromosomes was a human/hamster hybrid cell line X11-4A (Chang et al.,
[0240] Total RNA was extracted from five regions of postmortem human brain (caudate, putamen, hippocampus, amygdala, frontal cortex) and from human placenta by acid-guanidine, phenol/chloroform method (Chomzynski and Sacchi,
[0241] Direct cDNA selection was performed using the magnetic bead capture technique described previously (Lovett et al.
[0242] Approximately 40,000 clones from a normalized infant brain library constructed by Soares et al. (1994),
[0243] Each pool of amplified secondary-selected cDNA was labeled with gamma-
[0244] Another set of hybridizations was performed using a mixture of two pools of secondary-selected cDNA. The hybridization pattern yielded by the secondary selected cDNAs was compared with that produced by human placental DNA. The clones corresponding to positive spots common to both filters were not picked due to the possibility that the signals were from repeat hybridization. In addition, the hybridization pattern obtained with the cDNA subpools were compared with that produced using a combination of all secondary selected cDNA pools. All high and medium intensity clones were chosen, and clones that gave low intensity signals but were common to two or more filters were also picked. The insert sizes were determined using the colony PCR method described previously (Yoshikawa et al.
[0245] The microtiter plate addresses of the positive clones chosen for further analysis were determined, and this allowed us to search the EST database (dbEST) (Boguski et al.,
[0246] Genomic DNA was extracted from a panel of 20 somatic cell hybrids, one of which included the entire human chromosome 18 and the rest containing various segments of the chromosome (Overhauser et al.
[0247] PCR was performed using the Perkin Elmer Cetus GeneAmp System 9600. Amplification was done in a 20 μl reaction containing either 80 ng (somatic cell hybrid) or 30 ng (human or hamster genomic DNA) template DNA, 5 μM of each primer, 200 μM of each dNTP and 0.75 unit of AmpliTaq (Perkin Elmer Cetus) in a standard PCR I buffer
TABLE 2 TABLE 2: Primers used for PCR mapping. Clone Primer Sequence Product Number Forward SEQ ID NO: Reverse SEQ ID NO: Size (bp) 1 5′-AGGAGTGGTGTACATTTCT-3′ 110 5′-ACCTGCAACACATTAGAAAC-3′ 111 134 2 GGTTTCTTCAAAATTTTATTAACAA 112 TCCTCCACTCATCTGTTTCT 113 175 3 CCTGACCTGATCAAGTTTA 114 GGTAAAGGAACAAGCTGC 115 125 4* TGATCACAOAGTCAGCACTGT 116 GGGCAGAAGTTTCCAATTACC 117 131 5 TATTGAGACCTAAGTCAGCATCC 118 GACAGAAAGCAGGTTAGAGGT 119 192 6 GAAACTTTACATCAGGTGTCTC 120 ATGGACTAGGAGTTTAAGC 121 283 7 GGAACAGTGTACACTTTCC 122 TATATAGCCTCGATGATGAGAG 123 185 8 CATGAGAGGAAGAGGTCTTTAT 124 GGGTAATGTCTTAGTCGAG 125 275 9 TCAGTAGAAACTCAAGCTGCTTC 126 CTCCCTCTCAGTGTGAGGCT 127 230 10 CCTGACCTGATCAAGTTTAA 128 TGTACACCACTCCTCATGT 129 179 11 CGACGACTCATACAACATATC 130 GGTTACAGCTGAAGTGTAT 131 177 12 TATTCAGGAACAGTGTACAC 132 TCGATGATGAGAGGGTTAC 133 174 13 GAACACTTATCTCCTTCTTCAG 134 TCCACTCCTTTCACCTCTTCT 135 243 14 AGACAAGAGCAAAACACAAC 136 CTCTTTGCAGTTCAGTCTA 137 169 15 AGGTGAACCATTTGACTGGTTT 138 GCTTGTGTGTGGCTGTCCTT 139 148 16 GGCTAAACTTACAGTATGTAAGGAG 140 CTGTAAGGACAGACTACTCA 141 152 17 CCAGGAGGTTTCAGCGGT 142 CGCAAAGCCATGATAAACCG 143 115 18 TCAGGAACAGTGTACACTTTC 144 TGTGGGCTTAATACCATGTCT 145 207 19 GGAATCTCTGTACTTGCT 146 GTGACACATTACAAAGCCA 147 154 20 TCAGTAGAAACTCAAGCTGC 148 CCTCTTCCTCTTTAAGTGT 149 101 21 TCACTTCAGAATCACTACTC 150 ACCCATCCTATATGAAAAGC 151 228 22 TACAAAAGAGGACAAAGCAC 30 GGTGCCTGTATATAAGTTGA 32 157 23 GGGATCATACTAAAGAGAAG 152 GGATAAACAGAGAGCTTGAT 153 193 24 CTACAGAATAGAATACATGGCG 34 GAGCTCTGAACTGTATTCAGA 36 224 25 GTCAGTTACTCTATTTGCTGTG 154 AACCTGTGCTGTAAAGTTCA 155 233 26 CTTAAGAGGAAGAGGCCAT 156 CTCTCCCTCTCAGTGTGAG 157 145 27 ACAATTAGGCATTGTTGATGG 158 CAGTTCTTGCACATACAAGACA 159 112 28 ACCTTTGGCAAGGGGTATGA 160 TGTGAAGGCTGGGAAACACT 161 207 29 TCTCAGCTTACTCAACCT 38 GATGAGGTGGAACAATCAC 40 138 30 AACACTCAGCTCTGTAGAA 162 CGAGTCATCAATAGGACAA 163 212 31 GGTCTGTACAGTGTAATAAACC 42 CTACTGCAAAATGTGTCCTGTC 44 124 32 GAGCCAAGTGGAACTCTTGAA 164 GTCAGGAAAGAGGTTGTGAGC 165 156 33 ACACATATGTACACAGGAAC 166 TGTGTACAGCGAGTGAATTA 167 103 34 TTGTTCACACACAATCTAGG 168 ACTAGCATATCTGAATTCCCA 169 159 35 CTACAGAATAGAATACATGGCG 170 TTGAAACCAGACCCTGTAGT 171 166 36 CATTTAGTCCAGAGGCTCTT 172 TCCTCGAAGAGGTTGCAGC 173 161 37 CACATTAGCCAGTCTGATAAAG 46 AAGTTACACACAGTAGCTGA 48 107 38 CATTCAGCACACATAGAGTCTA 174 CCCTGTCCCTTGTATATGTA 175 189 39 AGTGTATCTACAACCTCAACTGTC 176 GTAAAGGCCCAATCAATGCACT 177 109 40 GOCAGATTCACAATTGATAG 178 CTGAAGGCACTTTATGTAC 179 139 41 CTGGAGCAGGTTAGATACACC 180 CTTCCCTCTTAACCTTTAGTGC 181 143 42 GTGTCTTGTATGTGCAAGAAC 182 GACTGGGTATCCTAGCTTAC 183 157 43 TTAGTCAGACCCATTCAGTC 184 CCAGACTGCTTTATGTTAG 185 103 44 GTGTCTTGTATGTGCAAGAAC 186 CCTAGCCTTACTGTTTTAAC 187 146 45* ACGATGCGATCCTGGAAG 188 CTGGCTTGAGTTTGTCTG 189 113 46† CCTTTCTGTGTGAAGATCAC 190 AAGAAAGTCCCAAGGGTGGA 191 123 47† GGAATGAGGGTTAGAGTCC 192 AGTGCTTCTGTAGCTCTT 193 114 48 TGAGGGTGTGAACCACTCTG 194 GAATCCTGGTGTGCCCAAGT 195 137
[0248] (Perkin Elmer Cetus). “Touchdown” PCR was done as follows: 30 sec at 94EC, 30 sec at (T+11−n)EC (T is listed in Table 2 and n is cycle number), 1 min at 72EC for the first 10 cycles, and 30 sec at 94EC, 30 sec at TEC, 1 min at 72EC in the subsequent 25 cycles. The PCR products were separated on 3% Nusieve:Seakem agarose gels.
[0249] The Stanford G3 radiation hybrid panel (Cox et al.,
[0250] For radiation hybrid mapping, 40 ng of DNA from each of the 83 radiation hybrid cell lines were used as template, and PCR was performed with primers specific for a given cDNA clone (Table 3).
[0251] PCR was done in a 10 μl volume, and conditions were identical to those previously described for mapping with the chromosome 18 regional panel of somatic cell hybrids. Fifteen ng of human genomic DNA was used as positive control. The size of a PCR product, amplified from each radiation hybrid cell line, and a given pair of primers was determined by electrophoresis on a 3% Nusieve:Seakem agarose gel. For a given primer pair, the raw data indicating the presence or absence of an amplified product in each of the 83 radiation hybrid cell lines was submitted to the Stanford radiation hybrid e-mail server (http://shgc.stanford.edu/rhserver/intro.html). If linkage to reference markers was found, the mapping data transmitted from Stanford included a list of linked markers (STSs), lod scores and distances in cR
[0252] To isolate brain-expressed transcripts that map specifically to chromosome 18, we performed direct cDNA selection with pools of chromosome 18 biotinylated cosmid clones and primary cDNAs derived from human brain and placenta. After two cycles of selection, the secondary selected cDNA was PCR amplified, and this was found to have an average size of about 400 bp. Longer cognate cDNA clones were isolated by using labeled amplified pools of secondary selected cDNAs to probe high density filters of an arrayed, normalized infant brain library (Soares et al.,
[0253] Initially, we focused our analysis on clones that had these partial sequences to facilitate rapid chromosomal localization by PCR. The availability of these sequences also permitted comparison with sequences in the databases for homology to known genes, and evaluation of possible redundancies between the selected transcripts.
[0254] To determine the chromosomal location of the positive cDNA clones we designed PCR primers from the 3′ end sequence, whenever possible. Since the infant brain cDNA library was constructed by oligo (dT) priming and directional cloning this would most likely correspond to the 3′ untranslated region (UTR), which is usually unique and uninterrupted by introns (Sikela and Auffray,
[0255] In the initial step of the clone-based physical mapping, a panel of template DNAs was used for PCR amplification. These included: human placental DNA, somatic cell hybrid DNAs for the entire human chromosome 18 (HHW 324,
[0256] We found that the use of primers derived from 48 cDNA clones successfully chromosome into amplified unique bands of the expected size, specifically on chromosome 18 somatic cell hybrid DNA (Table 2). Further analysis using the same primer pairs (Table 2) revealed that each of these 48 clones mapped to a specific chromosome 18 cytogenetic bin (Table 3 and
[0257] Interestingly, most of 48 brain transcripts appeared to cluster within discrete cytogenetic regions on chromosome 18; bins A and B, in the short arm and bins M and S, in the long arm (Table 3 and
[0258] To determine the identity and uniqueness of each of the 48 chromosome 18-specific transcripts, a homology search against sequence databases was conducted. By comparison using a BLASTN similarity search with GenBank (Altschul,
[0259] Myelin basic protein (MBP, Kamholz et al.,
[0260] A FASTA (Pearson and Lipman, TABLE 4 Chromosome 18 specific brain derived cDNAs homologous to known genes. Clone BLASTN/TIGR Cytogenetic Percentage Identical Number sequence homology Location 5′ 2′ Reference 1 63 kDa protein kinase related to ERK3 (H863KDAP) 18q21.2-18q21.3 99.4 97.5 Li et al. 1994 3 63 kDa protein kinase related to ERK3 (H863KDAP) 18q21.2-18q21.3 95.3 96.2 Li et al. 1994 7 Myelin basic protein (MBP) 18q23 95.8 93.5 Kambols et al. 1986 10 63 kDa protein kinase related to ERK3 (H863KDAP) 18q21.2-18q21.3 99.6 99.4 Li at al. 1994 11 Protein tyrosine phosphatase, receptor-type, mu polypeptide (PTPRM) 18q11.2 none 89.2 Suijkerbuijk et al. 1993 12 Myelin basic protein (MBP) 18q23 95.7 92.1 Kambolz et al. 1986 18 Myelin basic protein (MBP) 18q23 94.5 99.1 Kambolz et al. 1986 31 Guanine nucleotide-binding protein, alpha-subunit, olfactory type (GNAL) 18q11.22-p11.21 98.9 98.6 Zigman et al. 1993 40 5′ 18q21.3-18qter 99.4 none Nomura et al. 1995 43 Protein tyrosine phosphatase, receptor-type, mu polypeptide (PTPRM) 18p11.2 94.0 90.0 Suijkerbuijk et al. 1993 46 Myelin basic protein (MBP) 18q23 95.3 none Kambolz et al. 1986
[0261] the transcripts for the known genes, we have identified a total of 30 unique transcripts, of which 25 did not exhibit homology to previously known genes. The insert sizes of the cDNA clones that were determined to be chromosome 18-specific ranged from 1 to 2 kb (Table 3). To explore the presence of an open reading frame (ORF) in each clone and to further examine any homology to known genes, we determined the remaining sequence of the unique clones (sequences were deposited in the Genbank, with the following accession numbers: U55777 and U55962 to U55991). We found potential polyadenylation signals in some of the clones. So far, no ORFs have been detected suggesting that a major portion of the cDNA clones corresponded to 3′ UTRs. More importantly, comparison of the longer sequences of the cDNAs with sequences in the databases failed to reveal significant homology with any known genes, supporting the idea that these transcripts were derived from novel genes.
[0262] To achieve a higher resolution map for each of the transcripts by PCR, we used the Stanford G3 radiation hybrid series and primers specific for each cDNA. Of the 25 unique transcripts, 19 were successfully linked to chromosome 18 STSs (see Table 5, below and
[0263] The positions of the cDNAs in the radiation hybrid framework map were consistent with their subchromosomal assignments (
[0264] Radiation hybrid mapping was also used to position the known genes identified in this study against the 25 non-redundant transcripts. We found that HS63 KDAP formed a high resolution linkage group with clones 2, 4, 19 and 33 (Table 5 and TABLE 5 Radiation hybrid mapping of unique cDNA clones* Test Reference Distance to Reference Marker Marker LOD Marker (cR8000) 14 D18S476 10.2 25.78 D18S481 13.3 15.04 D18S54 8.9 32.31 D18S63 10.9 22.50 D18S459 10.2 25.78 D18S1132 7.0 42.16 34 D18S476 8.3 32.61 D18S481 12.4 16.08 D18S54 12.7 15.96 D18563 24.6 9.61 D185459 14.6 9.61 14 7.6 38.31 D18S1132 6.8 41.79 24 D18S464 11.0 16.07 D18853 6.1 45.87 GNAL D18S4B2 7.8 36.15 D8S71 9.0 31.62 D18S53 D18S464 7.5 35.77 D18S482 7.0 39.77 D18S71 6.9 42.39 37 D18S73 7.1 40.23 D18S71 6.0 38.03 22 D18S73 7.7 37.31 D18S40 13.3 26.92 D18S40 D18S73 11.2 21.40 D18S71 7.3 40.07 D18837 D18S73 13.5 16.28 D18S71 7.6 39.76 39 D18S1101 7.8 37.31 13 D18S1160 7.8 28.72 D18S475 7.7 19.22 25 D18S454 10.3 10.73 8 D18S460 7.8 28.65 D18S72 7.8 28.72 15 D18S460 7.2 30.83 D18S72 8.5 24.10 8 12.5 8.60 4 D18S470 6.1 42.49 19 6.6 35.80 2 19 14.3 4.08 D18S470 12.6 11.47 4 6.0 40.67 D18S474 8.3 24.21 D18S69 8.2 27.02 HS63KDAP 2 10.5 17.02 D18S474 9.8 18.18 18 9.1 22.46 D18S470 7.7 33.44 D18S69 6.5 38.01 19 D18S470 10.3 19.96 D18S474 8.4 24.51 D18S69 7.1 33.41 33 D18S69 7.1 33.41 41 D18S436 6.5 33.52 D18S53 7.2 30.83 23 15.1 3.85 23 D18S58 6.6 35.71 43 23 14.4 7.28 41 12.7 11.44 D18S58 6.5 35.88 MRP D18S554 7.8 25.97 5 D18S70 7.0 21.37 6 D18S70 6.3 28.01 9 NL 16 NL 29 NL 30 NL 36 NL 47 NL HUMKIAAN NL PTPRM NL
[0265] unique transcripts showed that at least six radiation hybrid linkage groups were evident (
[0266] In sum, using direct cDNA selection and physical mapping by PCR, we have identified and positionally catalogued 48 chromosome 18-specific cDNAs that are expressed in infant brain. Sequence database comparison revealed a level of redundancy in the 48 clones, yielding a total of 30 unique transcripts. Five genes previously assigned to chromosome 18 were represented in these transcripts. Additional sequence analysis of the remaining 25 non-redundant cDNA clones and database comparisons failed to elicit any significant homology to known genes indicating that these brain-expressed transcripts represent novel genes.
[0267] So far, we have no evidence for possible redundancies among the unique transcripts due to alternative splicing or the presence of pseudogenes, but these probably are very minor components of the cDNA library. Polymeropoulos et al., Chromosomal distribution of 320 genes from a brain cDNA library,
[0268] In the pedigree series described in Berrettini et al., Psychiatric. Gene. 2:125-160 (1991), incorporated herein by reference) linkage disequilibrium with manic-depressive illness is observed for genes within the region of the radiation hybrid map (
[0269] The second pedigree series is the manic-depressive pedigree series recently made publicly available by the Nationals Institutes of Mental Health as part of its Genetics
TABLE 6 TRANSMISSION/DISEQUILIBRIUM TEST (TDT) ON CLONE 22 IN TWO PEDIGREE SERIES Allele Freq Transmitted Not Transmitted P-value Bethesda Bipolar Pedigree Series 1 0.344 37 59 0.025 2 0.656 59 37 NIMH Genetics Initiative Collaborative Series 1 0.376 83 116 0.019 2 0.622 118 83 0.014 3 0.002 0 2 N/A
[0270] Initiative. The statistical test is the transmission/disequilibrium test (TDT) of Spielman R. et al.,
[0271] This example sets forth the discovery, characterization and isolation of the novel inositol monophosphatase gene and protein of the invention, designated IMP.18p.
[0272] Linkage of manic depression/bipolar disorder to the broad pericentromeric region of chromosome 18 (Berrettini (1994) supra; Stine (1995)
[0273] As described in Example 11, above, markers within 18p11.2 showing increased sharing were mapped using a radiation hybrid (RH) panel to an approximately 6 megabase (Mb) region. These lines of evidence indicated this region on 18p11.2 is a site for the identification of transcripts and genes associated with bipolar disorder. Electronic databanks were searched for ESTs, sequence tag sites (STSs) and genes which had been mapped as being encoded in the general area of 18p11.2.
[0274] This search identified a human STS of 145 base pairs, identified as A006N05 (GenBank), localized between D18S464 and D18S71, markers that map to 18p11.2. This STS had been isolated and mapped by The Institute of Genome Research (TIGR) and was included in an approximately 1 kb TIGR EST, designated contig THC98649, described by Boguski (1997)
[0275] The STS sequence of A006N05 was searched using the transcript database of Schuler (1996) Science 274:540-546, http://www.ncbi.nlm.nih.gov/SCIENCE96/, and, updates in Unigene at http://www.tigr.org/tigr_home/index.html, of the National Center for Biotechnology Information. Using the method (described in Altschul (1990)
[0276] To identify which human cells express this or a message closely related to clone ID #39740, Northern blots of various human tissues was performed. Northern blots of multiple human tissues were purchased from Clontech (Palo Alto, Calif.). The Northern's probe was prepared by amplifying the insert of the cDNA clone #39740 with M13 forward and reverse primers, then
[0277] The amplified probe from cDNA clone ID #39740 was found to detect a major band of approximately 1.5 kb in multiple tissues through Northern hybridization, as shown in
[0278] cDNA clone #39740 was sequenced by conventional techniques. Analysis of this sequence showed that clone #39740 was missing its 5′-end coding sequence. Additional upstream coding sequence was acquired using rapid amplification of cDNA 5′ ends (5′ RACE) PCR, as described above. Marathon-Ready cDNA derived from human skeletal muscle (Clontech, Palo Alto, Calif.) and the clone-specific primer designated p1: 5′-ACGTCGGGCTGTGGGTGAGCACACACTTG (SEQ ID NO:24) (corresponding to nucleotides number 405 to 433 of clone #39740). PCR was performed using an initial one minute denaturation at 94° C., followed by 5 amplification cycles at 94° C. for 15 sec, 72° C. for 2 min; and, 30 cycles of 94° C. for 15 sec, 65° C. for 30 sec, 72° C. for 2 min, and final extension period at 72° C. for 5 min, using Taq DNA polymerase (Perkin Elmer) and MasterAmp 2× PCR PreMix I (Epicentre Technologies, Madison, Wis.). Sequencing was conducted using a dye terminator cycle sequencing kit with Taq FS (Perkin Elmer Applied Biosystems, Foster City, Calif.) and the ABI 373 DNA sequencer (Applied Biosystems, supra). Each nucleotide sequence was verified using at least two independent sequence reactions including both strands. Sequence similarity search, alignment and motif detection were done using the Genetics Computer Group, Inc. (GCG, Madison, Wis.) computer package.
[0279] The RACE method extended the upstream region of clone #39740 by 278 base pairs and included the potential initiation methionine.
[0280] The location of this IMP gene on chromosome 8 was established using ESTs from the Unigene databank (Boguski (1995) supra).
[0281] The 1447 bp full-length cDNA has a predicted open reading frame encoding a protein with 288 amino acids and a G-C rich 5′-untranslated region (UTR) (
[0282] Two protein motifs characteristic of the myo-inositol monophosphatase protein family, which includes animal inositol phosphatases, fungal and bacterial regulatory proteins of unknown enzymatic activity as found by Neuwald (1991)
[0283] In IMP.18p, motif A and motif B correspond to amino acids number 98 to 111 and 230 to 244, respectively, see
[0284] Northern hybridization was conducted under high stringency conditions to minimize cross hybridization with homologous mRNAs (i.e., wash conditions that minimized cross hybridization were used: 0.1×SSC, 0.1% SDS, 65° C.). The human chromosome 8 IMP of McAllister, (1992) supra, expresses a transcript that is 2.2 kb (Pollack (1993) supra), which distinguishes it from the novel IMP of the invention, IMP.18p, whose primary mRNA transcript, as determined by Northern blot, is 1.5 kb in length (see
[0285] To achieve a fine physical localization on chromosome 18, IMP.18p was further mapped utilizing radiation hybrid (RH) mapping (using the Stanford Human Genome Center's (SHGC) G3 panel, as described by Cox (1990) “Radiation hybrid mapping: a somatic cell genetic method for constructing high resolution maps of mammalian chromosomes.”
[0286] This example sets forth the discovery and characterization of the promoter region of the novel inositol monophosphatase gene, IMP.18p, of the invention.
[0287] To facilitate screening of the entire IMP.18p genomic sequence and to provide for a monophosphatase-specific transcriptional regulatory element for use in the construction of, for example, tissue-specific expression vectors, transgenic animal expression cassettes, and targets for expression-regulating nucleotide sequences, the promoter of IMP.18p was identified and characterized.
[0288] Cosmid clones from a chromosome 18-specific cosmid library LL18NC02, from Lawrence Berkeley Laboratory, were isolated by spotting the library onto nylon membranes to generate high density filters. These filters were hybridized with a IMP.18p cDNA probe (SEQ ID NO:16). Three clones, designated 119C4, 97A4 and 69E10, which hybridized to the cDNA probe were isolated. Sequencing was performed using the dye terminator cycle sequence kit with TaqFS from Perkin Elmer-Applied Biosystems, Inc. (ABI, Foster City, Calif.) and an ABI 373 DNA sequencer.
[0289] The transcriptional initiation site was determined by primer extension using a “Primer Extension System” from Promega (Madison, Wis.). An IMP18.p-specific antisense oligonucleotide primer (underlined coding sequence in
[0290] The major extension product was 183 base pairs (SEQ ID NO:29) corresponding to 160 base pairs upstream of the initiation ATG. as shown in
[0291] The sequence around the transcription initiation site did not indicate the presence of TATA and CAAT boxes. However, there were multiple, potential recognition sites for Spl, in addition to consensus sites for other transcription factors, as indicated in
[0292] All publications and patents mentioned in this specification are herein expressly incorporated by reference into the specification for all purposes to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference.
(Upstream) SEQ ID NO: 1 (Downstream) SEQ ID NO: 2 Clone 22 primer sequence: upstream primer: 5′-CAA GTT TAT GTT ACT GCC AGG G-3′ downstream primer: 5′-GCA GCT TOC TAA TGC ATC CAG-3′ (unspliced protein) SEQ ID NO: 3 (alternatively spliced protein) SEQ ID NO: 4 (spliced portion) SEQ ID NO: 5 1 MPEAGFQATN AFTECKFTCT SGKCLYLGSL VCNQQNDCGD NSDEENCLLV 51 TEHPPPGIFN SELEFAQIII IVVVVTVMVV VIVCLLNHYK VSTRSFINRP 101 NQSRRREDGL PQEGCLWPSD SAAPRLGASE IMHAPRSRDR FTAPSFIQRD 151 RFSRFQPTYP YVQHEIDLPP TLSLSDGEEP PPYQGPCTLQ LRDPEQQMEL 201 NRESVRAPPN RTIFDSDLID IAMYSGGPCP PSSNSGISAS TCSSNGRMEG 251 PPPTYSEVMG HHPGASFLHH QRSNAHRGSR LQFQQNNAES TIVPIKGKDR 301 KPGNLV (comprising SEQ ID NO: 7, coding far protein of SEQ ID NO: 6 SEQ ID NO:3) and (coding for protein cf SEQ ID NO: 4) ) SEQ ID NO: 8 Clone 22 common region nucleotide sequence Length: 8065 CCCAGCAGAG CGATGGACTT GGACAGGCTA AGATGGAAGT GACCTGAGCC 51 TCGCCCGCCG GCTTCCTCGA CGGGACAGCG CAAGAGTTGG AGCACAGGCT 101 TGTCCGGGGA GCAGTATGCC GGAAGCTGGT TTTCAGGCCA CAAATGCTTT 151 CACAGAGTGC AAATTCACCT GCACCAGTGG TAAATGCTTG TATCTTGGTT 201 CGCTGGTCTG TAACCAACAG AACGACTGTG GGGACAACAG TGACGAAGAG 251 AACTGTCTCC TGGTGACCGA GCACCCGCCT CCGGGCATCT TCAACTCGGA 301 GCTGGAGTTC GCCCAAATCA TCATCATGGT CGTGGTGGTC ACGGTGATGG 351 TGGTGGTCAT CGTCTGCCTG CTGAACCACT ACAAAGTCTC CACGCGGTCC 401 TTCATCAACC GCCCGAACCA GAGCCGGAGG CGGGAGGACG GGCTGCCGCA 451 GGAAGGGTGC CTGTGGCCTT CAGACAGCGC CGCACCGCGG CTGGGCGCCT 501 CGGACATCAT GCATGCCCCG CGGTGGAGGG ACAGGTTCAC AGCGCCGTCC 551 TTCATCCAGA GGGATCGCTT CAGCCGCTTC CAGCCCACCT ACCCCTATGT 601 GCAGCACGAG ATTGATCTTC CTCCCACCAT CTCCCTGTCC GACGGTGAAG 651 AGCCACCTCC TTACCAGGGG CCCTGCACCC TGCAGCTCCG GGACCCTGAA 701 CAGCAGATGG AACTCAACCG AGAGTCCGTG AGGGCCCCAC CCAACCGAAC 751 CATATTTGAC AGTGATTTAA TAGACATTGC TATGTATAGC GGGGGTCCAT 801 GCCCACCCAG CAGCAACTCG GGCATCAGTG CAAGCACCTG CAGCAGTAAC 851 GGGAGGATGG AGGGGCCACC CCCCACATAC AGCGAGGTGA TGGGCCACCA 901 CCCAGGCGCC TCTTTCCTCC ATCACCAGCG CAGCAACGCA CACAGGGGCA 951 GCAGACTGCA GTTTCAGCAG AACAATGCAG AGAGCACAAT AGTACCCATC 1001 AAAGGCAAAG ATAGGAAGCC TGGGAACCTG GTCTGATTCC TTCCAACGTG 1051 CACTTCAGCT GGAGAAAGAA ACCAAGAAGG GAAGCGGCCG CTGGGCCCCT 1101 CCTGGGCACA GTGTTGTTCA GTTTCACATG GTACAAATAA GTAAAACCAA 1151 ATGAGCAAAC ACGGTCTTTG TTTCTGATTC CTTTTAGGGG AATTGCATGC 1201 AAACTAGACT GAAATGATAC AAACTTCCAT CTGGTCTGAC CGCAAACAGT 1251 GTTTATTTGG GGACAGGGGT TGGGATGGGG GTGTGGGCAG GGGAAAACAG 1301 AGAACGGGAT GCTTTGAAGA TACCATGAAA TAAAACCCAC AGAGGTATTT 1351 GATGTATTTA ATTGTGAAAG GAGACTTTGC AGATAAATGA GGCCAGAATG 1401 GCATGTTTTA TAATTAACTG AATAAAGAAG GAAGCATTAT TATATATTAT 1451 TGTGGGGAAG AACCAGCCAG TTCGCTTTTT CTCCTAAGGT GTGGACTTTT 1501 ATTTTGTTTT AAAAATATGA ATCAAAATTC CTGTGTTGTG TGCCAAGGTA 1551 TAAAGTGGAG AAGTTAGATG AGTGCAAGGA GCTCCTTTGT GTTGTGATGA 1601 TGTGTTTTAA AAGTTGCACT ATCTTAATGT TGAAAATATT TACAAGGGAA 1651 CTGTTTTACG TGAAGTTCTG TATGTTGTCT TTTCACCTGT GGATTGTAAT 1701 CAGGCCCAAG GAATATCCTG GAGTGGTCCC CAGAAGCATC CAAGAAAAGA 1751 TATTTGGGGA CGTAGCCTAA CATTTTACCA ACTTACGTAA ATCAAAAAAG 1801 TCATTATTGT TGCAGGAGTT TGCATCAAAT AGCAGTGCAT CGCTGAAGCT 1851 TTTGGAGACT TTTGGATGGA AGATAAGATA GGGAAGATTA AGTTCCAGCA 1901 TTTCTGACTT GTTATTTTGA GTTACTCTGC TACTCTTAGG CTGCATAGTT 1951 TATGAGAAAA TGAACACATG CATTTATGGA TCCAGTATCA TGCAGTGCTG 2001 CCCTCATCCT CCAGCAGTGC AATTTCTTCA GTAATTTAGA TTTTTTTCAC 2051 TATAGCATGA AATATATTCA AATACATACC TTATTTTATG CAATAAATTG 2101 TTTAAAATGC AAGGTGGTTA TTCTGCATAC TGTTGAAATA TGTGACTCCT 2151 CAGTATATTC CCATTGCCTC TCCCCCTTTC CTCGACAGCT TAGTTCAGTT 2201 CTGCAGGGCT GCTCAGTTCA CAGGAGGCTC CCAGCAGCCA CCCCACATCC 2251 AGCCTACACA GAACTTTCGT GTGGGAGTGG TGTGGGTGGT GGTTTTCTTA 2301 TGCTTTGGAA GCCCCTAGAA ATAATGACGG AAGAATGCCA TGTTGCTGAT 2351 CGTGGTAATA AGCCATTGTG GGTTATTGTA TGTCACTAGT ATTAGCATAG 2401 CATTCTTAAA GGAATGCAGT GTTCAAAACC TACCCAAATT CCCCGCAGGA 2451 TTTTACCAAA CCCTTCCCCA GGCCAGTTTT GTACTGAAGG CAAGAACTGG 2501 ACAGTCAGAG AACAGTGGAG GGGGCAAGTG ACTGAAGAGC ACCGGGTAAA 2551 AAGCACAACA TGCAGTTAAA ATGCAAACTA GAAAACTAAT TTTAAATATT 2601 GTTAGTTTTA ATATTTCCTG ATATTTACAA ATATTCATTC TTATATACAA 2651 TGAAAAAAAT AACTTTCTTC TGCAGATGTA AGCACTGGCT TTTATAAGAG 2710 CAGCAGCCAA CACGTTTAGC AGACACTGCG CGTGGAGAAG GGCTTATCTG 2751 CAGTACACTC TGCCATGTGG AGGGTGGGCC TCTGTGGCCT CTTCACATAA 2801 CAAGATGAGC TGGAATGATG ATTCCATGAC TCCCACCTAT GCAGCCTTAA 2851 AGCCAAATCC GCGTGTGTGT GTTTGTGTCT GTCTGTGGGT CTCGAAGGTG 2901 ATCCGTCGGT GCGGTGGCTC TGTGCTGTAA CTGGAGAGAC TGTTCCAAAC 2951 CCCAAGAGTT GTCTGATCCT AGTCTGTTCC CTTCTGCTTC TTACCTCTGT 3001 AGATAGGTCA CTGGTTTTTG TTTGTTTGTT TTGAGGATTG GAATTTCCAT 3051 TACATTCATC CTTTGCACAC AGTAACATCC ACAGAACTAG TCCAACTCTT 3101 AAAAGGAGAG AGGAAAAACA CAGGCACCAG TTGTCAGCTC ATCGTTACAA 3151 CCTGTGTGGA AGTATATACA GTTGAGAGTC ACAGTGGAGG TTCTGAGACT 3201 GGATTCAGTC TTGTTCCAGT GACAGTTGGA AGGCCTCTGC TGGAGAGACA 3251 CCAGCTCTCA GGGCAGAGAT TGGCTTGGGG CCAGAAGGAC CCTCCCCAAC 3301 CCTGGAGACA CCCTGAAGGT TCACTGGCTC TCCAGATTAG CCTCTCTTCC 3351 TCTGTCAGGC AAAGATGAGG AGCCCGTGTT CCCATCGGGC CCTGCTGGCA 3401 GGGACTTGCA GTGGATTCTT GGTCAGGTGT GCCCACAGAT GCGGAGGCGA 3451 GGTGAGTGAT TCCATCATTT CAGTTCTCAC CTGCAGTTTT GGTGAAGCAG 3501 GAGATGCACC CCACAGCTCT AGCTCTCAAA TGGCTTCACA GTCCTTACTT 3551 CTCTACCTGC CTCAAGAAGG GGCTCAGAGC AGAGACTTGT GAATTCCTTA 3601 GTAACTGTGA GTATATGAAT GTGTTGCACA TGTCCACAGT ATTGGCCAGA 3651 TAATTACATA ATTCAGATAC CTTTAATCAT CTTTCAAGAA AGAGGCTCCT 3701 CCCATTCAAC CACCCTAGAG AACTGCCTTT GTTAAATAGT TATTTAAAGA 3751 CTCATACATA TCAAACCATG ACTTTGAAAG GTCTTCGAGG CTGGGGCTCT 3801 GTAATGAATT AGTTTAAAAG CCAAGGTCAT AACATGAATT GATGGTCAAT 3851 TTCCCTTCAG CAGAAGGAAA AGGTGATTTA GATCAGTAGC TCTTTTGAAG 3901 GTTGTGGCTG ACCTGTTCAT ACCGTGTCGC CTCATGGCTA GTGTGGCGTT 3951 GAAAGAGTAG CGACTGGGAA GATACAACTT ACACAGTGGG GCCTATTGTT 4001 CTTTCAAGAA CCCTTTTTTT AGCTTATAGA ACCCATGGGT CCAGTTTAGT 4051 AACGAGTGAT TTAGGCAATC AATGATAGGT TTATAATCTT AGATTATTCC 4101 AGCAAAGTGT GGATTGCATT GTTAGGAAGA ACATTTGGTG GGAATGAACA 4151 CTCCTGGGCA TACCGCTGAC TTTTGTCCCT TGTTCCCGGT GTAGGAGACC 4201 CAAGGCATCT TGAATCCCAT CTATAAGAAC ACAATCTTCC AGCATACGTT 4251 TGCTTTTTCA GAAACTCTAG CATTCTCTTT AAATACTGAC GCAATCCTTA 4301 ATGGAAAAGA GATTTCATGA AGCAAATTAT GTATTTCAAT AGTTCTTCTA 4351 TTTTTAGTGT CCAAAATTTA CTAATACAGA AGCTTGACAA GCATGTCCTC 4401 ACCCTCCCCA CCACATAAAC ACATGGACAC ACACCCAAGC CACAAGAAAT 4451 CCCAAGAGAG CAGAAGCGAA TTTTTAAAAG ATTTATCGTG AGGACTGCAT 4501 TTCCATTCAC TAATTTTGGC TCAAACTTAT GAGGCAGGAA ATAGGGGCCA 4551 ACAGTAAATG GGGGAGGCCT CCTGACACCA GCAGAGGAAT TTTGTACCCA 4601 GGCGAGCACT TCTTGAACTT CTGCGTATCT CCGTTTGATC TCTTTCACCT 4651 TTATTTCATC TTCATAAGAA TGAGAAAGGC TCAAAAGGAA GCACTTTTAG 4701 AAATCTTCTC TGACCTAGAA GAATCCATCC AAATCCCTGC CTTCCTCTCT 4751 GAACCAACAG TTCCCTTCTC TGACAGGGGG CCATCCTCTA TATTCCATCC 4801 AGCGGCTCTT CCTTTTAGGA AGGCTCTGGT GCAGAGCACT TCAAATATGT 4851 CCTCAGGCCA GATACTGATT GCTAGTAGAG AGACACCCGG CACCCAGTCC 4901 GAAGCCCTCC CTCAAAGGAC CGGCTTATGG CGTTGGTCAC TGGCAGGCTC 4951 AGAGACATTC TACTGTGGGC GCAGGGAGCC CGGCCCCCCA TGCAGCCATG 5001 ACTGGATGCG CCCCCATCTC GGGGGCTTGC TGCACTGCTT GTTTATTGAA 5051 TTTTGCTACT TAGAATGGCA ACATTAACTT TGTGTACCAT TCATTTTTTA 5101 AAAATTTTCC AAAGCTCGGC AGTGTATGAA AGAAAAAACT GGGAAAGATA 5151 CTTGGTTTCT GTTAACTTTT GTGTTGCTTG CTTAAGTGAT TAAAGCCAGT 5201 GCTTGGAGCC AAGCCTTCAT GCCACGAACA TGCTCCACAG CCTGCCCTTT 5251 GCTCTCCTGC TCACACTGAC CAAGAATGCC GCGTGCTTGG CCTACTGAGG 5301 TGAAAGGACA ATTGAATGAC AGGTGGGCAA AGGGAGAACT TCCCCTTCTT 5351 GGTGCGAGGA AAGTCACAAA TTTAAAAATG TTGCTTCCAG CCCAGATCCT 5401 AAATGCTAGT TCTCAGCAGC TGCGTGGCTT ACCGTTCGCC ATTTCCACCA 5451 CCGCCAGCTG CCAGCACCGC TACAGATCAC AGAGATGTGA ACAGACAATC 5501 GAAAGCACTC TTAGCCTTGC AGTGGTCTAC ATTTTTTAGG AACCAATATT 5551 TCAGCATTCT TTATTACCCG GCACGCTGTG TCCTTTGCAG AGTTCAAGTT 5601 TATGTTACTC CCAGGGTCAG ACAGTCATTT GTCGCTGCTG CTGCTGCTGC 5651 TGCTGCTTCT CGAACTGGAT GCATTAGGAA GCTGCTGTCT GAGTGTAGGA 5701 ATGTCTTGCT AAGAAAGCAA TGTCTTCCTT CATCCTTTTC TTTCTTCCCT 5751 CTGCGTGTCC TTGTTTTTGT GTAATGCGGG AGAGGGTTAG AGCTATAGAG 5801 ATTATATATA CACTATCCGT GCACATTATA TATATGTAGA TATACCCCTA 5851 TCATGTCAGA GATCTGCATG TCAGTTTTTC AGCAACTAAG GTGCCTCATG 5901 TTCTGAGTTC AGCAGATATA CGAACCAAGC CGCCCCCTCC TGCACTTGAT 5951 GCTCCCACCT TTGTTGTGCC TCACTTAAAA TGGTGCTTTT TTCAGTTGTC 6001 TGTCTTTTCT TATGTTTTTA TTTGTAAGGT GCTGTATATA AGTTGAATAT 6051 ATTATGCACA TATCCTACCC AATGGGTAGA ACAAAAAGTT GTTAATACTG 6101 TAATATAATG TATAGATGAT ACCAATTTTA ACAGAAATGG CATAGAATTT 6151 GTGAATGCCT ATGTGCTTTG TCCTCTTTTG TAAGGAAATT TGCAAATGGA 6201 TGCATACAGA TTAAAGTCTA TGTAGTTTAT TTTCCTATTA AATATCAATA 6251 TTATAACACA AGAGAAAGAA GTGTGAACAA ACAAGCAACA GTTTATGACC 6301 AGCGTATATA TAGCAATGGA AAGTTGCATC TTTGCTGTGA AAACACTTTA 6351 AAGAAAATAC TTTTTAAAAA ATCCCACAGC TTTTTGGTTG CCACTAGACG 6401 CTTCTTATTT TAATCATTTT AGTAATGCTC AGCTGGACCA GTGTTAGTTA 6451 TATTTGAGTC AGAAAAATGT TGTTTTTCAA CTTGCTTTAT AATCTCCTGC 6501 ATCTATCTCC TGCTGTAGCA TCAyGAAGGT GTCAGGCAAC AGTGAAAAGT 6551 GCACATTTTT GTTGTTGCAG AAACTGTGTC AGAGGAATAA GTAAATCAGC 6601 CTGCAGCAGA AGACTTTGTT CAGCTCCAGA GGCATCTGTG ACCGTCTGTG 6651 TCCAAGTCTC TCTGTGCCTT TTTCTTTTAC AAACTGAAGC TGTGGAGCCA 6701 ATGAAGTAAC AGTAGAGATT GTAGGGAAAG AATACCTCAG GAAAAACAAA 6751 TACACTTACA AGAAGACCCT GTTCTTAGAA AATGTGTTTA GTTATGGGTT 6801 AGCACTAGAA GAGACTTGGC TGTCAGCCAG CCAAGTGAAG GACCTCTCAT 6851 CCATTCCCAT TCATGTCCCA TCATAATACG GACmCAAAAA GCAAACTCGG 6901 TTTTGCCATC AGTTAGAAAT TACGTTTTGG ATTGTATATT GTTACATCTC 6951 TCTTCCAGCT TAGTTTTTAG TGTCTGATTG TGACCTCTGC ATTTATCTTC 7001 AAATACCCTA ATTTTAAAAC AAAAGAACAA GAAAAGTTTA TAACACCATG 7051 TTCACTAAAA CCACGGTTGA ATCTTGGGTG TGGGCATCCT TTCGAGTGTT 7101 GTCCATAAGA GCAGTTCGTG GAATTTTGCC CATCTGACCC ATATTATCAG 7151 CTTATTCTGC CACCAGAGTA GAGTCTAATA AATTCCAAAG TTTTTATTTG 7201 CTCCATGGTG TATGTTCTGA CTTTGAAAAT GTCAGATTCT ATAATCATAC 7251 CCCTAACATC CAGGAGACAA ATGACAGATT ATCTTTAAAC TGAAATTGAC 7301 TCTACAATGC AACCCTTAAT GCTGAATGGA TTAAAAAAGT CAGCCCTTTT 7351 AGTATCTGTT TGAAAGGGCC GTAAAAAGTT GACACTTTTG TTGTTGTGGA 7401 TCCTGCGTGT CTAGACCCAC GTGTTGTTTC CATCGTATAC TGTAGGGTGC 7451 ACCCCTTGGG ATTCATCATT AAGAACTGAG GCTCACTGTT GTCAGAAACA 7501 AAGCTCCCAC CCCCCAGGTT CAACCTTGTG GGAGAACTGT TGAGCATGAG 7551 AATGTTCTAG ACTCAGAGGT ACTAAAATTT GTTACCACAT CATTGCTTCC 7601 TTTCTACAGG ACGAATTGAG GCTTAAACTT TACTGTTAAT GATACTGGTT 7651 CATTTTAATG TGCTTGTTGG TATGTTGCTA TTTTTCATTT CATAGCTTTC 7701 AAAAATCATG CTAATTGTAT ACTTGTCTAN TTTAAGGCTA TTTTAAAATA 7751 TGTACAATAC TATTCACAGC ATTTAGTTCG TTTAATTTTT ATTATAAAGC 7801 AATCTACTAA AAAAGTACAA CTGTATTTGA ACTTTTCAAT AGTTGTTTGT 7851 GAGCTATGAT AATCAAAAGT CATTAAAGTC TTTTTTAACA AACATTCGTG 7901 CTTACTTTTC AACATAATTC CCAGTTATAT ACAGAAAAAG ATTTCCACCT 7951 GTCACGTATC TGCCTCTTTT ACCTGAGCAA TGGTGTAGTT CTTANACCTA 8001 AGGTCTGTAA TTGCAATACT TTTAAAGAAA GAGTTGCTCT AAGTGCTGTT 8051 TGTTAGTTAT GAAAC SEQ ID NO: 9 PRIMER A: 5′ATGCC GGA AGC TGG TTT TCA GG 3′ SEQ ID NO: 10 PRIMER B: 5′TCC AGC TGA ACT GCA CGT TGGCT3′ (Primers for nucleic acid encoding protein of SEQ ID NO: 3) SEQ ID NO:12 1 TGCGAGAGCC GGGCAGGTGG GCCGCGGATG CTCCCAGAGG CCGG SEQ ID NO:13 1 ATTTCCAGTA GAGGTGGATA GAGATGGTGA GCAGCATTGA CTCTCAAAAA 51 TAGGGTCCTA TGGCTGGTAA GGAGGTTGGT GCCTTCTCGA AGGGCTAGTG 101 CTGGGAAGCT TCCTTTTAAA AACGGCCCTT TCTGCCGGTT TGGCTAGCCA 151 AGAATGGCAT CCTCCTCTCT GTATCTTCCC TGGAGCTTCA GGACTGAGTA 201 TTGAATGACA GAGAAGGTTC TGCAAAGTCT GCACAGGGAG ACTGCCATTG 251 CATCAAGTCA TGTCTGCATT CTGTATATGC GGTTCAAGCT CTACGTTCGT 301 GACATCAAAC CTCCTGTTCG GCCATTTCCG AGAACTCCCA TCAGTTTCTG 351 TATAGTGTAA AAGTTTCAGA GGCGGAGCAC AGAGAGCTGC GGCTGGGACA 401 AGGAGCACCC GCGTGCAGGT GCGACCCTGC AGGATGCTGG CAGCGGCGTG 451 GCCAGGGGCG CCCGTGTTCT GAGGGCCTGA CGGCCAGCCC C (allele 1) SEQ ID NO: 14 (allele 2) SEQ ID NO: 15 Clone 22 Allele 1 CAAGTTTATGTTACTGCCAGGCTCAGACAGTCATTT Clone 22 Allele 2 CAAGTTTATGTTACTGCCAGGGTCAGACAGTCATTT
[0293] Underline shows the polymorphic repeat sequence.