cDNAs coding for members of the carcinoembryonic antigen family

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns nucleic acid sequences which code for carcinoembryonic antigen (CEA) antigen family peptide sequences.
2. Background Information
Carcinoembryonic antigen was first described by Gold and Freedman, J. Exp. Med., 121, 439-462, (1965). CEA is characterized as a glycoprotein of approximately 200,000 molecular weight with 50-60% by weight of carbohydrate. CEA is present during normal human fetal development, but only in very low concentration in the normal adult intestinal tract. It is produced and secreted by a number of different tumors.
CEA is a clinically useful tumor marker for the management of colorectal cancer patients. CEA can be measured using sensitive immunoassay methods. When presurgical serum levels of CEA are elevated, a postsurgical drop in serum CEA to the normal range typically indicates successful resection of the tumor. Postsurgical CEA levels that do not return to normal often indicate incomplete resection of the tumor or the presence of additional tumor sites in the patient. After returning to normal levels, subsequent rapid rises in serum CEA levels usually indicate the presence of metastages. Slower postsurgical rises from the normal level are most often interpreted to indicate the presence of new primary tumors not previously detected. Post surgical management of colon cancer patients is thus facilitated by the measurement of CEA.
CEA is a member of an antigen family. Because of this, the immunoassay of CEA by presently available methods is complicated by the fact that CEA is but one of several potentially reactive antigens. There have been at least sixteen CEA-like antigens described in the literature. Since some of these appear to be the same antigen described by different investigators, the actual number of different antigens is somewhat less than this number. Nonetheless, there is a complex array of cross-reactive antigens which can potentially interfere with an immunoassay of the CEA released by tumors. It is known that serum levels of CEA-like antigens are elevated in many non-cancerous conditions such an inflammatory liver diseases and also in smokers. It is important that immunoassays used for the monitoring of cancer patient status not be interfered with by these other CEA-like antigens. Conversely, it is important to be able to distinguish the antigens by immunoassays because of the possibility that different tumor types may preferentially express different forms of CEA. If so, then the ability to reliably measure the different forms of CEA can provide the means to diagnose or more successfully treat different forms of cancer.
The members of the "CEA family" share some antigenic determinants. These common epitopes are not useful in distinguishing the members of the antigen family and antibodies recognizing them are of little use for measuring tumor-specific CEA levels.
U.S. Pat. No. 3,663,684, entitled "Carcinoembryonic Antigen and Diagnostic Method Using Radioactive Iodine", concerns purification and radioiodination of CEA for use in a RIA.
U.S. Pat. No. 3,697,638 describes that CEA is a mixture of antigens (components A and B in this case). U.S. Pat. No. 3,697,638 mentions methods for separating and radioiodinating each component and their use in specific RIA's.
U.S. Pat. No. 3,852,415, entitled "Compositions for Use in Radioimmunoassay, as Substitute for Blood Plasma Extract in Determination of Carcinoembryonic Antigen" relates to the use of a buffer containing EDTA and bovine serum albumin as a substitute for plasma as a diluent for CEA RIA's.
U.S. Pat. No. 3,867,363, entitled "Carcinoembryonic Antigens", is directed to the isolation of CEA components A and B, their labelling and use in a RIA.
U.S. Pat. No. 3,927,193, entitled "Localization of Tumors by Radiolabelled Antibodies", concerns the use of radiolabelled anti-CEA antibodies in whole body tumor imaging.
U.S. Pat. No. 3,956,258, entitled "Carcinoembryonic Antigens", relates to the isolation of CEA components A and B.
U.S. Pat. No. 4,086,217, entitled "Carcinoembryonic Antigens", is directed to the isolation of CEA components A and B.
U.S. Pat. No. 4,140,753, entitled "Diagnostic Method and Reagent", concerns the purification of a CEA isomer called CEA-S1 and its use in a RIA.
U.S. Pat. No. 4,145,336, entitled "Carcinoembryonic Antigen Isomer", relates to the antigen CEA-S1.
U.S. Pat. No. 4,180,499, entitled "Carcinoembryonic Antigens", describes a process for producing CEA component B.
U.S. Pat. No. 4,228,236, entitled "Process of Producing Carcinoembryonic Antigen", is directed to the use of the established cell lines LS-174T and LS-180 or clones or derivatives thereof for the production of CEA.
U.S. Pat. No. 4,272,504, entitled "Antibody Adsorbed Support Method for Carcinoembryonic Antigen Assay", concerns two concepts for the radioimmunoassay of CEA. First, U.S. Pat. No. 4,272,504 relates to a sample pretreatment in the form of heating to 65 to 85.degree. C. at pH 5 to precipitate and eliminate extraneous protein. Second, it describes the use of a solid phase antibody (either on beads or tubes) as a means to capture analyte and radiolabelled CEA tracer.
U.S. Pat. No. 4,299,815, entitled "Carcinoembryonic Antigen Determination", concerns diluting a CEA sample with water and pretreating by heating to a temperature below which precipitation of protein will occur. The pretreated sample is then immunoassayed using RIA, EIA, FIA or chemiluminescent immunoassay.
U.S. Pat. No. 4,349,528, entitled "Monoclonal Hybridoma Antibody Specific for High Molecular Weight Carcinoembryonic Antigen", is directed to a monoclonal antibody reacting with 180 kD CEA, but not with other molecular weight forms.
U.S. Pat. No. 4,467,031, entitled "Enzyme-Immunoassay for Carcinoembryonic Antigen", relates to a sandwich enzyme immunoassay for CEA in which the first of two anti-CEA monoclonal antibodies is attached to a solid phase and the second monoclonal is conjugated with peroxidase.
U.S. Pat. No. 4,489,167, entitled "Methods and Compositions for Cancer Detection", describes that CEA shares an antigenic determinant with alpha-acid glycoprotein (AG), which is a normal component of human serum. The method described therein concerns a solid-phase sandwich enzyme immunoassay using as one antibody an antibody recognizing AG and another antibody recognizing CEA, but not AG.
U.S. Pat. No. 4,578,349, entitled "Immunoassay for Carcinoembryonic Antigen (CEA)", is directed to the use of high salt containing buffers as diluents in CEA immunoassays.
EP 113072-A, entitled "Assaying Blood Sample for Carcinoembryonic Antigen--After Removal of Interfering Materials by Incubation with Silica Gel", relates to the removal from a serum of a plasma sample of interfering substances by pretreatment with silica gel. The precleared sample is then subjected to an immunoassay.
EP 102008-A, entitled "Cancer Diagnostics Carcinoembryonic Antigen--Produced from Perchloric Acid Extracts Without Electrophoresis", relates to a procedure for the preparation of CEA from perchloric acid extracts, without the use of an electrophoresis step.
EP 92223-A, entitled "Determination of Carcinoembryonic Antigen in Cytosol or Tissue--for Therapy Control and Early Recognition of Regression", concerns an immunoassay of CEA, not in serum or plasma, but in the cytosol fraction of the tumor tissue itself.
EP 83103759.6, entitled "Cytosole-CEA-Measurement as Predictive Test in Carcinoma, Particularly Mammacarcinoma", is similar to EP 92223-A.
EP 83303759, entitled "Monoclonal Antibodies Specific to Carcinoembryonic Antigen", relates to the production of "CEA specific" monoclonal antibodies and their use in immunoassays.
WO 84/02983, entitled "Specific CEA-Family Antigens, Antibodies Specific Thereto and Their Methods of Use", is directed to the use of monoclonal antibodies to CEA-meconium (MA)-, and NCA-specific epitopes in immunoassays designed to selectively measure each of these individual components in a sample.
All of the heretofore CEA assays utilize either monoclonal or polyclonal antibodies which are generated by immunizing animals with the intact antigen of choice. None of them address the idea of making sequence specific antibodies for the detection of a unique primary sequence of the various antigens. They do not cover the use of any primary amino acid sequence for the production of antibodies to synthetic peptides or fragments of the natural product. They do not include the concept of using primary amino acid sequences to distinguish the CEA family members. None of them covers the use of DNA or RNA clones for isolating the genes with which to determine the primary sequence.
______________________________________DEFINITIONS______________________________________Nucleic Acid Abbreviations A adenine G guanine C cytosine T thymidine U uracilAmino Acid Abbreviations: Asp aspartic acid Asn asparagine Thr threonine Ser serine Glu glutamic acid Gln glutamine Pro proline Gly glycine Ala alanine Cys cysteine Val valine Met methionine Ile isoleucine Leu leucine Tyr tyrosine Phe phenylalanine Trp tryptophan Lys lysine His histidine Arg arginine______________________________________
Nucleotide--A monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C"), and thymine ("T"). The four RNA bases are A, G, C and uracil ("U").
DNA Sequence--A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
Functional equivalents--It is well known in the art that in a DNA sequence some nucleotides can be replaced without having an influence on the sequence of the expression product. With respect to the peptide this term means that one or more amino acids which have no function in a particular use can be deleted or replaced by another one.
Codon--A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode the amino acid leucine ("Leu"), TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.
Reading Frame--The grouping of codons during translation of mRNA into amino acid sequences. During translation, the proper reading frame must be maintained. For example, the sequence GCTGGTTGTAAG SEQ ID NO: 10 may be translated in three reading frames or phases, each of which affords a different amino acid sequence
GCT GGT TGT AAG Ala--Gly--Cys--Lys SEQ ID NO: 11 - G CTG GTT GTA AG Leu--Val--Val SEQ ID NO: 12 - GC TGG TTG TAA G Trp--Leu-(STOP) SEQ ID NO: 13.
Polypeptide--A linear array of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.
Genome--The entire DNA of a cell or a virus. It includes inter alia the structural genes coding for the polypeptides of the cell or virus, as well as its operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine-Dalgarno sequences.
Structural Gene--A DNA sequence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific polypeptide.
Transcription--The process of producing mRNA from a structural gene.
Translation--The process of producing a polypeptide from mRNA.
Expression--The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
Plasmid--A non-chromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (Tet.sup.R) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant".
Phage or Bacteriophage--Bacterial virus, many of which consist of DNA sequences encapsulated in a protein envelope or coat ("capsid protein").
Cloning Vehicle--A plasmid, phage DNA or other DNA sequence which is capable of replicating in a host cell, which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.
Cloning--The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
Recombinant DNA Molecule or Hybrid DNA--A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and have the capacity to infect some host cell and be maintained therein.
cDNA Expression Vector--A procaroytic cloning vehicle which also contains sequences of nucleotides that facilitate expression of cDNA sequences in eucaroytic cells. These nucleotides include sequences that function as eucaryotic promoter, alternative splice sites and polyadenylation signals.
Transformation/Transfection--DNA or RNA is introduced into cells in such a way as to allow gene expression. "Infected" referred to herein concerns the introduction of RNA or DNA by a viral vector into the host.
"Injected" referred to herein concerns the microinjection (use of a small syringe) of DNA into a cell.
CEA antigen family (CEA gene family)--a set of genes (gene family) and their products (antigen family) that share nucleotide sequences homologous to partial cDNA LV-7 (CEA-(a)) and as a result of theses similarities also share a subset of their antigenic epitopes. Examples of the CEA antigen family include CEA (.dbd.CEA-(b)), transmembrane CEA (TMCEA).dbd.CEA-(c) and normal crossreacting antigen NCA (.dbd.CEA-(d)).
SUMMARY OF THE INVENTION
The present invention concerns the following DNA sequences designated as TM-2 (CEA-(e)), TM-3 (CEA-(f)), TM-4 (CEA-(g)), KGCEA1 and KGCEA2, which code for CEA antigen family peptide sequences or nucleic acids having a base sequence (DNA or RNA) that are hybridizable therewith:
SEQUENCE AND TRANSLATION OF cDNA OF TM-2(SEQ ID NO:1) 10 30 50 . . . . . . CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA - 70 90 110 . . . . . . GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG MetGlyHisLeuSerAlaProLeuHisArgValArgValProTrpGln - 130 150 170 . . . . . . GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu - 190 210 230 . . . . . . ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCAC ThrThrGluSerMetProPheAsnValAlaGluGlyLysGluValLeuLeuLeuValHis - 250 270 290 . . . . . . AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAAC AsnLeuProGlnGlnLeuPheGlyTyrSerTrpTyrLysGlyGluArgValAspGlyAsn - 310 330 350 . . . . . . CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer - 370 390 410 . . . . . . GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp - 430 450 470 . . . . . . ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA ThrGlyPheTyrThrLeuGlnValIleLysSerAspLeuValAsnGluGluAlaThrGly - 490 510 530 . . . . . . CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro - 550 570 590 . . . . . . GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr - 610 630 650 . . . . . . CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly - 670 690 710 . . . . . . AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu - 730 750 770 . . . . . . ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC IleGlnAsnProValSerAlaAsnArgSerAspProValThrLeuAsnValThrTyrGly - 790 810 830 . . . . . . CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer - 850 870 890 . . . . . . CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr - 910 930 950 . . . . . . TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC PheGlnGlnSerThrGlnGluLeuPheIleProAsnIleThrValAsnAsnSerGlySer - 970 990 1010 . . . . . . TATACCTGCCACGCCAATAACTCAGTCACTGGCTGCAACAGGACCACAGTCAAGACGATC TyrThrCysHisAlaAsnAsnSerVAlThrGlyCysAsnArgThrThrValLysThrIle - 1030 1050 1070 . . . . . . ATAGTCACTGATAATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGC IleValThrAspAsnAlaLeuProGlnGluAsnGlyLeuSerProGlyAlaIleAlaGly - 1090 1110 1130 . . . . . . ATTGTGATTGGAGTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTG IleValIleGlyValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeu - 1150 1170 1190 . . . . . . CATTTCGGGAAGACCGGCAGGGCAAGCGACCAGCGTGATCTCACAGAGCACAAACCCTCA HisPheGlyLysThrGlyArgAlaSerAspGlnArgAspLeuThrGluHisLysProSer - 1210 1230 1250 . . . . . . GTCTCCAACCACACTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGAAGTTACT ValSerAsnHisThrGlnAspHisSerASnAspProProAsnLysMetAsnGluValThr - 1270 1290 1310 . . . . . . TATTCTACCCTGAACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTCCCCATCC TyrSerThrLeuAsnPheGluAlaGlnGlnProThrGlnProThrSerAlaSerProSer - 1330 1350 1370 . . . . . . CTAACAGCCACAGAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCTGTCCTGC LeuThrAlaThrGluIleIleTyrSerGluValLysLysGln - 1390 1410 1430 . . . . . . TCACTGCAGTGCTGATGTATTTCAAGTCTCTCACCCTCATCACTAGGAGATTCCTTTCCC - 1450 1470 1490 . . . . . . CTGTAGGGTAGAGGGGTGGGGACAGAAACAACTTTCTCCTACTCTTCCTTCCTAATAGGC - 1510 1530 1550 . . . . . . ATCTCCAGGCTGCCTGGTCACTGCCCCTCTCTCAGTGTCAATAGATGAAAGTACATTGGG - 1570 1590 1610 . . . . . . AGTCTGTAGGAAACCCAACCTTCTTGTCATTGAAATTTGGCAAAGCTGACTTTGGGAAAC - 1630 1650 1670 . . . . . . AGGGACCAGAACTTCCCCTCCCTTCCCCTTTTCCCAACCTGGACTTGTTTTAAACTTGCC - 1690 1710 1730 . . . . . . TGTTCAGAGCACTCATTCCTTCCCACCCCCAGTCCTGTCCTATCACTCTAATTCGGATTT - 1750 1770 1790 . . . . . . GCCATAGCCTTGAGGTTATGTCCTTTTCCATTAAGTACATGTGCCAGGAAACAGCGAGAG - 1810 1830 1850 . . . . . . AGAGAAAGTAAACGGCAGTAATGCTTCTCCTATTTCTCCAAAGCCTTGTGTGAACTAGCA - 1870 1890 1910 . . . . . . AAGAGAAGAAAATCAAATATATAACCAATAGTGAAATGCCACAGGTTTGTCCACTGTCAG - 1930 1950 1970 . . . . . . GGTTGTCTACCTGTAGGATCAGGGTCTAAGCACCTTGGTGCTTAGCTAGAATACCACCTA - 1990 2010 2030 . . . . . . ATCCTTCTGGCAAGCCTGTCTTCAGAGAACCCACTAGAAGCAACTAGGAAAAATCACTTG - 2050 2070 2090 . . . . . . CCAAAATCCAAGGCAATTCCTGATGGAAAATGCAAAAGCACATATATGTTTTAATATCTT - 2110 2130 2150 . . . . . . TATGGGCTCTGTTCAAGGCAGTGCTGAGAGGGAGGGGTTATAGCTTCAGGAGGGAACCAG - 2170 2190 2210 . . . . . . CTTCTGATAAACACAATCTGCTAGGAACTTGGGAAAGGAATCAGAGAGCTGCCCTTCAGC - 2230 2250 2270 . . . . . . GATTATTTAAATTGTTAAAGAATACACAATTTGGGGTATTGGGATTTTTCTCCTTTTCTC - 2290 2310 2330 . . . . . . TGAGACATTCCACCATTTTAATTTTTGTAACTGCTTATTTATGTGAAAAGGGTTATTTTT - 2350 2370 2390 . . . . . . ACTTAGCTTAGCTATGTCAGCCAATCCGATTGCCTTAGGTGAAAGAAACCACCGAAATCC - 2410 2430 2450 . . . . . . CTCAGGTCCCTTGGTCAGGAGCCTCTCAAGATTTTTTTTGTCAGAGGCTCCAAATAGAAA - 2470 2490 2510 . . . . . . ATAAGAAAAGGTTTTCTTCATTCATGGCTAGAGCTAGATTTAACTCAGTTTCTAGGCACC - 2530 2550 2570 . . . . . . TCAGACCAATCATCAACTACCATTCTATTCCATGTTTGCACCTGTGCATTTTCTGTTTGC - 2590 2610 2630 . . . . . . CCCCATTCACTTTGTCAGGAAACCTTGGCCTCTGCTAAGGTGTATTTGGTCCTTGAGAAG - 2650 2670 2690 . . . . . . TGGGAGCACCCTACAGGGACACTATCACTCATGCTGGTGGCATTGTTTACAGCTAGAAAG - 2710 2730 2750 . . . . . . CTGCACTGGTGCTAATGCCCCTTGGGAAATGGGGCTGTGAGGAGGAGGATTATAACTTAG - 2770 2790 2810 . . . . . . GCCTAGCCTCTTTTAACAGCCTCTGAAATTTATCTTTTCTTCTATGGGGTCTATAAATGT - 2830 2850 2870 . . . . . . ATCTTATAATAAAAAGGAAGGACAGGAGGAAGACAGGCAAATGTACTTCTCACCCAGTCT - 2890 2910 2930 . . . . . . TCTACACAGATGGAATCTCTTTGGGGCTAAGAGAAAGGTTTTATTCTATATTGCTTACCT - 2950 2970 2990 . . . . . . GATCTCATGTTAGGCCTAAGAGGCTTTCTCCAGGAGGATTAGCTTGGAGTTCTCTATACT - 3010 3030 3050 . . . . . . CAGGTACCTCTTTCAGGGTTTTCTAACCCTGACACGGACTGTGCATACTTTCCCTCATCC - 3070 3090 3110 . . . . . . ATGCTGTGCTGTGTTATTTAATTTTTCCTGGCTAAGATCATGTCTGAATTATGTATGAAA - 3130 3150 3170 . . . . . . ATTATTCTATGTTTTTATAATAAAAATAATATATCAGACATCGAAAAAAAAAA
SEQUENCE AND TRANSLATION OF cDNA OF TM-3(SEQ ID NO:2) 10 30 50 . . . . . . CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA - 70 90 110 . . . . . . GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG MetGlyHisLeuSerAlaProLeuHisArgValArgValProTrpGln - 130 150 170 . . . . . . GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu - 190 210 230 . . . . . . ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCAC ThrThrGluSerMetProPheAsnValAlaGluGlyLysGluBalLeuLeuLeuValHis - 250 270 290 . . . . . . AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAAC AsnLeuProGlnGlnLeuPheGlyTyrSerTrpTyrLysGlyGluArgValAspGlyAsn - 310 330 350 . . . . . . CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer - 370 390 410 . . . . . . GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp - 430 450 470 . . . . . . ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA ThrGlyPheTyrThrLeuGlnValIleLysSerAspLeuValAsnGluGluAlaThrGly - 490 510 530 . . . . . . CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro - 550 570 590 . . . . . . GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr - 610 630 650 . . . . . . CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly - 670 690 710 . . . . . . AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu - 730 750 770 . . . . . . ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC IleGlnAsnProValSerAlaAsnArgSerAspProValThrLeuAsnValThrTyrGly - 790 810 830 . . . . . . CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer - 850 870 890 . . . . . . CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr - 910 930 950 . . . . . . TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC PheGlnGlnSerThrGlnGluLeuPheIleProAsnIleThrValAsnAsnSerGlySer - 970 990 1010 . . . . . . TATACCTGCCACGCCAATAACTCAGTCACTGGCTGCAACAGGACCACAGTCAAGACGATC TyrThrCysHisAlaAsnAsnSerValThrGlyCysAsnArgThrThrValLysThrIle - 1030 1050 1070 . . . . . . ATAGTCACTGAGCTAAGTCCAGTAGTAGCAAAGCCCCAAATCAAAGCCAGCAAGACCACA IleValThrGluLeuSerProValValAlaLysProGlnIleLysAlaSerLysThrThr - 1090 1110 1130 . . . . . . GTCACAGGAGATAAGGACTCTGTGAACCTGACCTGCTCCACAAATGACACTGGAATCTCC ValThrGlyAspLysAspSerValAsnLeuThrCysSerThrAsnAspThrGlyIleSer - 1150 1170 1190 . . . . . . ATCCGTTGGTTCTTCAAAAACCAGAGTCTCCCGTCCTCGGAGAGGATGAAGCTGTCCCAG IleArgTrpPhePheLysAsnGlnSerLeuProSerSerGluArgMetLysLeuSerGln - 1210 1230 1250 . . . . . . GGCAACACCACCCTCAGCATAAACCCTGTCAAGAGGGAGGATGCTGGGACGTATTGGTGT GlyAsnThrThrLeuSerIleAsnProValLysArgGluAspAlaGlyTHrTyrTrpCys - 1270 1290 1310 . . . . . . GAGGTCTTCAACCCAATCAGTAAGAACCAAAGCGACCCCATCATGCTGAACGTAAACTAT GluValPheAsnProIleSerLysAsnGlnSerAspProIleMetLeuAsnValAsnTyr - 1330 1350 1370 . . . . . . AATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGCATTGTGATTGGA AsnAlaLeuProGlnGluAsnGlyLeuSerProGlyAlaIleAlaGlyIleValIleGly - 1390 1410 1430 . . . . . . GTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTGCATTTCGGGAAG ValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeuHisPheGlyLys - 1450 1470 1490 . . . . . . ACCGGCAGCTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGAAGTTACTTATTC ThrGlySerSerGlyProLeuGln - 1510 1530 1550 . . . . . . TACCCTGAACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTCCCCATCCCTAAC - 1570 1590 1610 . . . . . . AGCCACAGAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCTGAAAAAAAAAAA - 1630 . AAAAAAAAAA
SEQUENCE AND TRANSLATION OF cDNA OF TM-4(SEQ ID NO:3) 10 30 50 . . . . . . CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA - 70 90 110 . . . . . . GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG MetGlyHisLeuSerAlaProLeuHisArgValArgValProTrpGln - 130 150 170 . . . . . . GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu - 190 210 230 . . . . . . ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCAC ThrThrGluSerMetProPheAsnValAlaGluGlyLysGluValLeuLeuLeuValHis - 250 270 290 . . . . . . AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAAC AsnLeuProGlnGlnLeuPheGlyTyrSerTrpTyrLysGlyGluArgValAspGlyAsn - 310 330 350 . . . . . . CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer - 370 390 410 . . . . . . GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp - 430 450 470 . . . . . . ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA ThrGlyPheTyrThrLeuGlnValIleLysSerAspLeuValAsnGluGluAlaThrGly - 490 510 530 . . . . . . CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro - 550 570 590 . . . . . . GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr - 610 630 650 . . . . . . CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly - 670 690 710 . . . . . . AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu - 730 750 770 . . . . . . ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC IleGlnAsnProValSerAlaAsnArgSerAspProValThrLeuAsnValThrTyrGly - 790 810 830 . . . . . . CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer - 850 870 890 . . . . . . CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr - 910 930 950 . . . . . . TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC PheGlnGlnSerThrGlnGluLeuPheIleProAsnIleThrValAsnAsnSerGlySer - 970 990 1010 . . . . . . TATACCTGCCACGCCAATAACTCAGTCACTGGCTGCAACAGGACCACAGTCAAGACGATC TyrThrCysHisAlaAsnAsnSerVAlThrGlyCysAsnArgThrThrValLysThrIle - 1030 1050 1070 . . . . . . ATAGTCACTGATAATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGC IleValThrAspAsnAlaLeuProGlnGluAsnGlyLeuSerProGlyAlaIleAlaGly - 1090 1110 1130 . . . . . . ATTGTGATTGGAGTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTG IleValIleGlyValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeu - 1150 1170 1190 . . . . . . CATTTCGGGAAGACCGGCAGCTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGA HisPheGlyLysThrGlySerSerGlyProLeuGln - 1210 1230 1250 . . . . . . AGTTACTTATTCTACCCTGAACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTC - 1270 1290 1310 . . . . . . CCCATCCCTAACAGCCACAGAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCT - 1330 . GAAAAAAAAAAAAAAAAAA
The present invention is also directed to a replicable recombinant cloning vehicle ("vector") having an insert comprising a nucleic acid, e.g., DNA, which comprises a base sequence which codes for a CEA peptide or a base sequence hybridizable therewith.
This invention also relates to a cell that is transformed/transfected, infected or injected with the above described replicable recombinant cloning vehicle or nucleic acid hybridizable with the aforementioned cDNA. Thus the invention also concerns the transfection of cells using free nucleic acid, without the use of a cloning vehicle.
Still further, the present invention concerns a polypeptide expressed by the above described transfected, infected or injected cell, which polypeptide exhibits immunological cross-reactivity with a CEA, as well as labelled forms of the polypeptide. The invention also relates to polypeptides having an amino acid sequence, i.e., synthetic peptides, or the expression product of a cell that is transfected, injected, infected with the above described replicable recombinant cloning vehicles, as well as labelled forms thereof. Stated otherwise, the present invention concerns a synthetic peptide having an amino acid sequence corresponding to the entire amino acid sequence or a portion thereof having no less than five amino acids of the aforesaid expression product.
The invention further relates to an antibody preparation specific for the above described polypeptide.
Another aspect of the invention concerns an immunoassay method for detecting CEA or a functional equivalent thereof in a test sample comprising
(a) contacting the sample with the above described antibody preparation, and
(b) determining binding thereof to CEA in the sample.
The invention also is directed to a nucleic acid hybridization method for detecting a CEA or a related nucleic acid (DNA or RNA) sample in a test sample comprising
(a) contacting the test sample with a nucleic acid probe comprising a nucleic acid, which comprises a base sequence which codes for a CEA peptide sequence or a base sequence that is hybridizable therewith, and
(b) determining the formation of the resultant hybridized probe.
The present invention also concerns a method for detecting the presence of carcinoembryonic antigen or a functional equivalent thereof in an animal or human patient in vivo comprising
a) introducing into said patient a labeled (e.g., a radio-opaque material that can be detected by X-rays, radiolabeled or labeled with paramagnetic materials that can be detected by NMR) antibody preparation according to the present invention and
b) detecting the presence of such antibody preparation in the patient by detecting the label.
In another aspect, the present invention relates to the use of an antibody preparation according to the present invention for therapeutic purposes, namely, attaching to an antibody preparation radionuclides, toxins or other biological effectors to form a complex and introducing an effective amount of such complex into an animal or human patient, e.g., by injection or orally. The antibody complex would attach to CEA in a patient and the radionuclide, toxin or other biological effector would serve to destroy the CEA expressing cell.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the transmembrane CEA's

DETAILED DESCRIPTION OF THE INVENTION
In parent applications, applicants described the following CEA's:
______________________________________ ATCC No.______________________________________CEA-(a) partial CEA (pcLV7)CEA-(b) full coding CEA (pc 15LV7) 67709 - CEA-(c) TM-1 (FL-CEA; pc 19-22) 67710 - CEA-(d) NCA (pcBT 20) 67711______________________________________
In the present application, applicants described the following CEA's:
______________________________________ ATTC No.______________________________________CEA-(e) TM-2 (pc E22) 67712CEA-(f) TM-3 (pc HT-6) 67708 - CEA-(g) TM-4.______________________________________
ATCC Nos. 67708, 67709, 67710, 67711 and 67712 were all deposited with the American Type Culture Collection on May 25, 1988.
The sequences for CEA-(a), CEA-(b), CEA-(c) and CEA-(d) are given hereinbelow:
CEA-(a)(SEQ ID NO:4): - GG GGT TTA CAC AAC CAC CAC CCC ATC AAA CCC TTC ATC ACC AGC AAC AAC TCC AAC CCC GTG - GAG GAT GAG GAT GCT GTA GCC TTA ACC TGT GAA CCT GAG ATT CAG AAC ACA ACC TAC CTG - TGG TGG GTA AAT AAT CAG AGC CTC CCG GTC AGT CCC AGG CTG CAG CTG TCC AAT GAC AAC - AGG ACC CTC ACT CTA CTC AGT GTC ACA AGG AAT GAT GTA GGA CCC TAT GAG TGT GGA ATC - CAG AAC GAA TTA AGT GTT GAC CAC AGC GAC CCA GTC ACC CAG GCA TTC CTC TAT GGC CCA - GAC GAC CCC ACC ATT TCC CCC TCA TAC ACC TAT TAC CGT CCA GGG GTG GAA CCT CAG CCT - CTC TGC CAT GCA GCC TCT AAC CCA CCT GCA CAG TAT TCT TGG CTG ATT GAT GGG ACC GTC - CAG CAA CAC ACA CAA GAG CTC TTT ATC TCC AAC ATC ACT GAG AAG AAC AGC GGA CTC TAT - ACC TGC CAG GCC AAT AAC TCA GCC AGT GGC ACA GCA GGA CTA CAG TCA AGA CAA TCA CAG - TCT CTG CGG ATG CCC AAG CCC TCC ATC TCC AGC AAC AAC TCC AAA CCC GTG GAG GAC AAG - GAT CGC TGT GGC CTT CAC TGT GAA CCT GAG GCT CAG AAC ACA ACC TAC CTG TGG TGG GTA - AAT GGT CAG AGC CTC CCA GTC AGT CCC AGG CTG CAG CTG TCC AAT GGC AAC AGG ACC CTC - ACT CTA TTC AAT GTC ACA AGA AAT GAC GCA AGA GCC TAT GTA TGT GGA ATC CAG AAC TCA - GTG AGT GCA AAC CGC AGT GAC CCA GTC ACC CTG GAT GTC CTC TAT GGG CCG GAC ACC CCC - ATC ATT TCC CCC CCC CC - CEA-(b)(SEQ ID NO:5): - 10 30 50 . . . . . . CACCATGGAGTCTCCCTCGGCCCCTCCCCACAGATGGTGCATCCCCTGGCAGAGGCTCCTG MetGluSerProSerAlaProProHisArgTrpCysIleProTrpGlnArgLeuLeu - 70 90 110 . . . . . . CTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCAAGCTCACTATTGAA LeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaLysLeuThrIleGlu - 130 150 170 . . . . . . TCCACGCCGTTCAATGTCGCAGAGGGGAAGGAGGTGCTTCTACTTGTCCACAATCTGCCC SerThrProPheAsnValAlaGluGlyLysGluValLeuLeuLeuValHisAsnLeuPro - 190 210 230 . . . . . . CAGCATCTTTTTGGCTACAGCTGGTACAAAGGTGAAAGAGTGGATGGCAACCGTCAAATT GlnHisLeuPheGlyTyrSerTrpTyrLysGlyGluArgValAspGlyAsnArgGlnIle - 250 270 290 . . . . . . ATAGGATATGTAATAGGAACTCAACAAGCTACCCCAGGGCCCGCATACAGTGGTCGAGAG IleGlyTyrValIleGlyThrGlnGlnAlaThrProGlyProAlaTyrSerGlyArgGlu - 310 330 350 . . . . . . ATAATATACCCCAATGCATCCCTGCTGATCCAGAACATCATCCAGAATGACACAGGATTC IleIleTyrProAsnAlaSerLeuLeuIleGlnAsnIleIleGlnAsnAspThrGlyPhe - 370 390 410 . . . . . . TACACCCTACACGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGCCAGTTCCGG TyrThrLeuHisValIleLysSerAspLeuValAsnGluGluAlaThrGlyGlnPheArg - 430 450 470 . . . . . . GTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGAC ValTyrProGluLeuProLysproSerIleSerSerAsnAsnSerLysProValGluAsp - 490 510 530 . . . . . . AAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACGCAACCTACCTGTGGTGG LysAspAlaValAlaPheThrCysGluProGluThrGlnAspAlaThrTyrLeuTrpTrp - 550 570 590 . . . . . . GTAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACC ValAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGlyAsnArgThr - 610 630 650 . . . . . . CTCACTCTATTCAATGTCACAAGAAATGACACAGCAAGCTACAAATGTGAAACCCAGAAC LeuThrLeuPheAsnValThrArgAsnAspThrAlaSerTyrLysCysGluThrGlnAsn - 670 690 710 . . . . . . CCAGTGAGTGCCAGGCGCAGTGATTCAGTCATCCTGAATGTCCTCTATGGCCCGGATGCC ProValSerAlaArgArgSerAspSerValIleLeuAsnValLeuTyrGlyProAspAla - 730 750 770 . . . . . . CCCACCATTTCCCCTCTAAACACATCTTACAGATCAGGGGAAAATCTGAACCTCTCCTGC ProThrIleSerProLeuAsnThrSerTyrArgSerGlyGluAsnLeuAsnLeuSerCys - 790 810 830 . . . . . . CACGCAGCCTCTAACCCACCTGCACAGTACTCTTGGTTTGTCAATGGGACTTTCCAGCAA HisAlaAlaSerAsnProProAlaGlnTyrSerTrpPheValAsnGlyThrPheGlnGln - 850 870 890 . . . . . . TCCACCCAAGAGCTCTTTATCCCCAACATCACTGTGAATAATAGTGGATCCTATACGTGC SerThrGlnGluLeuPheIleProAsnIleThrValAsnAsnSerGlySerTyrThrCys - 910 930 950 . . . . . . CAAGCCCATAACTCAGACACTGGCCTCAATAGGACCACAGTCACGACGATCACAGTCTAT GlnAlaHisAsnSerAspThrGlyLeuAsnArgThrThrValThrThrIleThrValTyr - 970 990 1010 . . . . . . GCAGAGCCACCCAAACCCTTCATCACCAGCAACAACTCCAACCCCGTGGAGGATGAGGAT AlaGluProProLysProPheIleThrSerAsnAsnSerAsnProValGluAspGluAsp - 1030 1050 1070 . . . . . . GCTGTAGCCTTAACCTGTGAACCTGAGATTCAGAACACAACCTACCTGTGGTGGGTAAAT AlaValAlaLeuThrCysGluProGluIleGlnAsnThrThrTyrLeuTrpTrpValAsn - 1090 1110 1130 . . . . . . AATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGACAACAGGACCCTCACT AsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnAspAsnArgThrLeuThr - 1150 1170 1190 . . . . . . CTACTCAGTGTCACAAGGAATGATGTAGGACCCTATGAGTGTGGAATCCAGAACGAATTA LeuLeuSerValThrArgAsnAspValGlyProTyrGluCysGlyIleGlnAsnGluLeu - 1210 1230 1250 . . . . . . AGTGTTGACCACAGCGACCCAGTCATCCTGAATGTCCTCTATGGCCCAGACGACCCCACC SerValAspHisSerAspProValIleLeuAsnValLeuTyrGlyProAspAsnProThr - 1270 1290 1310 . . . . . . ATTTCCCCCTCATACACCTATTACCGTCCAGGGGTGAACCTCAGCCTCTCCTGCCATGCA IleSerProSerTyrThrTyrTyrArgProGlyValAsnLeuSerLeuSerCysHisAla - 1330 1350 1370 . . . . . . GCCTCTAACCCACCTGCACAGTATTCTTGGCTGATTGATGGGAACATCCAGCAACACACA AlaSerAsnProProAlaGlnTyrSerTrpLeuIleAspGlyAsnIleGlnGlnHisThr - 1390 1410 1430 . . . . . . CAAGAGCTCTTTATCTCCAACATCACTGAGAAGAACAGCGGACTCTATACCTGCCAGGCC GlnGluLeuPheIleSerAsnIleThrGluLysAsnSerGlyLeuTyrThrCysGlnAla - 1450 1470 1490 . . . . . . AATAACTCAGCCAGTGGCCACAGCAGGACTACAGTCAAGACAATCACAGTCTCTGCGGAG AsnAsnSerAlaSerGlyHisSerArgThrThrValLysThrIleThrValSerAlaGlu - 1510 1530 1550 . . . . . . CTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGACAAGGATGCTGTG LeuProLysProSerIleSerSerAsnAsnSerLysProValGluAspLysAspAlaVal - 1570 1590 1610 . . . . . . GCCTTCACCTGTGAACCTGAGGCTCAGAACACAACCTACCTGTGGTGGGTAAATGGTCAG AlaPheThrCysGluProGluAlaGlnAsnThrThrTyrLeuTrpTrpValAsnGlyGln - 1630 1650 1670 . . . . . . AGCCTCCCAGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTATTC SerLeuProValSerProArgLeuGlnLeuSerAsnGlyAsnArgThrLeuThrLeuPhe - 1690 1710 1730 . . . . . . AATGTCACAAGAAATGACGCAAGAGCCTATGTATGTGGAATCCAGAACTCAGTGAGTGCA AsnValThrArgAsnAspAlaArgAlaTyrValCysGlyIleGlnAsnSerValSerAla - 1750 1770 1790 . . . . . . AACCGCAGTGACCCAGTCACCCTGGATGTCCTCTATGGGCCGGACACCCCCATCATTTCC AsnArgSerAspProValThrLeuAspValLeuTyrGlyProAspThrProIleIleSer - 1810 1830 1850 . . . . . . CCCCCAGACTCGTCTTACCTTTCGGGAGCGAACCTCAACCTCTCCTGCCACTCGGCCTCT ProProAspSerSerTyrLeuSerGlyAlaAsnLeuAsnLeuSerCysHisSerAlaSer - 1870 1890 1910 . . . . . . AACCCATCCCCGCAGTATTCTTGGCGTATCAATGGGATACCGCAGCAACACACACAAGTT AsnProSerProGlnTyrSerTrpArgIleAsnGlyIleProGlnGlnHisThrGlnVal - 1930 1950 1970 . . . . . . CTCTTTATCGCCAAAATCACGCCAAATAATAACGGGACCTATGCCTGTTTTGTCTCTAAC LeuPheIleAlaLysIleThrProAsnAsnAsnGlyThrTyrAlaCysPheValSerAsn - 1990 2010 2030 . . . . . . TTGGCTACTGGCCGCAATAATTCCATAGTCAAGAGCATCACAGTCTCTGCATCTGGAACT LeuAlaThrGlyArgAsnAsnSerIleValLysSerIleThrValSerAlaSerGlyThr - 2050 2070 2090 . . . . . . TCTCCTGGTCTCTCAGCTGGGGCCACTGTCGGCATCATGATTGGAGTGCTGGTTGGGGTT SerProGlyLeuSerAlaGlyAlaThrValGlyIleMetIleGlyValLeuValGlyVal - 2110 2130 2150 . . . . . . GCTCTGATATAGCAGCCCTGGTGTAGTTTCTTCATTTCAGGAAGACTGACAGTTGTTTTG AlaLeuIleEnd - 2170 2190 2210 . . . . . . CTTCTTCCTTAAAGCATTTGCAACAGCTACAGTCTAAAATTGCTTCTTTACCAAGGATAT - 2230 2250 2270 . . . . . . TTACAGAAAAGACTCTGACCAGAGATCGAGACCATCCTAGCCAACATCGTGAAACCCCAT - 2290 2310 2330 . . . . . . CTCTACTAAAAATACAAAAATGAGCTGGGCTTGGTGGCGCGCACCTGTAGTCCCAGTTAC - 2350 2370 2390 . . . . . . TCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGTGGAGATTGCAGTGAGCCCA - 2410 2430 2450 . . . . . . GATCGCACCACTGCACTCCAGTCTGGCAACAGAGCAAGACTCCATCTCAAA - 2460 2470 2480 2490 2500 * * * * * AAG AAA AGA AAA GAA GAC TCT GAC CTG TAC TCT TGA ATA CAA GTT TCT GAT ACC ACT - 2510 2520 2530 2540 2550 2560 * * * * * * GCA CTG TCT GAG AAT TTC CAA AAC TTT AAT GAA CTA ACT GAC AGC TTC ATG AAA CTG - 2570 2580 2590 2600 2610 2620 * * * * * * TCC ACC AAG ATC AAG CAG AGA AAA TAA TTA ATT TCA TGG GGA CTA AAT GAA CTA ATG - 2630 2640 2650 2660 2670 2680 * * * * * * AGG ATA ATA TTT TCA TAA TTT TTT ATT TGA AAT TTT GCT GAT TCT TTA AAT GTC TTG - 2690 2700 2710 2720 2730 * * * * * TTT CCC AGA TTT CAG GAA ACT TTT TTT CTT TTA AGC TAT CCA CTC TTA CAG CAA TTT - 2740 2750 2760 2770 2780 2790 * * * * * * GAT AAA ATA TAG TTT TGT GAA CAA AAA TTG AGA CAT TTG CAT TTT ATC CCT ATG TGG - 2800 2810 2820 2830 * * * * TCG CTC CAG ACT TGG GAA ACT ATT CAT GAA TAT TTA TAT TGT ATG - CEA-(c)(SEQ ID NO:6): - 10 30 50 . . . . . . CAGCCGTGCTCGAAGCGTTCCTGGAGCCCAAGCTCTCCTCCACAGGTGAAGACAGGGCCA - 70 90 110 . . . . . . GCAGGAGACACCATGGGGCACCTCTCAGCCCCACTTCACAGAGTGCGTGTACCCTGGCAG MetGlyHisLeuSerAlaProLeuHisArgValArgValProTrpGln - 130 150 170 . . . . . . GGGCTTCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCCAGCTC GlyLeuLeuLeuThrAlaSerLeuLeuThrPheTrpAsnProProThrThrAlaGlnLeu - 190 210 230 . . . . . . ACTACTGAATCCATGCCATTCAATGTTGCAGAGGGGAAGGAGGTTCTTCTCCTTGTCCAC ThrThrGluSerMetProPheAsnValAlaGluGlyLysGluValLeuLeuLeuValHis - 250 270 290 . . . . . . AATCTGCCCCAGCAACTTTTTGGCTACAGCTGGTACAAAGGGGAAAGAGTGGATGGCAAC AsnLeuProGlnGlnLeuPheGlyTyrSerTrpTyrLysGlyGluArgValAspGlyAsn - 310 330 350 . . . . . . CGTCAAATTGTAGGATATGCAATAGGAACTCAACAAGCTACCCCAGGGCCCGCAAACAGC ArgGlnIleValGlyTyrAlaIleGlyThrGlnGlnAlaThrProGlyProAlaAsnSer - 370 390 410 . . . . . . GGTCGAGAGACAATATACCCCAATGCATCCCTGCTGATCCAGAACGTCACCCAGAATGAC GlyArgGluThrIleTyrProAsnAlaSerLeuLeuIleGlnAsnValThrGlnAsnAsp - 430 450 470 . . . . . . ACAGGATTCTACACCCTACAAGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGA ThrGlyPheTyrThrLeuGlnValIleLysSerAspLeuValAsnGluGluAlaThrGly - 490 510 530 . . . . . . CAGTTCCATGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAACCCT GlnPheHisValTyrProGluLeuProLysProSerIleSerSerAsnAsnSerAsnPro - 550 570 590 . . . . . . GTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACACAACCTAC ValGluAspLysAspAlaValAlaPheThrCysGluProGluThrGlnAspThrThrTyr - 610 630 650 . . . . . . CTGTGGTGGATAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGC LeuTrpTrpIleAsnAsnGlnSerLeuProValSerProArgLeuGlnLeuSerAsnGly - 670 690 710 . . . . . . AACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGACACAGGACCCTATGAGTGTGAA AsnArgThrLeuThrLeuLeuSerValThrArgAsnAspThrGlyProTyrGluCysGlu - 730 750 770 . . . . . . ATACAGAACCCAGTGAGTGCGAACCGCAGTGACCCAGTCACCTTGAATGTCACCTATGGC IleGlnAsnProValSerAlaAsnArgSerAspProvalThrLeuAsnValThrTyrGly - 790 810 830 . . . . . . CCGGACACCCCCACCATTTCCCCTTCAGACACCTATTACCGTCCAGGGGCAAACCTCAGC ProAspThrProThrIleSerProSerAspThrTyrTyrArgProGlyAlaAsnLeuSer - 850 870 890 . . . . . . CTCTCCTGCTATGCAGCCTCTAACCCACCTGCACAGTACTCCTGGCTTATCAATGGAACA LeuSerCysTyrAlaAlaSerAsnProProAlaGlnTyrSerTrpLeuIleAsnGlyThr - 910 930 950 . . . . . . TTCCAGCAAAGCACACAAGAGCTCTTTATCCCTAACATCACTGTGAATAATAGTGGATCC PheGlnGlnSerThrGlnGluLeuPheIleProAsnIleThrValAsnAsnSerGlySer - 970 990 1010 . . . . . . TATACCTGCCACGCCAATAACTCAGTCACTGGCTGCAACAGGACCACAGTCAAGACGATC TyrThrCysHisAlaAsnAsnSerValThrGlyCysAsnArgThrThrValLysThrIle - 1030 1050 1070 . . . . . . ATAGTCACTGAGCTAAGTCCAGTAGTAGCAAAGCCCCAAATCAAAGCCAGCAAGACCACA IleValThrGluLeuSerProValValAlaLysProGlnIleLysAlaSerLysThrThr - 1090 1110 1130 . . . . . . GTCACAGGAGATAAGGACTCTGTGAACCTGACCTGCTCCACAAATGACACTGGAATCTCC ValThrGlyAspLysAspSerValAsnLeuThrCysSerThrAsnAspThrGlyIleSer - 1150 1170 1190 . . . . . . ATCCGTTGGTTCTTCAAAAACCAGAGTCTCCCGTCCTCGGAGAGGATGAAGCTGTCCCAG IleArgTrpPhePheLysAsnGlnSerLeuProSerSerGluArgMetLysLeuSerGln - 1210 1230 1250 . . . . . . GGCAACACCACCCTCAGCATAAACCCTGTCAAGAGGGAGGATGCTGGGACGTATTGGTGT GlyAsnThrThrLeuSerIleAsnProValLysArgGluAspAlaGlyThrTyrTrpCys - 1270 1290 1310 . . . . . . GAGGTCTTCAACCCAATCAGTAAGAACCAAAGCGACCCCATCATGCTGAACGTAAACTAT GluValPheAsnProIleSerLysAsnGlnSerAspProIleMetLeuAsnValAsnTyr - 1330 1350 1370 . . . . . . AATGCTCTACCACAAGAAAATGGCCTCTCACCTGGGGCCATTGCTGGCATTGTGATTGGA AsnAlaLeuProGlnGluAsnGlyLeuSerProGlyAlaIleAlaGlyIleValIleGly - 1390 1410 1430 . . . . . . GTAGTGGCCCTGGTTGCTCTGATAGCAGTAGCCCTGGCATGTTTTCTGCATTTCGGGAAG ValValAlaLeuValAlaLeuIleAlaValAlaLeuAlaCysPheLeuHisPheGlyLys - 1450 1470 1490 . . . . . . ACCGGCAGGGCAAGCGACCAGCGTGATCTCACAGAGCACAAACCCTCAGTCTCCAACCAC ThrGlyArgAlaSerAspGlnArgAspLeuThrGluHisLysProSerValSerAsnHis - 1510 1530 1550 . . . . . . ACTCAGGACCACTCCAATGACCCACCTAACAAGATGAATGAAGTTACTTATTCTACCCTG ThrGlnAspHisSerAsnAspProProAsnLysMetAsnGluValThrTyrSerThrLeu - 1570 1590 1610 . . . . . . AACTTTGAAGCCCAGCAACCCACACAACCAACTTCAGCCTCCCCATCCCTAACAGCCACA AsnPheGluAlaGlnGlnProThrGlnProThrSerAlaSerProSerLeuThrAlaThr - 1630 1650 1670 . . . . . . GAAATAATTTATTCAGAAGTAAAAAAGCAGTAATGAAACCTGTCCTGCTCACTGCAGTGC GluIleIleTyrSerGluValLysLysGln - 1690 1710 1730 . . . . . . TGATGTATTTCAAGTCTCTCACCCTCATCACTAGGAGATTCCTTTCCCCTGTAGGGTAGA - 1750 1770 1790 . . . . . . GGGGTGGGGACAGAAACAACTTTCTCCTACTCTTCCTTCCTAATAGGCATCTCCAGGCTG - 1810 1830 1850 . . . . . . CCTGGTCACTGCCCCTCTCTCAGTGTCAATAGATGAAAGTACATTGGGAGTCTGTAGGAA - 1870 1890 1910 . . . . . . ACCCAACCTTCTTGTCATTGAAATTTGGCAAAGCTGACTTTGGGAAAGAGGGACCAGAAC - 1930 1950 1970 . . . . . . TTCCCCTCCCTTCCCCTTTTCCCAACCTGGACTTGTTTTAAACTTGCCTGTTCAGAGCAC - 1990 2010 2030 . . . . . . TCATTCCTTCCCACCCCCAGTCCTGTCCTATCACTCTAATTCGGATTTGCCATAGCCTTG - 2050 2070 2090 . . . . . . AGGTTATGTCCTTTTCCATTAAGTACATGTGCCAGGAAACAGCGAGAGAGAGAAAGTAAA - 2110 2130 2150 . . . . . . CGGCAGTAATGCTTCTCCTATTTCTCCAAAGCCTTGTGTGAACTAGCAAAGAGAAGAAAA - 2170 2190 2210 . . . . . . TCAAATATATAACCAATAGTGAAATGCCACAGGTTTGTCCACTGTCAGGGTTGTCTACCT - 2230 2250 2270 . . . . . . GTAGGATCAGGGTCTAAGCACCTTGGTGCTTAGCTAGAATACCACCTAATCCTTCTGGCA - 2290 2310 2330 . . . . . . AGCCTGTCTTCAGAGAACCCACTAGAAGCAACTAGGAAAAATCACTTGCCAAAATCCAAG - 2350 2370 2390 . . . . . . GCAATTCCTGATGGAAAATGCAAAAGCACATATATGTTTTAATATCTTTATGGGCTCTGT - 2410 2430 2450 . . . . . . TCAAGGCAGTGCTGAGAGGGAGGGGTTATAGCTTCAGGAGGGAACCAGCTTCTGATAAAC - 2470 2490 2510 . . . . . . ACAATCTGCTAGGAACTTGGGAAAGGAATCAGAGAGCTGCCCTTCAGCGATTATTTAAAT - 2530 2550 2570 . . . . . . TGTTAAAGAATACACAATTTGGGGTATTGGGATTTTTCTCCTTTTCTCTGAGACATTCCA - 2590 2610 2630 . . . . . . CCATTTTAATTTTTGTAACTGCTTATTTATGTGAAAAGGGTTATTTTTACTTAGCTTAGC - 2650 2670 2690 . . . . . . TATGTCAGCCAATCCGATTGCCTTAGGTGAAAGAAACCACCGAAATCCCTCAGGTCCCTT - 2710 2730 2750 . . . . . . GGTCAGGAGCCTCTCAAGATTTTTTTTGTCAGAGGCTCCAAATAGAAAATAAGAAAAGGT - 2770 2790 2810 . . . . . . TTTCTTCATTCATGGCTAGAGCTAGATTTAACTCAGTTTCTAGGCACCTCAGACCAATCA - 2830 2850 2870 . . . . . . TCAACTACCATTCTATTCCATGTTTGCACCTGTGCATTTTCTGTTTGCCCCCATTCACTT - 2890 2910 2930 . . . . . . TGTCAGGAAACCTTGGCCTCTGCTAAGGTGTATTTGGTCCTTGAGAAGTGGGAGCACCCT - 2950 2970 2990 . . . . . . ACAGGGACACTATCACTCATGCTGGTGGCATTGTTTACAGCTAGAAAGCTGCACTGGTGC - 3010 3030 3050 . . . . . . TAATGCCCCTTGGGAAATGGGGCTGTGAGGAGGAGGATTATAACTTAGGCCTAGCCTCTT - 3070 3090 3110 . . . . . . TTAACAGCCTCTGAAATTTATCTTTTCTTCTATGGGGTCTATAAATGTATCTTATAATAA - 3130 3150 3170 . . . . . . AAAGGAAGGACAGGAGGAAGACAGGCAAATGTACTTCTCACCCAGTCTTCTACACAGATG - 3190 3210 3230 . . . . . . GAATCTCTTTGGGGCTAAGAGAAAGGTTTTATTCTATATTGCTTACCTGATCTCATGTTA - 3250 3270 3290 . . . . . . GGCCTAAGAGGCTTTCTCCAGGAGGATTAGCTTGGAGTTCTCTATACTCAGGTACCTCTT - 3310 3330 3350 . . . . . . TCAGGGTTTTCTAACCCTGACACGGACTGTGCATACTTTCCCTCATCCATGCTGTGCTGT - 3370 3390 3410 . . . . . . GTTATTTAATTTTTCCTGGCTAAGATCATGTCTGAATTATGTATGAAAATTATTCTATGT - 3430 3450 . . . . TTTTATAATAAAAATAATATATCAGACATCGAAAAAAAAAA - CEA-(d)(SEQ ID NO:7): - 10 30 50 . . . . . . CCGGGGGACACGCAGGGCCAACAGTCACAGCAGCCCTGACCAGAGCATTCCTGGAGCTCAAG - 70 90 110 . . . . . . CTCTCTACAAAGAGGTGGACAGAGAAGACAGCAGAGACCATGGGACCCCCCTCAGCCCCT MetGlyProProSerAlaPro - 130 150 170 . . . . . . CCCTGCAGATTGCATGTCCCCTGGAAGGAGGTCCTGCTCACAGCCTCACTTCTAACCTTC ProCysArgLeuHisValProTrpLysGluValLeuLeuThrAlaSerLeuLeuThrPhe - 190 210 230 . . . . . . TGGAACCCACCCACCACTGCCAAGCTCACTATTGAATCCACGCCATTCAATGTCGCAGAG TrpAsnProProThrThrAlaLysLeuThrIleGluSerThrProPheAsnValAlaGlu - 250 270 290 . . . . . . GGGAAGGAGGTTCTTCTACTCGCCCACAACCTGCCCCAGAATCGTATTGGTTACAGCTGG GlyLysGluValLeuLeuLeuAlaHisAsnLeuProGlnAsnArgIleGlyTyrSerTrp - 310 330 350 . . . . . . TACAAAGGCGAAAGAGTGGATGGCAACAGTCTAATTGTAGGATATGTAATAGGAACTCAA TyrLysGlyGluArgValAspGlyAsnSerLeuIleValGlyTyrValIleGlyThrGln - 370 390 410 . . . . . . CAAGCTACCCCAGGGCCCGCATACAGTGGTCGAGAGACAATATACCCCAATGCATCCCTG GlnAlaThrProGlyProAlaTyrSerGlyArgGluThrIleTyrProAsnAlaSerLeu - 430 450 470 . . . . . . CTGATCCAGAACGTCACCCAGAATGACACAGGATTCTACACCCTACAAGTCATAAAGTCA LeuIleGlnAsnValThrGlnAsnAspThrGlyPheTyrThrLeuGlnValIleLysSer - 490 510 530 . . . . . . GATCTTGTGAATGAAGAAGCAACCGGACAGTTCCATGTATACCCGGAGCTGCCCAAGCCC AspLeuValAsnGluGluAlaThrGlyGlnPheHisValTyrProGluLeuProLysPro - 550 570 590 . . . . . . TCCATCTCCAGCAACAACTCCAACCCCGTGGAGGACAAGGATGCTGTGGCCTTCACCTGT SerIleSerSerAsnAsnSerAsnProValGluAspLysAspAlaValAlaPheThrCys - 610 630 650 . . . . . . GAACCTGAGGTTCAGAACACAACCTACCTGTGGTGGGTAAATGGTCAGAGCCTCCCGGTC GluProGluValGlnAsnThrThrTyrLeuTrpTrpValAsnGlyGlnSerLeuProVal - 670 690 710 . . . . . . AGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTACTCAGCGTCAAAAGG SerProArgLeuGlnLeuSerAsnGlyAsnArgThrLeuThrLeuLeuSerValLysArg - 730 750 770 . . . . . . AACGATGCAGGATCGTATGAATGTGAAATACAGAACCCAGCGAGTGCCAACCGCAGTGAC AsnAspAlaGlySerTyrGluCysGluIleGlnAsnProAlaSerAlaAsnArgSerAsp - 790 810 830 . . . . . . CCAGTCACCCTGAATGTCCTCTATGGCCCAGATGGCCCCACCATTTCCCCCTCAAAGGCC ProValThrLeuAsnValLeuTyrGlyProAspGlyProThrIleSerProSerLysAla - 850 870 890 . . . . . . AATTACCGTCCAGGGGAAAATCTGAACCTCTCCTGCCACGCAGCCTCTAACCCACCTGCA AsnTyrArgProGlyGluAsnLeuAsnLeuSerCysHisAlaAlaSerAsnProProAla - 910 930 950 . . . . . . CAGTACTCTTGGTTTATCAATGGGACGTTCCAGCAATCCACACAAGAGCTCTTTATCCCC GlnTyrSerTrpPheIleAsnGlyThrPheGlnGlnSerThrGlnGluLeuPheIlePro - 970 990 1010 . . . . . . AACATCACTGTGAATAATAGCGGATCCTATATGTGCCAAGCCCATAACTCAGCCACTGGC AsnIleThrValAsnAsnSerGlySerTyrMetCysGlnAlaHisAsnSerAlaThrGly - 1030 1050 1070 . . . . . . CTCAATAGGACCACAGTCACGATGATCACAGTCTCTGGAAGTGCTCCTGTCCTCTCAGCT LeuAsnArgThrThrValThrMetIleThrValSerGlySerAlaProValLeuSerAla - 1090 1110 1130 . . . . . . GTGGCCACCGTCGGCATCACGATTGGAGTGCTGGCCAGGGTGGCTCTGATATAGCAGCCC ValAlaThrValGlyIleThrIleGlyValLeuAlaArgValAlaLeuIleEnd - 1150 1160 1170 1180 1190 . . . . . TGG TGT ATT TTC GAT ATT TCA GGA AGA CTG GCA GAT TGG ACC AGA CCC TGA ATT CTT - 1200 1210 1220 1230 1240 1250 . . . . . . CTA GCT CCT CCA ATC CCA TTT TAT CCC ATG GAA CCA CTA AAA ACA AGG TCT GCT CTG - 1260 1270 1280 1290 1300 1310 . . . . . . CTC CTG AAG CCC TAT ATG CTG GAG ATG GAC AAC TCA ATG AAA ATT TAA AAG AAA AAC - 1320 1330 1340 1350 1360 1370 . . . . . . CCT CAG GCC TGA GGT GTG TGC CAC TCA GAG ACT TCA CCT AAC TAG AGA CAG GCA AAC - 1380 1390 1400 1410 1420 . . . . . TGC AAA CCA nnC CTC TTT CGC TTG GCA GGA TGA TGG TGT CAT TAG TAT TTC ACA AGA - 1430 1440 1450 1460 1470 1480 . . . . . . AGT AGC TTC AGA GGG TAA CTT AAC AGA GTA TCA GAT CTA TCT TGT CAA TCC CAA CGT - 1490 1500 1510 1520 1530 1540 . . . . . . TTT ACA TAA AAT AAG CGA TCC TTT AGT GCA CCC AGT GAC TGA CAT TAG CAG CAT CTT - 1550 1560 1570 1580 1590 . . . . . TAA CAC AGC CGT GTG TTC AAG TGT ACA GTG GTC CTT TTC AGA GTT GGn nnT ACT CCA - 1600 1610 1620 1630 1640 1650 . . . . . . ACT GAA ATG TTA AGG AAG AAG ATA GAT CCA ATT AAA AAA AAT TAA AAC CAA TTT AAA - 1660 1670 1680 1690 1700 1710 . . . . . . AAA AAA AAA GAA CAC AGG AGA TTC CAG TCT ACT TGA GTT AGC ATA ATA CAG AAG TCC - 1720 1730 1740 1750 1760 . . . . . CCT CTA CTT TAA CTT TTA CAA AAA AGT AAC CTG AAC TAA TCT GAT GTT AAC CAA TGT 1770 1780 1790 1800 1810 1820 . . . . . . ATT TAT TTG TCT GGT TCT GTT TCC TTG TTC CAA TTT GAC AAA ACC CAC TGT TCT TGT - 1830 1840 1850 1860 1870 1880 . . . . . . ATT GTA TTG CCC AGG GGG AGC TAT CAC TGT ACT TGT AGA GTG GTG CTG CTT TAA GTT - 1890 1900 1910 1920 1930 1940 . . . . . . CAT AAA TCA CAA ATA AAA GCC AAT TAG CTC TAT AAC TAA AAA AAA AAA AAA AAA AAA - 1950 1960 . . AAA AAA AAA AAA AAA AAA AAA AAA
A schematic relationship of the transmembrane CEA's, namely TM-1 (CEA-(c)), TM-2 (CEA-(e)), TM-3 (CEA-(f)) and TM-4 (CEA-(g)) is depicted in FIG. 1:
Assuming TM-1 is composed of five sections as depicted in FIG. 1, namely 10, 12, 14, 16 and 18, TM-2 differs from TM-1 in that the 100 amino acid (100 AA) section 14 is deleted and at splice point 20 betweeen sections 12 and 16, surprisingly an extra amino acid, namely Asp occurs.
TM-3 is the same as TM-1 except that section 18 is truncated at splice point 22, i.e., a section of 70 amino acids is deleted and results in a new section made up of subsections 24+26. Surprisingly, however, six new amino acids (section 26) occur. Another example of formation of a novel amino acid sequence resulting from a deletion of nucleic acid sequence is for platelet derived growth factor-A.
TM-4 is the same as TM-2 up until the end of subsection 24.
Subsection 24 is contained in section 18 of TM-1 and TM-2, but is not depicted in FIG. 1 for TM-1 and TM-2.
Some CEA epitopes are unique. These are the epitopes which have been useful for distinguishing the various CEA-like antigens immunologically. Peptide epitopes are defined by the linear amino acid sequence of the antigen and/or features resulting from protein folding. The information required for protein folding is encoded in the primary amino acid sequence. Therefore, antigenic differences ultimately result from differences in the primary structure of the different CEA molecules. The differences residing in the CEA protein in the CEA species can thus be determined by determining the primary amino acid sequences. This can be most readily accomplished by cloning and sequencing each of the genes for CEA. To determine which gene products will be most useful for cancer diagnosis, unique probes can be selected for each gene and expression of each gene can be determined in different tumor types by nucleic acid hybridization techniques. The present invention provides a tool with which to identify potential genes coding for different members of the CEA family and to determine the theoretical primary amino acid sequences for them. Using the method of automated peptide synthesis, peptides can then be synthesized corresponding to unique sequences in these antigens. With these peptides, antibodies to these sequences can be produced which, in the intact CEA molecule, might not be recognized by the animal being immunized. Having accomplished this, advantage can then be taken of the differences in these antigens to generate specific immunoassays for the measurement of each antigen.
A wide variety of host/cloning vehicle combinations may be employed in cloning the double-stranded nucleic acid prepared in accordance with this invention. For example, useful cloning vehicles may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from E. coli including col E1, pCR1, pBR322, pMB89 and their derivatives, wider host range plasmids, e.g., RP4, and phage DNAs, e.g., the numerous derivatives of phage, e.g., NM989, and other DNA phages, e.g., M13 and Filamenteous single-stranded DNA phages and vectors derived from combinations of plasmids and phage DNAs such as plasmids which have been modified to employ phage DNA or other expression control sequences or yeast plasmids such as the 2.mu. plasmid or derivatives thereof. Useful hosts may include bacterial hosts such as strains of E. coli, such as E. coli HB 101, E. coli X1776, E. coli X2282, E. coli MRC1 and strains of Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus and other E. coli, bacilli, yeasts and other fungi, animal or plant hosts such as animal (including human) or plant cells in culture or other hosts. Of course, not all host/vector combinations may be equally efficient. The particular selection of host/cloning vehicle combination may be made by those of skill in the art after due consideration of the principles set forth without departing from the scope of this invention.
Furthermore, within each specific cloning vehicle, various sites may be selected for insertion of the nucleic acid according to the present invention. These sites are usually designated by the restriction endonuclease which cuts them. For example, in pBR322 the Pstl site is located in the gene for beta-lactamase, between the nucleotide triplets that code for amino acids 181 and 182 of that protein. One of the two HindII endonuclease recognition sites is between the triplets coding for amino acids 101 and 102 and one of the several Taq sites at the triplet coding for amino acid 45 of beta-lactamase in pBR322. In similar fashion, the EcoRI site and the PVUII site in this plasmid lie outside of any coding region, the EcoRI site being located between the genes coding for resistance to tetracycline and ampicillin, respectively. These sites are well recognized by those of skill in the art. It is, of course, to be understood that a cloning vehicle useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead, the vehicle could be cut and joined to the fragment by alternative means.
The vector or cloning vehicle and in particular the site chosen therein for attachment of a selected nucleic acid fragment to form a recombinant nucleic acid molecule is determined by a variety of factors, e.g., the number of sites susceptible to a particular restriction enzyme, the size of the protein to be expressed, the susceptibility of the desired protein to proteolytic degradation by host cell enzymes, the contamination of the protein to be expressed by host cell proteins difficult to remove during purification, the expression characteristics, such as the location of start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art. The choice of a vector and an insertion site for a particular gene is determined by a balance of these factors, not all sections being equally effective for a given case.
Methods of inserting nucleic acid sequences into cloning vehicles to form recombinant nucleic acid molecules include, for example, dA-dT tailing, direct ligation, synthetic linkers, exonuclease and polymerase-linked repair reactions followed by ligation, or extension of the nucleic acid strand with an appropriate polymerase and an appropriate single-stranded template followed by ligation.
It should also be understood that the nucleotide sequences or nucleic acid fragments inserted at the selected site of the cloning vehicle may include nucleotides which are not part of the actual structural gene for the desired polypeptide or mature protein or may include only a fragment of the complete structural gene for the desired protein or mature protein.
The cloning vehicle or vector containing the foreign gene is employed to transform an appropriate host so as to permit that host to replicate the foreign gene and to express the protein coded by the foreign gene or portion thereof. The selection of an appropriate host is also controlled by a number of factors recognized by the art. These include, for example, the compatibility with the chosen vector, the toxicity of proteins encoded by the hybrid plasmid, the ease of recovery of the desired protein, the expression characteristics, biosafety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for expression of a particular recombinant DNA molecule.
The level of production of a protein is governed by two major factors: the number of copies of its gene within the cell and the efficiency with which those gene copies are transcribed and translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon nucleotide sequences, normally situated ahead of the desired coding sequence. These nucleotide sequences or expression control sequences define inter alia, the location at which RNA polymerase interacts to initiate transcription (the promoter sequence) and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation. Not all such expression control sequences function with equal efficiency. It is thus of advantage to separate the specific coding sequences for the desired protein from their adjacent nucleotide sequences and fuse them instead to other known expression control sequences so as to favor higher levels of expression. This having been achieved, the newly engineered nucleic acid, e.g., DNA, fragment may be inserted into a multicopy plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further improve the yield of expressed protein.
Several expression control sequences may be employed as described above. These include the operator, promoter and ribosome binding and interaction sequences (including sequences such as the Shine-Dalgarno sequences) of the lactose operon of E. coli ("the lac system"), the corresponding sequences of the tryptophan synthetase system of E. coli ("the trp system"), the major operator and promoter regions of phage .lambda. (O.sub.L P.sub.L and O.sub.R P'.sub.R), the control region of Filamenteous single-stranded DNA phages, or other sequences which control the expression of genes of prokaryotic or eukaryotic cells and their viruses. Therefore, to improve the production of a particular polypeptide in an appropriate host, the gene coding for that polypeptide may be selected and removed from a recombinant nucleic acid molecule containing it and reinserted into a recombinant nucleic acid molecule closer or in a more appropriate relationship to its former expression control sequence or under the control of one of the above described expression control sequences. Such methods are known in the art.
As used herein "relationship" may encompass many factors, e.g., the distance separating the expression enhancing and promoting regions of the recombinant nucleic acid molecule and the inserted nucleic acid sequence, the transcription and translation characteristics of the inserted nucleic acid sequence or other sequences in the vector itself, the particular nucleotide sequence of the inserted nucleic acid sequence and other sequences of the vector and the particular characteristics of the expression enhancing and promoting regions of the vector.
Further increases in the cellular yield of the desired products depend upon an increase in the number of genes that can be utilized in the cell. This is achieved, for illustration purposes, by insertion of recombinant nucleic acid molecules engineered into the temperate bacteriophage .lambda. (NM989), most simply by digestion of the plasmid with a restriction enzyme, to give a linear molecule which is then mixed with a restricted phage .lambda. cloning vehicle (e.g., of the type described by N. E. Murray et al, "Lambdoid Phages That Simplify the Recovery of In Vitro Recombinants", Molec. Gen. Genet., 150, pp. 53-61 (1977) and N. E. Murray et al, "Molecular Cloning of the DNA Ligase Gene From Bacteriophage T4", J. Mol. Biol., 132, pp. 493-505 (1979)) and the recombinant DNA molecule recircularized by incubation with DNA ligase. The desired recombinant phage is then selected as before and used to lysogenize a host strain of E. coli.
Particularly useful .lambda. cloning vehicles contain a temperature-sensitive mutation in the repression gene cl and suppressible mutations in gene S, the product of which is necessary for lysis of the host cell, and gene E, the product of which is major capsid protein of the virus. With this system, the lysogenic cells are grown at 32.degree. C. and then heated to 45.degree. C. to induce excision of the prophage. Prolonged growth at 37.degree. C. leads to high levels of production of the protein, which is retained within the cells, since these are not lysed by phage gene products in the normal way, and since the phage gene insert is not encapsulated it remains available for further transcription. Artificial lysis of the cells then releases the desired product in high yield.
In addition, it should be understood that the yield of polypeptides prepared in accordance with this invention may also be improved by substituting different codons for some or all of the codons of the present DNA sequences, these substituted codons coding for amino acids identical to those coded for by the codons replaced.
Finally, the activity of the polypeptides produced by the recombinant nucleic acid molecules of this invention may be improved by fragmenting, modifying or derivatizing the nucleic acid sequences or polypeptides of this invention by well-known means, without departing from the scope of this invention.
The polypeptides of the present invention include the following:
(1) the polypeptides expressed by the above described cells,
(2) polypeptides prepared by synthetic means,
(3) fragments of polypeptides (1) or (2) above, such fragments produced by synthesis of amino acids or by digestion or cleavage.
Regarding the synthetic peptides according to the invention, chemical synthesis of peptides is described in the following publications: S. B. H. Kent, Biomedical Polymers, eds. Goldberg, E. P. and Nakajima, A. (Academic Press, New York), 213-242, (1980); A. R. Mitchell, S. B. H. Kent, M. Engelhard and R. B. Merrifield, J. Org. Chem., 43, 2845-2852, (1978); J. P. Tam, T.-W. Wong, M. Riemen, F.-S. Tjoeng and R. B. Merrifield, Tet. Letters, 4033-4036, (1979); S. Mojsov, A. R. Mitchell and R. B. Merrifield, J. Org. Chem., 45, 555-560, (1980); J. P. Tam, R. D. DiMarchi and R. B. Merrifield, Tet. Letters, 2851-2854, (1981); and S. B. H. Kent, M. Riemen, M. Le Doux and R. B. Merrifield, Proceedings of the IV International Symposium on Methods of Protein Sequence Analysis, (Brookhaven Press, Brookhaven, N.Y.) 1981.
In the Merrifield solid phase procedure, the appropriate sequence of L-amino acids is built up from the carboxyl terminal amino acid to the amino terminal amino acid. Starting with the appropriate carboxyl terminal amino acid attached to a polystyrene (or other appropriate) resin via chemical linkage to a chloromethyl group, benzhydrylamine group, or other reactive group of the resin, amino acids are added one by one using the following procedure. The peptide-resin is:
(a) washed with methylene chloride;
(b) neutralized by making for 10 minutes at room temperature with 5% (v/v) diisopropylethylamine (or other hindered base) in methylene chloride;
(c) washed with methylene chloride;
(d) an amount of amino acid equal to six times the molar amount of the growing peptide chain is activated by combining it with one-half as many moles of a carbodiimide (e.g., dicyclohexylcarbodiimide, or diisopropylcarbodiimide) for ten minutes at 0.degree. C., to form the symmetric anhydride of the amino acid. The amino acid used should be provided originally as the N-alpha-tert.-butyloxycarbonyl derivative, with side chains protected with benzyl esters (e.g., aspartic or glutamic acids), benzyl ethers (e.g., serine, threonine, cysteine or tyrosine), benzyloxycarbonyl groups (e.g., lysine) or other protecting groups commonly used in peptide synthesis;
(e) the activated amino acid is reacted with the peptide-resin for two hours at room temperature, resulting in addition of the new amino acid to the end of the growing peptide chain;
(f) the peptide-resin is washed with methylene chloride;
(g) the N-alpha-(tert.-butyloxycarbonyl) group is removed from the most recently added amino acid by reacting with 30 to 65%, preferably 50% (v/v) trifluoroacetic acid in methylene chloride for 10 to 30 minutes at room temperature;
(h) the peptide-resin is washed with methylene chloride;
(i) steps (a) through (h) are repeated until the required peptide sequence has been constructed.
The peptide is then removed from the resin and simultaneously the side-chain protecting groups are removed, by reaction with anhydrous hydrofluoric acid containing 10% v/v of anisole or other suitable (aromatic) scavenger. Subsequently, the peptide can be purified by gel filtration, ion exchange, high pressure liquid chromatography, or other suitable means.
In some cases, chemical synthesis can be carried out without the solid phase resin, in which case the synthetic reactions are performed entirely in solution. The reactions are similar and well known in the art, and the final product is essentially identical.
Digestion of the polypeptide can be accomplished by using proteolytic enzymes, especially those enzymes whose substrate specificity results in cleavage of the polypeptide at sites inmmediately adjacent to the desired sequence of amino acids.
Cleavage of the polypeptide can be accomplished by chemical means. Particular bonds between amino acids can be cleaved by reaction with specific reagents. Examples include the following: bonds involving methionine are cleaved by cyanogen bromide; asparaginyl-glycine bonds are cleaved by hydroxylamine.
The present invention has the following advantages:
(1) The nucleic acids coding for TM-1, TM-2 and TM-3 can be used as probes to isolate other members of the CEA gene family.
(2) The nucleic acids coding for TM-1, TM-2 and TM-3 can be used to derive oligonucleotide probes to determine the expression of TM-1, TM-2, TM-3 and other CEA genes in various tumor types.
(3) TM-1, TM-2, TM-3 and TM-4 nucleotide sequences can be used to predict the primary amino acid sequence of the protein for production of synthetic peptides.
(4) Synthetic peptides derived from the above sequences can be used to produce sequence-specific antibodies.
(5) Imnunoassays for each member of the CEA antigen family can be produced with these sequence-specific antibodies and synthetic peptides.
(6) The aforementioned immunoassays can be used as diagnostics for different types of cancer if it is determined that different members of the CEA family are clinically useful markers for different types of cancer.
Peptides according to the present invention can be labelled by conventional means using radioactive moieties (e.g., .sup.125 I), enzymes, dyes and fluorescent compounds, just to name a few.
Several possible configurations for immunoassays according to the present invention can be used. The readout systems capable of being employed in these assays are numerous and non-limiting examples of such systems include fluorescent and calorimetric enzyme systems, radioisotopic labelling and detection and chemiluminescent systems. Two examples of immunoassay methods are as follows:
(1) An enzyme linked immunoassay (ELISA) using an antibody preparation according to the present invention (including Fab or F(ab)' fragments derived therefrom) to a solid phase (such as a microtiter plate or latex beads) is attached a purified antibody of a specificity other than that which is conjugated to the enzyme. This solid phase antibody is contacted with the sample containing CEA antigen family members. After washing, the solid phase antibody-antigen complex is contacted with the conjugated anti-peptide antibody (or conjugated fragment). After washing away unbound conjugate, color or fluorescence is developed by adding a chromogenic or fluorogenic substrate for the enzyme. The amount of color or fluorescence developed is proportional to the amount of antigen in the sample.
(2) A competitive fluorometric immunoassay using fluorescently labelled peptide or synthetic peptides of the sequences for TM-2, TM-2, TM-3 and TM-4. In this example, the purified peptide expressed by cells or synthetic peptides thereof are fluorescently labelled. To a solid phase is attached a purified antibody. This solid phase is then contacted with sample containing CEA antigen family members to which has been added fluorescent peptide probe. After binding, excess probe is washed away the amount of bound probe is quantitated. The amount of bound fluorescent probe will be inversely proportional to the amount of antigen in the sample.
In the nucleic acid hybridization method according to the present invention, the nucleic acid probe is conjugated with a label, for example, an enzyme, a fluorophore, a radioisotope, a chemiluminescent compound, etc. In the most general case, the probe would be contacted with the sample and the presence of any hybridizable nucleic acid sequence would be detected by developing in the presence of a chromogenic enzyme substrate, detection of the fluorophore by epifluorescence, by autoradiography of the radioisotopically labelled probe or by chemiluminescence. The detection of hybridizable RNA sequences can be accomplished by (1) a dot blot methodology or (2) an in situ hybridization methodology. Methods for these last two techniques are described by D. Gillespie and J. Bresser, "mRNA Immobilization in NaI: Quick Blots", Biotechniques, 184-192, November/December 1983 and J. Lawrence and R. Singer, "Intracellular Localization of Messenger RNAs for Cytosketal Proteins", Cell, 45, 407-415, May 9, 1986, respectively. The readout systems can be the same as described above, e.g., enzyme labelling, radiolabelling, etc.
As stated above, the invention also relates to the use in medicine of the aforementioned complex of the invention.
The invention further provides a pharmaceutical composition containing as an active ingredient a complex of the invention in the form of a sterile and/or physiologically isotonic aqueous solution.
For parenteral administration, solutions and emulsions containing as an active ingredient the complex of the invention should be sterile and, if appropriate, blood-isotonic.
It is envisaged that the active complex will be administered perorally, parenterally (for example, intramuscularly, intraperitoneally, or intravenously), rectally or locally.
EXAMPLE 1
Preparation of cDNA in pcE22 Which Codes for TM2-CEA [CEA-(e)]
Example 1a
RNA Preparation
Messenger RNA was prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methods in Enzymology, 65, 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3'-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides). To obtain approximately 100 .mu.g of poly A+ RNA, approximately 3.times.10.sup.8 cells of transfectant 23.411 (ATCC No. CRL 9731, deposited with the ATCC on Jun. 1, 1988), that expresses TM-1, TM-2, TM-3 and TM-4, Kamarck et al, Proc. Natl. Acad. Sci., USA, 84, 5350-5354, August 1987, were harvested from roller bottles after late logarithmic growth. Cells were lysed by homogenization in an ice-cold solution of 140 mM NaCl, 1.5 mM MgCl.sub.2, 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. Sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei were separated by centrifugation of the homogenate at 12,000.times.g for 20 minutes. The cytoplasmic fraction was mixed with an equal volume of 0.2M Tris-HCl, pH 7.8, 25 mM EDTA, 0.3M NaCl, 2% sodium dodecyl sulfate and 400 .mu.g/ml of proteinase K, incubated for 1 hour at 37.degree. C., then extracted once with an equal volume of phenol/cholorform (1:1/v:v) solution. Nucleic acids were obrtained by ethanol precipitation of the separated aqueous phase. Total RNA was enriched by passage in 0.5M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA was eluted in the same solution without sodium chloride.
Example 1b
Reverse Transcription of mRNA
Ten micrograms of poly A+ RNA were primed for reverse transcription with oligo dT(12-18) and pdN.sub.6 primers. One hundred microliter reaction was performed for 4 hours at 42.degree. C. with 200 units AMV reverse transcriptase (Life Science, Inc. St. Petersburg, Fla., U.S.A.). The RNA component of the cDNA/mRNA hybrids was replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12.degree. C. and 22.degree. C. for 1.5 hours each. Molecular ends were polished by treatment with T4 DNA polymerase. cDNA was phenol/chloroform extracted and purified over a "SEPHADEX G-50" spun column prepared in 10 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE).
Example 1c
Cloning of pcE22 (plasmid cDNA E22)
Synthetic DNA 5' pCCCGGG 3' (SEQ ID NO:14) linkers 3' GGGCCCTTAA 5'
were attached to the ends of cDNA by blunt end ligation with excess T4 DNA ligase. Excess linkers were removed by chromatography through "SEPHADEX G-50" (medium) in TE, and by fractionation on 0.8% low melting agarose gel. Based on Northern blot analysis of poly A+ RNA of the 23.411 cell line, the size of the CEA-related mRNA was estimated at 3.6 kb. Therefore, cDNA fragments between 2 and 4 kb were recovered from gel slices and fragments were ethanol precipitated. After resuspension of cDNA in TE, EcoRI-cleaved lambada gt10 arms were added to cDNA at an estimated molar ratio of 1:1. Ligation proceeded at 7.degree. C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction were added to commercially-obtained packaging mix (Stratagene, San Diego, Calif., U.S.A.). Five million phage particles were obtained ofter in vitro packaging and infection of E. coli host NM514.
Example 1d
Screening of Recombinant Library
Five hundred thousand packaged lambda particles were plated on lawns of E. coli NM514 and replicate patterns were lifted onto nitrocellulose sheets as described by W. D. Benton and R. W. Davis, Science 196, 180-182, (1977). Positive phage were selected by hybridization with .sup.32 P-labeled LV7 cDNA insert probe that contained a domain repeated amoung various CEA family members. By multiple rounds of screening. Phage from individual plaques were amplified and titered, and these were used to prepare small quantities of recombinant phage DNA.
Example 1e
DNA Manipulation
Phage DNA was prepared according to T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Habor, (1982). DNA segments were isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif., U.S.A.). DNA sequencing was performed by the dideoxy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977). The nucleic acid and translated sequence for cDNA in pcE22 is given hereinabove (TM-2 (CEA-(e)).
EXAMPLE 2
Preparation of cDNA in pcHT-6 Which Partically Codes for TM3-CEA [CEA-(f)]
Example 2a
RNA Preparation
Messenger RNA was prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methods in Enzymology, 65 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3'-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides). To obtain approximately 100 .mu.g of poly A+ RNA, approximately 3.times.10.sup.8 cells of HT-29 tumor cells (ATCC HTB38) were harvested form roller bottles after late logarithmic growth. Cells were lysed by homogenization in an ice-cold solution of 140 mM NaCl, 1.5 mM MgCl.sub.2, 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. Sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei were separated by centrifugation of the homogenate at 12,000.times.g for 20 minutes. The cytoplasmic fraction was mixed with an equal volume of 0.2M Tris-Hcl, pH 7.8, 25 mM EDTA, 0.3M NaCl, 2% sodium dodecyl sulfate and 400 .mu.g/ml of proteinase K, incubated for 1 hour at 37.degree. C., then extracted once with an equal volume of phenol/cholorform (1:1/v:v) solution. Nucleic acids were obrtained by ethanol precipitation of the separated aqueous phase. Total RNA was enriched by passage in 0.5M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA was eluted in the same solution without sodium chloride.
Example 2b
Reverse Transcription of mRNA
Ten micrograms of HT-29 poly A+ RNA were primed for reverse transcription with oligo dT(12-18) and pdN.sub.6 primers. One hundred microliter reaction was performed for 4 hours at 42.degree. C. with 200 units AMV reverse transcriptase (Life Science, Inc. St. Petersburg, Fla., U.S.A.). The RNA component of the cDNA/mRNA hybrids was replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12.degree. C. and 22.degree. C. for 1.5 hours each. Molecular ends were polished by treatment with T4 DNA polymerase. cDNA was phenol/chloroform extracted and purified over a "SEPHADEX G-50" spun column prepared in 10 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE).
Example 2c
Cloning of pcHT-6 (plasmid cDNA HT-6)
(SEQ ID NO:14)1 Synthetic DNA linkers 5' pCCCGGG 3' - 3' GGGCCCTTAA 5'
were attached to the ends of cDNA by blunt end ligation with excess T4 DNA ligase. Excess linkers were removed by chromatography through "SEPHADEX G-50" (medium) in TE, and by fractionation on 0.8% low melting agarose gel. Based on Northern blot analysis of poly A+ RNA of the HT-29 cell line, the size of the CEA-related mRNA was estimated at 2.2 kb. Therefore, cDNA fragments between 2 and 3 kb were recovered from gel slices and fragments were ethanol precipitated. After resuspension of cDNA in TE, EcoRI-cleaved lambada gt10 arms were added to cDNA at an estimated molar ratio of 1:1. Ligation proceeded at 7.degree. C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction were added to commercially-obtained packaging mix (Stratagene, San Diego, Calif., U.S.A.). Five million phage particles were obtained ofter in vitro packaging and infection of E. coli host NM514.
Example 2d
Screening of Recombinant Library
Five hundred thousand packaged lambda particles were plated on lawns of E. coli NM514 and replicate patterns were lifted onto nitrocellulose sheets as described by W. D. Benton and R. W. Davis, Science, 196, 180-182, (1977). Positive phage were selected by hybridization with .sup.32 P-labeled LV7 cDNA insert probe that contained a domain repeated amoung various CEA family members. By multiple rounds of screening. Phage from individual plaques were amplified and titered, and these were used to prepare small quantities of recombinant phage DNA.
Example 2e
DNA Manipulation
Phage DNA was prepared according to T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Habor, (1982). DNA segments were isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif., U.S.A.). DNA sequencing was performed by the dideoxy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977). The nucleic acid and translated sequence for cDNA in HT-6 not complete at the 5' end of its coding region, but the nucleotide sequenece and restriction map of the HT-6 insert indicates that it is related to nucleic acid sequences of cDNA clones coding for CEA-(c) and CEA-(e). The nucleotide sequence of HT-6 insert differs from these clones at only nucleotide position 1463 to 1515 and 1676 to 2429 of the CEA-(c) cDNA. It is inferred from this result that the pcHT-6 insert is a partial coding sequence for CEA-(f) and the presumed nucleic acid and translated sequence of CEA-(f) is given hereinabove (TM-3 (CEA-(f))
EXAMPLE 3
Preparation of cDNA Which Codes for TM4-CEA [CEA-(g)]
Example 3a
RNA Preparation
Messenger RNA is prepared by the proteinase K extraction method of J. Favolaro, R. Treisman and R. Kamen, Methos in Enzymology, 65, 718, Academic Press, Inc. (1980), followed by oligo dT cellulose chromatography to yield poly A+ RNA (3'-polyadenylated eukaryotic RNA containing most mRNA sequences that can be translated into polypeptides). To obtain approximately 100 .mu.g of poly A+ RNA, approximately 3.times.10.sup.8 cells of transfectant 23.411 or tumor cell line HT-29 (ATCC HTB 38) are harvested from roller bottles after late logarithmic growth. Cells are lysed by homogenization in an ice-cold solution of 140 mM NaCl, 1.5 mM MgCl.sub.2, 10 mM Tris-HCl, pH 8.0, 0.5% NP40, 4 mM dithiothreitol and 20 units of placental ribonuclease inhibitor/ml. Sodium deoxycholate was then added to 0.2%. Cytoplasm and nuclei are separated by centrifugation of the homogenate at 12,000.times.g for 20 minutes. The cytoplasmic fraction is mixed with an equal volume of 0.2M Tris-Hcl, pH 7.8, 25 mM EDTA, 0.3M NaCl, 2% sodium dodecyl sulfate and 400 .mu.g/ml of proteinase K, incubated for 1 hour at 37.degree. C., then extracted once with an equal volume of phenol/cholorform (1:1/v:v) solution. Nucleic acids are obtained by ethanol precipitation of the separated aqueous phase. Total RNA is enriched by passage in 0.5M NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% sarcosyl through an oligo dT(12-18) cellulose column. After washing, bound RNA is eluted in the same solution without sodium chloride.
Example 3b
Reverse Transcription of mRNA
Ten micrograms of 23.411 or HT 29 poly A+ RNA are primed for reverse transcription with oligo dT(12-18) and pdN.sub.6 primers. One hundred microliter reaction was performed for 4 hours at 42.degree. C. with 200 units AMV reverse transcriptase (Life Science, Inc. St. Petersburg, Fla., U.S.A.). The RNA component of the cDNA/mRNA hybrids is replaced with the second complementary strand by treatment with RNase H, E. coli DNA polymerase I and E. coli DNA ligase at 12.degree. C. and 22.degree. C. for 1.5 hours each. Molecular ends are polished by treatment with T4 DNA polymerase. cDNA is phenol/chloroform extracted and purified over a "SEPHADEX G-50" spun column prepared in 10 mM Tris-HCl, pH 7.8, 1 mM EDTA (TE).
Example 3c
Cloning of cDNA for CEA-(g)
Synthetic DNA linkers 5' pCCCGGG 3' - 3' GGGCCCTTAA 5' (SEQ ID NO: 14)
are attached to the ends of cDNA by blunt end ligation with excess T4 DNA ligase. Excess linkers are removed by chromatography through "SEPHADEX G-50" (medium) in TE, and by fractionation on 0.8% low melting agarose gel. Based on Northern blot analysis of poly A+ RNA of the 23.411 and HT-29 cell lines, the size of the CEA-related mRNA is estimated at 1.7 kb. Therefore, cDNA fragments between 1 and 2 kb are recovered from gel slices and fragments are ethanol precipitated. After resuspension of cDNA in TE, EcoRI-cleaved lambda gt10 arms are added to cDNA at an estimated molar ratio of 1:1. Ligation proceeds at 7.degree. C. for 2 days in the presence of T4 DNA ligase. Aliquots of the ligation reaction are added to commercially-obtained packaging mix (Stratagene, San Diego, Calif., U.S.A.). Phage particles are obtained after in vitro packaging and infection of E. coli host NM514.
Example 3d
Screening of Recombinant Library
Five hundred thousand to one million packaged lambda particles are plated on lawns of E. coli NM514 and replicate patterns are lifted onto nitrocellulose sheets as described by W. D. Benton and R. W. Davis, Science, 196, 180-182, (1977). Positive phage are selected by hybridization with .sup.32 P-labeled LV7 cDNA insert probe that contained a domain repeated amoung various CEA family members. By this selection method, positive phage are obtained after multiple rounds of screening. Phage from individual plaques are amplified and titered, and these are used to prepare small quantities of recombinant phage DNA.
Example 3e
DNA Manipulation
Phage DNA is prepared according to T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, (1982). DNA segments are isolated from low melting agarose gels and inserted for subcloning into Bluescript plasmid vectors (Stratagene, San Diego, Calif., U.S.A.). DNA sequencing is performed by the dideoxy termination method of F. Sanger, S. Nicklen and A. Coulson, Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, (1977). The nucleotide and translated sequence for a cDNA coding for CEA-(g) is given hereinabove (TM-4 (CEA-(g)).
EXAMPLE 4
Screening of a KG-1 cDNA Library with .sup.32 p-labelled CEA Probe, LV7 (CEA-(A))
A segment of cDNA coding for a portion of carcinoembryonic antigen [LV7 or CEA-(a)] was radiolabelled by random priming and used to detect homologous sequences on filter replicas of a commercial cDNA library prepared from KG-1 cells in bacteriophage vector .lambda. gt11 (Clontech Laboratories, Inc., Palo Alto, Calif., U.S.A.). Hybridizations were performed at 68.degree. C. in 2.times. SSSPE (1.times. SSPE--0.15M NaCl, 0.01M sodium phosphate and 1 mM EDTA, pH 7), 5.times. Denhardt's solution and 100 .mu.g of denatured salmon sperm DNA per ml, and post-hybridization washes were in 0.2.times. SSC, 0.25% sodium dodecyl sulfate.
Positive phage were picked, rescreened to homogeneity, and amplified for production of DNA. cDNA inserts were excised from phage DNA with EcoRI endonuclease and subcloned into the EcoRI site of the plasmid vector pBluescript KS. DNA sequencing on double-stranded DNA was by the method of Sanger et al, supra. The sequences of two different inserts from the KG-1 cDNA library are shown below:
pcKGCEA1(SEQ ID NO:8): - 1 acagcacagctgacagccgtactcaggaagcttctggatcctaggcttatctccacagag 60 - 61 gagaacacacaagcagcagagacc atggggcccctctcagcccctccctgcacac acctc 120 MetGlyProLeuSerAlaProProCysThrHisLeu - 121 atcacttggaagggggtcctgctcacagcatcacttttaaacttctggaatccgcccaca 180 IleThrTrpLysGlyValLeuLeuThrAlaSerLeuLeuAsnPheTrpAsnProProThr - 181 actgcccaagtcacgattgaagcccagccacccaaagtttctgaggggaaggatgttctt 240 ThrAlaGlnValThrIleGluAlaGlnProProLysValSerGluGlyLysAspValLeu - 241 ctacttgtccacaatttgccccagaatcttgctggctacatttggtacaaagggcaaatg 300 LeuLeuValHisASnLeuProGlnAsnLeuAlaGlyTyrIleTrpTyrLysGlyGlnMet - 301 acatacgtctaccattacattacatcatatgtagtagacggtcaaagaattatatatggg 360 ThrTyrValTyrHisTyrIleThrSerTyrValValAspGlyGlnArgIleIleTyrGly - 361 cctgcatacagtggaagagaaagagtatattccaatgcatccctgctgatccagaatgtc 420 ProAlaTyrSerGlyArgGluArgValTyrSerAsnAlaSerLeuLeuIleGlnAsnVal - 421 acgcaggaggatgcaggatcctacaccttacacatcataaagcgacgcgatgggactgga 480 ThrGlnGluAspAlaGlySerTyrThrLeuHisIleIleLysArgArgAspGlyThrGly - 481 ggagtaactggacatttcaccttcaccttacacctggagactcccaagccctccatctcc 540 GlyValThrGlyHisPheThrPheThrLeuHisLeuGluThrProLysProSerIleSer - 541 agcagcaacttaaatcccagggaggccatggaggctgtgatcttaacctgtgatcctgcg 600 SerSerAsnLeuAsnProArgGluAlaMetGluAlaValIleLeuThrCysAspProAla - 601 actccagccgcaagctaccagtggtggatgaatggtcagagcctccctatgactcacagg 660 ThrProAlaAlaSerTyrGlnTrpTrpMetAsnGlyGlnSerLeuProMetThrHisArg - 661 ttgcagctgtccaaaaccaacaggaccctctttatatttggtgtcacaaagtatattgca 720 LeuGlnLeuSerLysThrAsnArgThrLeuPheIlePheGlyValThrLysTyrIleAla - 721 ggaccctatgaatgtgaaatacggaacccagtgagtgccagccgcagtgacccagtcacc 780 GlyProTyrGluCysGluIleArgAsnProValSerAlaSerArgSerAspProValThr - 781 ctgaatctcctcccaaagctgtccaagccctacatcacaatcaacaacttaaaccccaga 840 LeuAsnLeuLeuProLysLeuSerLysProTyrIleThrIleAsnAsnLeuAsnProArg - 841 gagaataaggatgtcttaaccttcacctgtgaacctaagagtgagaactacacctacatt 900 GluAsnLysAspValLeuThrPheThrCysGluProLysSerGluAsnTyrThrTyrIle - 901 tggtggctaaatggtcagagcctccctgtcagtcccagggtaaagcgacccattgaaaac 960 TrpTrpLeuAsnGlyGlnSerLeuProValSerProArgValLysArgProIleGluAsn - 961 aggatcctcattctacccaatgtcacgagaaatgaaacaggaccttatcaatgtgaaata 1020 ArgIleLeuIleLeuProAsnValThrArgAsnGluThrGlyProTyrGlnCysCluIle - 1021 cgggaccgatatggtggcatccgcagtgacccagtcaccctgaatgtcctctatggtcca 1080 ArgAspArgTyrGlyGlyIleArgSerAspProValThrLeuAsnValLeuTyrGlyPro - 1081 gacctccccagcatttacccttcattcacctattaccgttcaggagaaaacctctacttt 1140 AspLeuProSerIleTyrProSerPheThrTyrTyrArgSerGlyGluAsnLeuTyrPhe - 1141 tcctgcttcggtgagtctaacccacgggcacaatattcttggacaattaatgggaagttt 1200 SerCysPheGlyGluSerAsnProArgAlaGlnTyrSerTrpThrIleAsnGlyLysPhe - 1201 cagctatcaggacaaaagctctctatcccccaaataactacaaagcatagtgggctctat 1260 GlnLeuSerGlyGlnLysLeuSerIleProGlnIleThrThrLysHisSerGlyLeuTyr - 1261 gcttgctctgttcgtaactcagccactggcaaggaaagctccaaatccatcacagtcaaa 1320 AlaCysSerValArgAsnSerAlaThrGlyLysGluSerSerLysSerIleThrValLys - 1321 gtctctgactggatattaccctgaattctactagttcctccaattccattttctcccatg 1380 ValSerAspTrpIleLeuProEnd - 1381 gaatcacgaagagcaagacccactctgttccagaagccctataatctggaggtggacaac 1440 - 1441 tcgatgtaaatttcatgggaaaacccttgtacctgacatgtgagccactcagaactcacc 1500 - 1501 aaaatgttcgacaccataacaacagctactcaaactgtaaaccaggataagaagttgatg 1560 - 1561 acttcacactgtggacagtttttcaaagatgtcataacaagactccccatcatgacaagg 1620 - 1621 ctccaccctctactgtctgctcatgcctgcctctttcacttggcaggataatgcagtcat 1680 - 1681 tagaatttcacatgtagtagcttctgagggtaacaacagagtgtcagatatgtcatctca 1740 - 1741 acctcaaacttttacgtaacatctcagggaaatgtggctctctccatcttgcatacaggg 1800 - 1801 ctcccaatagaaatgaacacagagatattgcctgtgtgtttgcagagaagatggtttcta 1860 - 1861 taaagagtaggaaagctgaaattatagtagagtctcctttaaatgcacattgtgtggatg 1920 - 1921 gctctcaccatttcctaagagatacagtgtaaaaacgtgacagtaatactgattctagca 1980 - 1981 gaataaacatgtaccacatttgcaaaaaa 2010 - pcKGCEA2(SEQ ID NO:9): - 1 gggtggatcctaggctcatctccataggggagaacacacatacagcagagaccatggga 59 MetGly - 60 cccctctcagcccctccctgcactcagcacatcacctggaaggggctcctgctcacagca 119 ProLeuSerAlaProProCysThrGlnHi sIleThrTrpLysGlyLeuLeuLeuThrAla - 120 tcacttttaaacttctggaacctgcccaccactgcccaagtaataattgaagcccagcca 179 SerLeuLeuAsnPheTrpAsnLeuProThrThrAlaGlnValIleIleGluAlaGlnPro - 180 cccaaagtttctgaggggaaggatgttcttctacttgtccacaatttgccccagaatctt 239 ProLysValSerGluGlyLysAspValLeuLeuLeuValHisAsnLeuProGlnAsnLeu - 240 actggctacatctggtacaaagggcaaatgacggacctctaccattacattacatcatat 299 ThrGlyTyrIleTrpTyrLysGlyGlnMetThrAspLeuTyrHisTyrIleThrSerTyr - 300 gtagtagacggtcaaattatatatgggcctgcctacagtggacgagaaacagtatattcc 359 ValValAspGlyGlnIleIleTyrGlyProAlaTyrSerGlyArgGluThrValTyrSer - 360 aatgcatccctgctgatccagaatgtcacacaggaggatgcaggatcctacaccttacac 419 AsnAlaSerLeuLeuIleGlnAsnValThrGlnGluAspAlaGlySerTyrThrLeuHis - 420 atcataaagcgaggcgatgggactggaggagtaactggatatttcactgtcaccttatac 479 IleIleLysArgGlyAspGlyThrGlyGlyValThrGlyTyrPheThrValThrLeuTyr - 480 tcggagactcccaagcgctccatctccagcagcaacttaaaccccagggaggtcatggag 539 SerGluThrProLysArgSerIleSerSerSerAsnLeuAsnProArgGluValMetGlu - 540 gctgtgcgcttaatctgtgatcctgagactccggatgcaagctacctgtggttgctgaat 599 AlaValArgLeuIleCysAspProGluThrProAspAlaSerTyrLeuTrpLeuLeuAsn - 600 ggtcagaacctccctatgactcacaggttgcagctgtccaaaaccaacaggaccctctat 659 GlyGlnAsnLeuProMetThrHisArgLeuGlnLeuSerLysThrAsnArgThrLeuTyr - 660 ctatttggtgtcacaaagtatattgcagggccctatgaatgtgaaatacggaggggagtg 719 LeuPheGlyValThrLysTyrIleAlaGlyProTyrGluCysGluIleArgArgGlyVal - 720 agtgccagccgcagtgacccagtcaccctgaatctcctcccgaagctgcccatgccttac 779 SerAlaSerArgSerAspProValThrLeuAsnLeuLeuProLysLeuProMetProTyr - 780 atcaccatcaacaacttaaaccccagggagaagaaggatgtgttagccttcacctgtgaa 839 IleThrIleAsnAsnLeuAsnProArgGluLysLysAspValLeuAlaPheThrCysGlu - 840 cctaagagtcggaactacacctacatttggtggctaaatggtcagagcctcccggtcagt 899 ProLysSerArgAsnTyrThrTyrIleTrpTrpLeuAsnGlyGlnSerLeuProValSer - 900 ccgagggtaaagcgacccattgaaaacaggatactcattctacccagtgtcacgagaaat 959 ProArgValLysArgProIleGluAsnArgIleLeuIleLeuProSerValThrArgAsn - 960 gaaacaggaccctatcaatgtgaaatacgggaccgatatggtggcatccgcagtaaccca 1019 GluThrGlyProTyrGlnCysGluIleArgAspArgTyrGlyGlyIleArgSerAsnPro - 1020 gtcaccctgaatgtcctctatggtccagacctccccagaatttacccttacttcacctat 1079 ValThrLeuAsnValLeuTyrGlyProAspLeuProArgIleTyrProTyrPheThrTyr - 1080 taccgttcaggagaaaacctcgacttgtcctgctttgcggactctaacccaccggcagag 1139 TyrArgSerGlyGluAsnLeuAspLeuSerCysPheAlaAspSerAsnProProAlaGlu - 1140 tatttttggacaattaatgggaagtttcagctatcaggacaaaagctctttatcccccaa 1199 TyrPheTrpThrIleAsnGlyLysPheGlnLeuSerGlyGlnLysLeuPheIleProGln - 1200 attactacaaatcatagcgggctctatgcttgctctgttcgtaactcagccactggcaag 1259 IleThrThrAsnHisSerGlyLeuTyrAlaCysSerValArgAsnSerAlaThrGlyLys - 1260 gaaatctccaaatccatgatagtcaaagtctctggtccctgccatggaaaccagacagag 1319 GluIleSerLysSerMetIleValLysValSerGlyProCysHisGlyAsnGlnThrGlu - 1320 tctcattaatggctgccacaatagagacactgagaaaaagaacaggttgataccttcatg 1379 SerHisEnd - 1380 aaattcaagacaaagaagaaaaaggctcaatgttattggactaaataatcaaaaggataa 1439 - 1440 tgttttcataatttttattggaaaatgtgctgattcttggaatgttttattctccagatt 1499 - 1500 tatgaactttttttcttcagcaattggtaaagtatacttttgtaaacaaaaattgaaaca 1559 - 1560 tttgcttttgctctctatctgagtgccccccc 1591
It will be appreciated that the instant specification and claims are set forth by way of illustration and not limitation and that various modifications and changes may be made without departing from the spirit and scope of the present invention.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 14 - - - - (2) INFORMATION FOR SEQ ID NO: 1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3173 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 1: - - CAGCCGTGCT CGAAGCGTTC CTGGAGCCCA AGCTGTCCTC - # - # 40 - - CACAGGTGAA GACAGGGCCA GCAGGAGACA CC ATG GGC - #- # 78 - # - #Met Gly - - CAC CTC TCA GCC CCA CTT CAC AGA GTG CGT GT - #A CCC TGG - # 117 His Leu Ser Ala Pro Leu His Arg Val Arg Va - #l Pro Trp 5 - # 10 - # 15 - - CAG GGG CTT CTG CTC ACA GCC TCA CTT CTA AC - #C TTC TGG - # 156 Gln Gly Leu Leu Leu Thr Ala Ser Leu Leu Th - #r Phe Trp 20 - # 25 - - AAC CCG CCC ACC ACT GCC CAG CTC ACT ACT GA - #A TCC ATG - # 195 Asn Pro Pro Thr Thr Ala Gln Leu Thr Thr Gl - #u Ser Met 30 - # 35 - # 40 - - CCA TTC AAT GTT GCA GAG GGG AAG GAG GTT CT - #T CTC CTT - # 234 Pro Phe Asn Val Ala Glu Gly Lys Glu Val Le - #u Leu Leu 45 - # 50 - - GTC CAC AAT CTG CCC CAG CAA CTT TTT GGC TA - #C AGC TGG - # 273 Val His Asn Leu Pro Gln Gln Leu Phe Gly Ty - #r Ser Trp 55 - # 60 - # 65 - - TAC AAA GGG GAA AGA GTG GAT GGC AAC CGT CA - #A ATT GTA - # 312 Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gl - #n Ile Val 70 - # 75 - # 80 - - GGA TAT GCA ATA GGA ACT CAA CAA GCT ACC CC - #A GGG CCC - # 351 Gly Tyr Ala Ile Gly Thr Gln Gln Ala Thr Pr - #o Gly Pro 85 - # 90 - - GCA AAC AGC GGT CGA GAG ACA ATA TAC CCC AA - #T GCA TCC - # 390 Ala Asn Ser Gly Arg Glu Thr Ile Tyr Pro As - #n Ala Ser 95 - # 100 - # 105 - - CTG CTG ATC CAG AAC GTC ACC CAG AAT GAC AC - #A GAA TTC - # 429 Leu Leu Ile Gln Asn Val Thr Gln Asn Asp Th - #r Gly Phe 110 - # 115 - - TAC ACC CTA CAA GTC ATA AAG TCA GAT CTT GT - #G AAT GAA - # 468 Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Va - #l Asn Glu 120 1 - #25 1 - #30 - - GAA GCA ACT GGA CAG TTC CAT GTA TAC CCG GA - #G CTG CCC - # 507 Glu Ala Thr Gly Gln Phe His Val Tyr Pro Gl - #u Leu Pro 135 - # 140 - # 145 - - AAG CCC TCC ATC TCC AGC AAC AAC TCC AAC CC - #T GTG GAG - # 546 Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pr - #o Val Glu 150 - # 155 - - GAC AAG GAT GCT GTG GCC TCC ACC TGT GAA CC - #T GAG ACT - # 585 Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pr - #o Glu Thr 160 - # 165 - # 170 - - CAG GAC ACA ACC TAC CTG TGG TGG ATA AAC AA - #T CAG AGC - # 624 Gln Asp Thr Thr Tyr Leu Trp Trp Ile Asn As - #n Gln Ser 175 - # 180 - - CTC CCG GTC AGT CCC AGG CTG CAG CTG TCC AA - #T GGC AAC - # 663 Leu Pro Val Ser Pro Arg Leu Gln Leu Ser As - #n Gly Asn 185 1 - #90 1 - #95 - - AGG ACC CTC ACT CTA CTC AGT GTC ACA AGG AA - #T GAC ACA - # 702 Arg Thr Leu Thr Leu Leu Ser Val Thr Arg As - #n Asp Thr 200 - # 205 - # 210 - - GGA CCC TAT GAG TGT GAA ATA CAG AAC CCA GT - #G AGT GCG - # 741 Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Va - #l Ser Ala 215 - # 220 - - AAC CTC AGT GAC CCA GTC ACC TTG AAT GTC AC - #C TAT GGC - # 780 Asn Arg Ser Asp Pro Val Thr Leu Asn Val Th - #r Tyr Gly 225 - # 230 - # 235 - - CCG GAC ACC CCC ACC ATT TCC CCT TCA GAC AC - #C TAT TAC - # 819 Pro Asp Thr Pro Thr Ile Ser Pro Ser Asp Th - #r Tyr Tyr 240 - # 245 - - CGT CCA GGG GCA AAC CTC AGC CTC TCC TGC TA - #T GCA GCC - # 858 Arg Pro Gly Ala Asn Leu Ser Leu Ser Cys Ty - #r Ala Ala 250 2 - #55 2 - #60 - - TCT AAC CCA CCT GCA CAG TAC TCC TGG CTT AT - #C AAT GGA - # 897 Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Il - #e Asn Gly 265 - # 270 - # 275 - - ACA TTC CAG CAA AGC ACA CAA GAG CTC TTT AT - #C CCT AAC - # 936 Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Il - #e Pro Asn 280 - # 285 - - ATC ACT GTG AAT AAT AGT GGA TCC TAT ACC TG - #G CAC GCC - # 975 Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cy - #s His Ala 290 - # 295 - # 300 - - AAT AAC TCA GTC ACT GGC TGC AAC AGG ACC AC - #A GTC AAG - # 1014 Asn Asn Ser Val Thr Gly Cys Asn Arg Thr Th - #r Val Lys 305 - # 310 - - ACG ATC ATA GTC ACT GAT AAT GCT CTA CCA CA - #A GAA AAT - # 1053 Thr Ile Ile Val Thr Asp Asn Ala Leu Pro Gl - #n Glu Asn 315 3 - #20 3 - #25 - - GGC CTC TCA CCT GGG GCC ATT GCT GGC ATT GT - #G ATT GGA - # 1092 Gly Leu Ser Pro Gly Ala Ile Ala Gly Ile Va - #l Ile Gly 330 - # 335 - # 340 - - GTA GTG GCC CTG GTT GCT CTG ATA GCA GTA GC - #C CTG GCA - # 1131 Val Val Ala Leu Val Ala Leu Ile Ala Val Al - #a Leu Ala 345 - # 350 - - TGT TTT CTG CAT TTC GGG AAG ACC GGC AGG GC - #A AGC GAC - # 1170 Cys Phe Leu His Phe Gly Lys Thr Gly Arg Al - #a Ser Asp 355 - # 360 - # 365 - - CAG CGT GAT CTC ACA GAG CAC AAA CCC TCA GT - #C TCC AAC - # 1209 Gln Arg Asp Leu Thr Glu His Lys Pro Ser Va - #l Ser Asn 370 - # 375 - - CAC ACT CAG GAC CAC TCC AAT GAC CCA CCT AA - #C AAG ATG - # 1248 His Thr Gln Asp His Ser Asn Asp Pro Pro As - #n Lys Met 380 3 - #85 3 - #90 - - AAT GAA GTT ACT TAT TCT ACC CTG AAC TTT GA - #A GCC CAG - # 1287 Asn Glu Val Thr Tyr Ser Thr Leu Asn Phe Gl - #u Ala Gln 395 - # 400 - # 405 - - CAA CCC ACA CAA CCA ACT TCA GCC TCC CCA TC - #C CTA ACA - # 1326 Gln Pro Thr Gln Pro Thr Ser Ala Ser Pro Se - #r Leu Thr 410 - # 415 - - GCC ACA GAA ATA ATT TAT TCA GAA GTA AAA AA - #G CAG- # 1362 Ala Thr Glu Ile Ile Tyr Ser Glu Val Lys Ly - #s Gln 420 - # 425 - - TAATGAAACC TGTCCTGCTC ACTGCAGTGC TGATGTATTT - # - # 1402 - - CAAGTCTCTC ACCCTCATCA CTAGGAGATT CCTTTCCCCT - # - # 1442 - - GTAGGGTAGA GGGGTGGGGA CAGAAACAAC TTTCTCCTAC - # - # 1482 - - TCTTCCTTCC TAATAGGCAT CTCCAGGCTG CCTGGTCACT - # - # 1522 - - GCCCCTCTCT CAGTGTCAAT AGATGAAAGT ACATTGGGAG - # - # 1562 - - TCTGTAGGAA ACCCAACCTT CTTGTCATTG AAATTTGGCA - # - # 1602 - - AAGCTGACTT TGGGAAAGAG GGACCAGAAC TTCCCCTCCC - # - # 1642 - - TTCCCCTTTT CCCAACCTGG ACTTGTTTTA AACTTGCCTG - # - # 1682 - - TTCAGAGCAC TCATTCCTTC CCACCCCCAG TCCTGTCCTA - # - # 1722 - - TCACTCTAAT TCGGATTTGC CATAGCCTTG AGGTTATGTC - # - # 1762 - - CTTTTCCATT AAGTACATGT GCCAGGAAAC AGCGAGAGAG - # - # 1802 - - AGAAAGTAAA CGGCAGTAAT GCTTCTCCTA TTTCTCCAAA - # - # 1842 - - GCCTTGTGTG AACTAGCAAA GAGAAGAAAA TCAAATATAT - # - # 1882 - - AACCAATAGT GAAATGCCAC AGGTTTGTCC ACTGTCAGGG - # - # 1922 - - TTGTCTACCT GTAGGATCAG GGTCTAAGCA CCTTGGTGCT - # - # 1962 - - TAGCTAGAAT ACCACCTAAT CCTTCTGGCA AGCCTGTCTT - # - # 2002 - - CAGAGAACCC ACTAGAAGCA ACTAGGAAAA ATCACTTGCC - # - # 2042 - - AAAATCCAAG GCAATTCCTG ATGGAAAATG CAAAAGCACA - # - # 2082 - - TATATGTTTT AATATCTTTA TGGGCTCTGT TCAAGGCAGT - # - # 2122 - - GCTGAGAGGG AGGGGTTATA GCTTCAGGAG GGAACCAGCT - # - # 2162 - - TCTGATAAAC ACAATCTGCT AGGAACTTGG GAAAGGAATC - # - # 2202 - - AGAGAGCTGC CCTTCAGCGA TTATTTAAAT TGTTAAAGAA - # - # 2242 - - TACACAATTT GGGGTATTGG GATTTTTCTC CTTTTCTCTG - # - # 2282 - - AGACATTCCA CCATTTTAAT TTTTGTAACT GCTTATTTAT - # - # 2322 - - GTGAAAAGGG TTATTTTTAC TTAGCTTAGC TATGTCAGCC - # - # 2362 - - AATCCGATTG CCTTAGGTGA AAGAAACCAC CGAAATCCCT - # - # 2402 - - CAGGTCCCTT GGTCAGGAGC CTCTCAAGAT TTTTTTTGTC - # - # 2442 - - AGAGGCTCCA AATAGAAAAT AAGAAAAGGT TTTCTTCATT - # - # 2482 - - CATGGCTAGA GCTAGATTTA ACTCAGTTTC TAGGCACCTC - # - # 2522 - - AGACCAATCA TCAACTACCA TTCTATTCCA TGTTTGCACC - # - # 2562 - - TGTGCATTTT CTGTTTGCCC CCATTCACTT TGTCAGGAAA - # - # 2602 - - CCTTGGCCTC TGCTAAGGTG TATTTGGTCC TTGAGAAGTG - # - # 2642 - - GGAGCACCCT ACAGGGACAC TATCACTCAT GCTGGTGGCA - # - # 2682 - - TTGTTTACAG CTAGAAAGCT GCACTGGTGC TAATGCCCCT - # - # 2722 - - TGGGAAATGG GGCTGTGAGG AGGAGGATTA TAACTTAGGC - # - # 2762 - - CTAGCCTCTT TTAACAGCCT CTGAAATTTA TCTTTTCTTC - # - # 2802 - - TATGGGGTCT ATAAATGTAT CTTATAATAA AAAGGAAGGA - # - # 2842 - - CAGGAGGAAG ACAGGCAAAT GTACTTCTCA CCCAGTCTTC - # - # 2882 - - TACACAGATG GAATCTCTTT GGGGCTAAGA GAAAGGTTTT - # - # 2922 - - ATTCTATATT GCTTACCTGA TCTCATGTTA GGCCTAAGAG - # - # 2962 - - GCTTTCTCCA GGAGGATTAG CTTGGAGTTC TCTATACTCA - # - # 3002 - - GGTACCTCTT TCAGGGTTTT CTAACCCTGA CACGGACTGT - # - # 3042 - - GCATACTTTC CCTCATCCAT GCTGTGCTGT GTTATTTAAT - # - # 3082 - - TTTTCCTGGC TAAGATCATG TCTGAATTAT GTATGAAAAT - # - # 3122 - - TATTCTATGT TTTTATAATA AAAATAATAT ATCAGACATC - # - # 3162 - - GAAAAAAAAA A - # - # - # 3173 - - - - (2) INFORMATION FOR SEQ ID NO: 2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1630 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 2: - - CAGCCGTGCT CGAAGCGTTC CTGGAGCCCA AGCTCTCCTC - # - # 40 - - CACAGGTGAA GACAGGGCCA GCAGGAGACA - # - # 70 - - CC ATG GGG CAC CTC TCA GCC CCA CTT CAC AGA - # GTG CGT - # 108 Met Gly His Leu Ser Ala Pro Leu His - #Arg Val Arg - # 5 - # 10 - - GTA CCC TGG CAG GGG CTT CTG CTC ACA GCC TC - #A CTT CTA - # 147 Val Pro Trp Gln Gly Leu Leu Leu Thr Ala Se - #r Leu Leu 15 - # 20 - # 25 - - ACC TTC TGG AAC CCG CCC ACC ACT GCC CAG CT - #C ACT ACT - # 186 Thr Phe Trp Asn Pro Pro Thr Thr Ala Gln Le - #u Thr Thr 30 - # 35 - - GAA TCC ATG CCA TTC AAT GTT GCA GAG GGG AA - #G GAG GTT - # 225 Glu Ser Met Pro Phe Asn Val Ala Glu Gly Ly - #s Glu Val 40 - # 45 - # 50 - - CTT CTC CTT GTC CAC AAT CTG CCC CAG CAA CT - #T TTT GGC - # 264 Leu Leu Leu Val His Asn Leu Pro Gln Gln Le - #u Phe Gly 55 - # 60 - - TAC AGC TGG TAC AAA GGG GAA AGA GTG GAT GG - #C AAC CGT - # 303 Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gl - #y Asn Arg 65 - # 70 - # 75 - - CAA ATT GTA GGA TAT GCA ATA GGA ACT CAA CA - #A GCT ACC - # 342 Gln Ile Val Gly Tyr Ala Ile Gly Thr Gln Gl - #n Ala Thr 80 - # 85 - # 90 - - CCA GGG CCC GCA AAC AGC GGT CGA GAG ACA AT - #A TAC CCC - # 381 Pro Gly Pro Ala Asn Ser Gly Arg Glu Thr Il - #e Tyr Pro 95 - # 100 - - AAT GCA TCC CTG CTG ATC CAG AAC GTC ACC CA - #G AAT GAC - # 420 Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gl - #n Asn Asp 105 - # 110 - # 115 - - ACA GGA TTC TAC ACC CTA CAA GTC ATA AAG TC - #A GAT CTT - # 459 Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Se - #r Asp Leu 120 - # 125 - - GTG AAT GAA GAA GCA ACT GGA CAG TTC CAT GT - #A TAC CCG - # 498 Val Asn Glu Glu Ala Thr Gly Gln Phe His Va - #l Tyr Pro 130 1 - #35 1 - #40 - - GAG CTG CCC AAG CCC TCC ATC TCC AGC AAC AA - #C TCC AAC - # 537 Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn As - #n Ser Asn 145 - # 150 - # 155 - - CCT GTG GAG GAC AAG GAT GCT GTG GCC TTC AC - #C TGT GAA - # 576 Pro Val Glu Asp Lys Asp Ala Val Ala Phe Th - #r Cys Glu 160 - # 165 - - CCT GAG ACT CAG GAC ACA ACC TAC CTG TGG TG - #G ATA AAC - # 615 Pro Glu Thr Gln Asp Thr Thr Tyr Leu Trp Tr - #p Ile Asn 170 - # 175 - # 180 - - AAT CAG AGC CTC CCG GTC AGT CCC AGG CTG CA - #G CTG TCC - # 654 Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gl - #n Leu Ser 185 - # 190 - - AAT GGC AAC AGG ACC CTC ACT CTA CTC AGT GT - #C ACA AGG - # 693 Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Va - #l Thr Arg 195 2 - #00 2 - #05 - - AAT GAC ACA GGA CCC TAT GAG TGT GAA ATA CA - #G AAC CCA - # 732 Asn Asp Thr Gly Pro Tyr Glu Cys Glu Ile Gl - #n Asn Pro 210 - # 215 - # 220 - - GTG AGT GCG AAC CGC AGT GAC CCA GTC ACC TT - #G AAT GTC - # 771 Val Ser Ala Asn Arg Ser Asp Pro Val Thr Le - #u Asn Val 225 - # 230 - - AAC TAT GGC CCG GAC ACC CCC ACC ATT TCC CC - #T TCA GAC - # 810 Thr Tyr Gly Pro Asp Thr Pro Thr Ile Ser Pr - #o Ser Asp 235 - # 240 - # 245 - - ACC TAT TAC CGT CCA GGG GCA AAC CTC AGC CT - #C TCC TGC - # 849 Thr Tyr Tyr Arg Pro Gly Ala Asn Leu Ser Le - #u Ser Cys 250 - # 255 - - TAT GCA GCC TCT AAC CCA CCT GCA CAG TAC TC - #C TGG CTT - # 888 Tyr Ala Ala Ser Asn Pro Pro Ala Gln Tyr Se - #r Trp Leu 260 2 - #65 2 - #70 - - ATC AAT GGA ACA TTC CAG CAA AGC ACA CAA GA - #G CTC TTT - # 927 Ile Asn Gly Thr Phe Gln Gln Ser Thr Gln Gl - #u Leu Phe 275 - # 280 - # 285 - - ATC CCT AAC ATC ACT GTG AAT AAT AGT GGA TC - #C TAT ACC - # 966 Ile Pro Asn Ile Thr Val Asn Asn Ser Gly Se - #r Tyr Thr 290 - # 295 - - TGC CAC GCC AAT AAC TCA GTC ACT GGC TGC AA - #C AGG ACC - # 1005 Cys His Ala Asn Asn Ser Val Thr Gly Cys As - #n Arg Thr 300 - # 305 - # 310 - - ACA GTC AAG ACG ATC ATA GTC ACT GAG CTA AG - #T CCA GTA - # 1044 Thr Val Lys Thr Ile Ile Val Thr Glu Leu Se - #r Pro Val 315 - # 320 - - GTA GCA AAG CCC CAA ATC AAA GCC AGC AAG AC - #C ACA GTC - # 1083 Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Th - #r Thr Val 325 3 - #30 3 - #35 - - ACA GGA GAT AAG GAC TCT GTG AAC CTG ACC TG - #C TCC ACA - # 1122 Thr Gly Asp Lys Asp Ser Val Asn Leu Thr Cy - #s Ser Thr 340 - # 345 - # 350 - - AAT GAC ACT GGA ATC TCC ATC CGT TGG TTC TT - #C AAA AAC - # 1161 Asn Asp Thr Gly Ile Ser Ile Arg Trp Phe Ph - #e Lys Asn 355 - # 360 - - CAG AGT CTC CCG TCC TCG GAG AGG ATG AAG CT - #G TCC CAG - # 1200 Gln Ser Leu Pro Ser Ser Glu Arg Met Lys Le - #u Ser Gln 365 - # 370 - # 375 - - GGC AAC ACC ACC CTC AGC ATA AAC CCT GTC AA - #G AGG GAG - # 1239 Gly Asn Thr Thr Leu Ser Ile Asn Pro Val Ly - #s Arg Glu 380 - # 385 - - GAT GCT GGG ACG TAT TGG TGT GAG GTC TTC AA - #C CCA ATC - # 1278 Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe As - #n Pro Ile 390 3 - #95 4 - #00 - - AGT AAG AAC CAA AGC GAC CCC ATC ATG CTG AA - #C GTA AAC - # 1317 Ser Lys Asn Gln Ser Asp Pro Ile Met Leu As - #n Val Asn 405 - # 410 - # 415 - - TAT AAT GCT CTA CCA CAA GAA AAT GGC CTC TC - #A CCT GGG - # 1356 Tyr Asn Ala Leu Pro Gln Glu Asn Gly Leu Se - #r Pro Gly 420 - # 425 - - GCC ATT GCT GGC ATT GTG ATT GGA GTA GTG GC - #C CTG GTT - # 1395 Ala Ile Ala Gly Ile Val Ile Gly Val Val Al - #a Leu Val 430 - # 435 - # 440 - - GCT CTG ATA GCA GTA GCC CTG GCA TGT TTT CT - #G CAT TTC - # 1434 Ala Leu Ile Ala Val Ala Leu Ala Cys Phe Le - #u His Phe 445 - # 450 - - GGG AAG ACC GGC AGC TCA GGA CCA CTC CAA - # - # 1464 Gly Lys Thr Gly Ser Ser Gly Pro Leu Gln 455 4 - #60 - - TGACCCACCT AACAAGATGA ATGAAGTTAC TTATTCTACC - # - # 1504 - - CTGAACTTTG AAGCCCAGCA ACCCACACAA CCAACTTCAG - # - # 1544 - - CCTCCCCATC CCTAACAGCC ACAGAAATAA TTTATTCAGA - # - # 1584 - - AGTAAAAAAG CAGTAATGAA ACCTGAAAAA AAAAAAAAAA - # - # 1624 - - AAAAAA - # - # -# 1630 - - - - (2) INFORMATION FOR SEQ ID NO: 3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1339 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 3: - - CAGCCGTGCT CGAAGCGTTC CTGGAGCCCA AGCTCTCCTC - # - # 40 - - CACAGGTGAA GACAGGGCCA GCAGGAGACA CC ATG - # -# 75 - # - #Met - - GGG CAC CTC TCA GCC CCA CTT CAC AGA GTG CG - #T GTA CCC - # 114 Gly His Leu Ser Ala Pro Leu His Arg Val Ar - #g Val Pro 5 - # 10 - - TGG CAG GGG CTT CTG CTC ACA GCC TCA CTT CT - #A ACC TTC - # 153 Trp Gln Gly Leu Leu Leu Thr Ala Ser Leu Le - #u Thr Phe 15 - # 20 - # 25 - - TGG AAC CCG CCC ACC ACT GCC CAG CTC ACT AC - #T GAA TCC - # 192 Trp Asn Pro Pro Thr Thr Ala Gln Leu Thr Th - #r Glu Ser 30 - # 35 - # 40 - - ATG CCA TTC AAT GTT GCA GAG GGG AAG GAG GT - #T CTT CTC - # 231 Met Pro Phe Asn Val Ala Glu Gly Lys Glu Va - #l Leu Leu 45 - # 50 - - CTT GTC CAC AAT CTG CCC CAG CAA CTT TTT GG - #C TAC AGC - # 270 Leu Val His Asn Leu Pro Gln Gln Leu Phe Gl - #y Tyr Ser 55 - # 60 - # 65 - - TGG TAC AAA GGG GAA AGA GTG GAT GGC AAC CG - #T CAA ATT - # 309 Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ar - #g Gln Ile 70 - # 75 - - GTA GGA TAT GCA ATA GGA ACT CAA CAA GCT AC - #C CCA GGG - # 348 Val Gly Tyr Ala Ile Gly Thr Gln Gln Ala Th - #r Pro Gly 80 - # 85 - # 90 - - CCC GCA AAC AGC GGT CGA GAG ACA ATA TAC CC - #C AAT GCA - # 387 Pro Ala Asn Ser Gly Arg Glu Thr Ile Tyr Pr - #o Asn Ala 95 - # 100 - # 105 - - TCC CTG CTG ATC CAG AAC GTC ACC CAG AAT GA - #C ACA GGA - # 426 Ser Leu Leu Ile Gln Asn Val Thr Gln Asn As - #p Thr Gly 110 - # 115 - - TTC TAC ACC CTA CAA GTC ATA AAG TCA GAT CT - #T GTG AAT - # 465 Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Le - #u Val Asn 120 - # 125 - # 130 - - GAA GAA GCA ACT GGA CAG TTC CAT GTA TAC CC - #G GAG CTG - # 504 Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pr - #o Glu Leu 135 - # 140 - - CCC AAG CCC TCC ATC TCC AGC AAC AAC TCC AA - #C CCT GTG - # 543 Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser As - #n Pro Val 145 1 - #50 1 - #55 - - GAG GAC AAG GAT GCT GTG GCC TTC ACC TGT GA - #A CCT GAG - # 582 Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Gl - #u Pro Glu 160 - # 165 - # 170 - - ACT CAG GAC ACA ACC TAC CTG TGG TGG ATA AA - #C AAT CAG - # 621 Thr Gln Asp Thr Thr Tyr Leu Trp Trp Ile As - #n Asn Gln 175 - # 180 - - AGC CTC CCG GTC AGT CCC AGG CTG CAG CTG TC - #C AAT GGC - # 660 Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Se - #r Asn Gly 185 - # 190 - # 195 - - AAC AGG ACC CTC ACT CTA CTC AGT GTC ACA AG - #G AAT GAC - # 699 Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Ar - #g Asn Asp 200 - # 205 - - ACA GGA CCC TAT GAG TGT GAA ATA CAG AAC CC - #A GTG AGT - # 738 Thr Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pr - #o Val Ser 210 2 - #15 2 - #20 - - GCG AAC CGC AGT GAC CCA GTC ACC TTG AAT GT - #C ACC TAT - # 777 Ala Asn Arg Ser Asp Pro Val Thr Leu Asn Va - #l Thr Tyr 225 - # 230 - # 235 - - GGC CCG GAC ACC CCC ACC ATT TCC CCT TCA GA - #C ACC TAT - # 816 Gly Pro Asp Thr Pro Thr Ile Ser Pro Ser As - #p Thr Tyr 240 - # 245 - - TAC CGT CCA GGG GCA AAC CTC AGC CTC TCC TG - #C TAT GCA - # 855 Tyr Arg Pro Gly Ala Asn Leu Ser Leu Ser Cy - #s Tyr Ala 250 - # 255 - # 260 - - GCC TCT AAC CCA CCT GCA CAG TAC TCC TGG CT - #T ATC AAT - # 894 Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Le - #u Ile Asn 265 - # 270 - - GGA ACA TTC CAG CAA AGC ACA CAA GAG CTC TT - #T ATC CCT - # 933 Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Ph - #e Ile Pro 275 2 - #80 2 - #85 - - AAC ATC ACT GTG AAT AAT AGT GGA TCC TAT AC - #C TGC CAC - # 972 Asn Ile Thr Val Asn Asn Ser Gly Ser Tyr Th - #r Cys His 290 - # 295 - # 300 - - GCC AAT AAC TCA GTC ACT GGC TGC AAC AGG AC - #C ACA GTC - # 1011 Ala Asn Asn Ser Val Thr Gly Cys Asn Arg Th - #r Thr Val 305 - # 310 - - AAG ACG ATC ATA GTC ACT GAT AAT GCT CTA CC - #A CAA GAA - # 1050 Lys Thr Ile Ile Val Thr Asp Asn Ala Leu Pr - #o Gln Glu 315 - # 320 - # 325 - - AAT GGC CTC TCA CCT GGG GCC ATT GCT GGC AT - #T GTG ATT - # 1089 Asn Gly Leu Ser Pro Gly Ala Ile Ala Gly Il - #e Val Ile 330 - # 335 - - GGA GTA GTG GCC CTG GTT GCT CTG ATA GCA GT - #A GCC CTG - # 1128 Gly Val Val Ala Leu Val Ala Leu Ile Ala Va - #l Ala Leu 340 3 - #45 3 - #50 - - GCA TGT TTT CTG CAT TTC GGG AAG ACC GGC AG - #C TCA GGA - # 1167 Ala Cys Phe Leu His Phe Gly Lys Thr Gly Se - #r Ser Gly 355 - # 360 - # 365 - - CCA CTC CAA TGACCCACCT AACAAGATGA ATGAAGTTAC - # - # 1206 Pro Leu Gln - - TTATTCTACC CTGAACTTTG AAGCCCAGCA ACCCACACAA - # - # 1246 - - CCAACTTCAG CCTCCCCATC CCTAACAGCC ACAGAAATAA - # - # 1286 - - TTTATTCAGA AGTAAAAAAG CAGTAATGAA ACCTGAAAAA - # - # 1326 - - AAAAAAAAAA AAA - # - # - # 1339 - - - - (2) INFORMATION FOR SEQ ID NO: 4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 862 nucleot - #ides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 4: - - GGGGTTTACA CAACCACCAC CCCATCAAAC CCTTCATCAC - # - # 40 - - CAGCAACAAC TCCAACCCCG TGGAGGATGA GGATGCTGTA - # - # 80 - - GCCTTAACCT GTGAACCTCA GATTCAGAAC ACAACCTACC - # - # 120 - - TGTGGTGGGT AAATAATCAG AGCCTCCCGG TCAGTCCCAG - # - # 160 - - GCTGCAGCTG TCCAATGACA ACAGGACCCT CACTCTACTC - # - # 200 - - AGTGTCACAA GGAATGATGT AGGACCCTAT GAGTGTGGAA - # - # 240 - - TCCAGAACGA ATTAAGTGTT GACCACAGCG ACCCAGTCAT - # - # 280 - - CCTGAATGTC CTCTATGGCC CAGACGACCC CACCATTTCC - # - # 320 - - CCCTCATACA CCTATTACCG TCCAGGGGTG AACCTCAGCC - # - # 360 - - TCTCCTGCCA TGCAGCCTCT AACCCACCTG CACAGTATTC - # - # 400 - - TTGGCTGATT GATGGGAACA TCCAGCAACA CACACAAGAG - # - # 440 - - CTCTTTATCT CCAACATCAC TGAGAAGAAC AGCGGACTCT - # - # 480 - - ATACCTGCCA GGCCAATAAC TCAGCCAGTG GCCACAGCAG - # - # 520 - - GACTACAGTC AAGACAATCA CAGTCTCTGC GGACGTGCCC - # - # 560 - - AAGCCCTCCA TCTCCAGCAA CAACTCCAAA CCCGTGGAGG - # - # 600 - - ACAAGGATGC TGTGGCCTTC CACTGTGAAC CTGAGGCTCA - # - # 640 - - GAACACAACC TACCTGTGGT GGGTAAATGG TCAGAGCCTC - # - # 680 - - CCAGTCAGTC CCAGGCTGCA GCTGTCCAAT GGCAACAGGA - # - # 720 - - CCCTCACTCT ATTCAATGTC ACAAGAAATG ACGCAAGAGC - # - # 760 - - CTATGTATGT GGAATCCAGA ACTCAGTGAG TGCAAACCGC - # - # 800 - - AGTGACCCAG TCACCCTGGA TGTCCTCTAT GGGCCGGACA - # - # 840 - - CCCCCATCAT TTCCCCCCCC CC - # - # 862 - - - - (2) INFORMATION FOR SEQ ID NO: 5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2839 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 5: - - CACC ATG GAG TCT CCC TCG GCC CCT CTC CAC A - #GA TGG TGC - # 40 Met Glu Ser Pro Ser Ala Pro Leu - # His Arg Trp Cys - # -30 - # -25 - - ATC CCC TGG CAG AGG CTC CTG CTC ACA GCC TC - #A CTT CTA - # 79 Ile Pro Trp Gln Arg Leu Leu Leu Thr Ala Se - #r Leu Leu -20 - # -15 - # -10 - - ACC TTC TGG AAC CCG CCC ACC ACT GCC AAG CT - #C ACT ATT - # 118 Thr Phe Trp Asn Pro Pro Thr Thr Ala Lys Le - #u Thr Ile -5 - # - #1 - - GAA TCC ACG CCG TTC AAT GTC GCA GAG GGG AA - #G GAG GTG - # 157 Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Ly - #s Glu Val 5 - # 10 - # 15 - - CTT CTA CTT GTC CAC AAT CTG CCC CAG CAT CT - #T TTT GGC - # 196 Leu Leu Leu Val His Asn Leu Pro Glu His Le - #u Phe Gly 20 - # 25 - # 30 - - TAC AGC TGG TAC AAA GGT GAA AGA GTG GAT GG - #C AAC CGT - # 235 Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gl - #y Asn Arg 35 - # 40 - - CAA ATT ATA GGA TAT GTA ATA GGA ACT CAA CA - #A GCT ACC - # 274 Gln Ile Ile Gly Tyr Val Ile Gly Thr Gln Gl - #n Ala Thr 45 - # 50 - # 55 - - CCA GGG CCC GCA TAC AGT GGT CGA GAG ATA AT - #A TAC CCC - # 313 Pro Gly Pro Ala Tyr Ser Gly Arg Glu Ile Il - #e Tyr Pro 60 - # 65 - - AAT GCA TCC CTG CTG ATC CAG AAC ATC ATC CA - #G AAT GAC - # 352 Asn Ala Ser Leu Leu Ile Gln Asn Ile Ile Gl - #n Asn Asn 70 - # 75 - # 80 - - ACA GGA TTC TAC ACC CTA CAC GTC ATA AAG TC - #A GAT CTT - # 391 Thr Gly Phe Tyr Thr Leu His Val Ile Lys Se - #r Asp Leu 85 - # 90 - # 95 - - GTG AAT GAA GAA GCA ACT GGC CAG TTC CGG GT - #A TAC CCG - # 430 Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Va - #l Tyr Pro 100 - # 105 - - GAG CTG CCC AAG CCC TCC ATC TCC AGC AAC AA - #C TCC AAA - # 469 Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn As - #n Ser Lys 110 - # 115 - # 120 - - CCC GTG GAG GAC AAG GAT GCT GTG GCC TTC AC - #C TGT GAA - # 508 Pro Val Glu Asp Lys Asp Ala Val Ala Phe Th - #r Cys Glu 125 - # 130 - - CCT GAG ACT CAG GAC GCA ACC TAC CTG TGG TG - #G GTA AAC - # 547 Pro Glu Thr Gln Asp Ala Thr Tyr Leu Trp Tr - #p Val Asn 135 1 - #40 1 - #45 - - AAT CAG AGC CTC CCG GTC AGT CCC AGG CTG CA - #G CTG TCC - # 586 Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gl - #n Leu Ser 150 - # 155 - # 160 - - AAT GGC AAC AGG ACC CTC ACT CTA TTC AAT GT - #C ACA AGA - # 625 Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Va - #l Thr Arg 165 - # 170 - - AAT GAA CAA GCA AGC TAC AAA TGT GAA ACC CA - #G AAC CCA - # 664 Asn Glu Gln Ala Ser Tyr Lys Cys Glu Thr Gl - #n Asn Pro 175 - # 180 - # 185 - - GTG AGT GCC AGG CGC AGT GAT TCA GTC ATC CT - #G AAT GTC - # 703 Val Ser Ala Arg Arg Ser Asp Ser Val Ile Le - #u Asn Val 190 - # 195 - - CTC TAT GGC CCG GAT GCC CCC ACC ATT TCC CC - #T CTA AAC - # 742 Leu Tyr Gly Pro Asp Ala Pro Thr Ile Ser Pr - #o Leu Asn 200 2 - #05 2 - #10 - - ACA TCT TAC AGA TCA GGG GAA AAT CTG AAC CT - #C TCC TGC - # 781 Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn Le - #u Ser Cys 215 - # 220 - # 225 - - CAC GCA GCC TCT AAC CCA CCT GCA CAG TAC TC - #T TGG TTT - # 820 His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Se - #r Trp Phe 230 - # 235 - - GTC AAT GGG ACT TTC CAG CAA TCC ACC CAA GA - #G CTC TTT - # 859 Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Gl - #u Leu Phe 240 - # 245 - # 250 - - ATC CCC AAC ATC ACT GTG AAT AAT AGT GGA TC - #C TAT ACG - # 898 Ile Pro Asn Ile Thr Val Asn Asn Ser Gly Se - #r Tyr Thr 255 - # 260 - - TGC CAA GCC CAT AAC TCA GAC ACT GGC CTC AA - #T AGG ACC - # 937 Cys Gln Ala His Asn Ser Asp Thr Gly Leu As - #n Arg Thr 265 2 - #70 2 - #75 - - ACA GTC ACG ACG ATC ACA GTC TAT GCA GAG CC - #A CCC AAA - # 976 Thr Val Thr Thr Ile Thr Val Tyr Ala Glu Pr - #o Pro Lys 280 - # 285 - # 290 - - CCC TTC ATC ACC AGC AAC AAC TCC AAC CCC GT - #G GAG GAT - # 1015 Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Va - #l Glu Asp 295 - # 300 - - GAG GAT GCT GTA GCC TTA ACC TGT GAA CCT GA - #G ATT CAG - # 1054 Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Gl - #u Ile Gln 305 - # 310 - # 315 - - AAC ACA ACC TAC CTG TGG TGG GTA AAT AAT CA - #G AGC CTC - # 1093 Asn Thr Thr Tyr Leu Trp Trp Val Asn Asn Gl - #n Ser Leu 320 - # 325 - - CCG GTC AGT CCC AGG CTG CAG CTG TCC AAT GA - #C AAC AGG - # 1132 Pro Val Ser Pro Arg Leu Gln Leu Ser Asn As - #p Asn Arg 330 3 - #35 3 - #40 - - ACC CTC ACT CTA CTC AGT GTC ACA AGG AAT GA - #T GTA GGA - # 1171 Thr Leu Thr Leu Leu Ser Val Thr Arg Asn As - #p Val Gly 345 - # 350 - # 355 - - CCC TAT GAG TGT GGA ATC CAG AAC GAA TTA AG - #T GTT GAC - # 1210 Pro Tyr Glu Cys Gly Ile Gln Asn Glu Leu Se - #r Val Asp 360 - # 365 - - CAC AGC GAC CCA GTC ATC CTG AAT GTC CTC TA - #T GGC CCA - # 1249 His Ser Asp Pro Val Ile Leu Asn Val Leu Ty - #r Gly Pro 370 - # 375 - # 380 - - GAC GAC CCC ACC ATT TCC CCC TCA TAC ACC TA - #T TAC CGT - # 1288 Asp Asp Pro Thr Ile Ser Pro Ser Tyr Thr Ty - #r Tyr Arg 385 - # 390 - - CCA GGG GTG AAC CTC AGC CTC TCC TGC CAT GC - #A GCC TCT - # 1327 Pro Gly Val Asn Leu Ser Leu Ser Cys His Al - #a Ala Ser 395 4 - #00 4 - #05 - - AAC CCA CCT GCA CAG TAT TCT TGG CTG ATT GA - #T GGG AAC - # 1366 Asn Pro Pro Ala Gln Tyr Ser Trp Leu Ile As - #p Gly Asn 410 - # 415 - # 420 - - ATC CAG CAA CAC ACA CAA GAG CTC TTT ATC TC - #C ACC ATC - # 1405 Ile Gln Gln His Thr Gln Glu Leu Phe Ile Se - #r Asn Ile 425 - # 430 - - ACT GAG AAG AAC AGC GGA CTC TAT ACC TGC CA - #G GCC AAT - # 1444 Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gl - #n Ala Asn 435 - # 440 - # 445 - - AAC TCA GCC AGT GGC CAC AGC AGG ACT ACA GT - #C AAG ACA - # 1483 Asn Ser Ala Ser Gly His Ser Arg Thr Thr Va - #l Lys Thr 450 - # 455 - - ATC ACA GTC TCT GCG GAC GTG CCC AAG CCC TC - #C ATC TCC - # 1522 Ile Thr Val Ser Ala Asp Val Pro Lys Pro Se - #r Ile Ser 460 4 - #65 4 - #70 - - AGC AAC AAC TCC AAA CCC GTG GAG GAC AAG GA - #T GCT GTG - # 1561 Ser Asn Asn Ser Lys Pro Val Glu Asp Lys As - #p Ala Val 475 - # 480 - # 485 - - GCC TTC ACC TGT GAA CCT GAG GCT CAG AAC AC - #A ACC TAC - # 1600 Ala Phe Thr Cys Glu Pro Glu Ala Gln Asn Th - #r Thr Tyr 490 - # 495 - - CTG TGG TGG GTA AAT GGT CAG AGC CTC CCA GT - #C AGT CCC - # 1639 Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Va - #l Ser Pro 500 - # 505 - # 510 - - AGG CTG CAG CTG TCC AAT GGC AAC AGG ACC CT - #C ACT CTA - # 1678 Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Le - #u Thr Leu 515 - # 520 - - TTC AAT GTC ACA AGA AAT GAC GCA AGA GCC TA - #T GTA TGT - # 1717 Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Ty - #r Val Cys 525 5 - #30 5 - #35 - - GGA ATC CAG AAC TCA GTG AGT GCA AAC CGC AG - #T GAC CCA - # 1756 Gly Ile Gln Asn Ser Val Ser Ala Asn Arg Se - #r Asp Pro 540 - # 545 - # 550 - - GTC ACC CTG GAT GTC CTC TAT GGG CCG GAC AC - #C CCC ATC - # 1795 Val Thr Leu Asp Val Leu Tyr Gly Pro Asp Th - #r Pro Ile 555 - # 560 - - ATT TCC CCC CCA GAC TCG TCT TAC CTT TCG GG - #A GCG AAC - # 1834 Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gl - #y Ala Asn 565 - # 570 - # 575 - - CTC AAC CTC TCC TGC CAC TCG GCC TCT AAC CC - #A TCC CCG - # 1873 Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pr - #o Ser Pro 580 - # 585 - - CAG TAT TCT TGG CGT ATC AAT GGG ATA CCG CA - #G CAA CAC - # 1912 Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gl - #n Gln His 590 5 - #95 6 - #00 - - ACA CAA GTT CTC TTT ATC GCC AAA ATC ACG CC - #A AAT AAT - # 1951 Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pr - #o Asn Asn 605 - # 610 - # 615 - - AAC GGG ACC TAT GCC TGT TTT GTC TCT AAC TT - #G GCT ACT - # 1990 Asn Gly Thr Tyr Ala Cys Phe Val Ser Asn Le - #u Ala Thr 620 - # 625 - - GGC CGC AAT AAT TCC ATA GTC AAG AGC ATC AC - #A GTC TCT - # 2029 Gly Arg Asn Asn Ser Ile Val Lys Ser Ile Th - #r Val Ser 630 - # 635 - # 640 - - GCA TCT GGA ACT TCT CCT GGT CTC TCA GCT GG - #G GCC ACT - # 2068 Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gl - #y Ala Thr 645 - # 650 - - GTC GGC ATC ATG ATT GGA GTG CTG GTT GGG GT - #T GCT CTG - # 2107 Val Gly Ile Met Ile Gly Val Leu Val Gly Va - #l Ala Leu 655 6 - #60 6 - #65 - - ATA TAGCAGCCCT GGTGTAGTTT CTTCATTTCA GGAAGACTGA - # 215 - #0 Ile - - CAGTTGTTTT GCTTCTTCCT TAAAGCATTT GCAACAGCTA - # - # 2190 - - CAGTCTAAAA TTGCTTCTTT ACCAAGGATA TTTACAGAAA - # - # 2230 - - ATACTCTGAC CAGAGATCGA GACCATCCTA GCCAACATCG - # - # 2270 - - TGAAACCCCA TCTCTACTAA AAATACAAAA ATGAGCTGGG - # - # 2310 - - CTTGGTGGCG CGCACCTGTA GTCCCAGTTA CTCGGGAGGC - # - # 2350 - - TGAGGCAGGA GAATCGCTTG AACCCGGGAG GTGGAGATTG - # - # 2390 - - CAGTGAGCCC AGATCGCACC ACTGCACTCC AGTCTGGCAA - # - # 2430 - - CAGAGCAAGA CTCCATCTCA AAAAGAAAAG AAAAGAAGAC - # - # 2470 - - TCTGACCTGT ACTCTTGAAT ACAAGTTTCT GATACCACTG - # - # 2510 - - CACTGTCTGA GAATTTCCAA AACTTTAATG AACTAACTGA - # - # 2550 - - CAGCTTCATG AAACTGTCCA CCAAGATCAA GCAGAGAAAA - # - # 2590 - - TAATTAATTT CATGGGGACT AAATGAACTA ATGAGGATAA - # - # 2630 - - TATTTTCATA ATTTTTTATT TGAAATTTTG CTGATTCTTT - # - # 2670 - - AAATGTCTTG TTTCCCAGAT TTCAGGAAAC TTTTTTTCTT - # - # 2710 - - TTAAGCTATC CACTCTTACA GCAATTTGAT AAAATATACT - # - # 2750 - - TTTGTGAACA AAAATTGAGA CATTTACATT TTATCCCTAT - # - # 2790 - - GTGGTCGCTC CAGACTTGGG AAACTATTCA TGAATATTTA - # - # 2830 - - TATTGTATG - # - #- # 2839 - - - - (2) INFORMATION FOR SEQ ID NO: 6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3461 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 6: - - CAGCCGTGCT CGAAGCGTTC CTGGAGCCCA AGCTCTCCTC - # - # 40 - - CACAGGTGAA GACAGGGCCA GCAGGAGACA CC ATG GGG - #- # 78 - # - #Met Gly - - CAC CTC TCA GCC CCA CTT CAC AGA GTC CGT GT - #A CCC TGG - # 117 His Leu Ser Ala Pro Leu His Arg Val Arg Va - #l Pro Trp 5 - # 10 - # 15 - - CAG GGG CTT CTG CTC ACA GCC TCA CTT CTA AC - #C TTC TGG - # 156 Gln Gly Leu Leu Leu Thr Ala Ser Leu Leu Th - #r Phe Trp 20 - # 25 - - AAC CCG CCC ACC ACT GCC CAG CTC ACT ACT GA - #A TCC ATG - # 195 Asn Pro Pro Thr Thr Ala Gln Leu Thr Thr Gl - #u Ser Met 30 - # 35 - # 40 - - CCA TTC AAT GTT GCA GAG GGG AAG GAG GTT CT - #T CTC CTT - # 234 Pro Phe Asn Val Ala Glu Gly Lys Glu Val Le - #u Leu Leu 45 - # 50 - - GTC CAC AAT CTG CCC CAG CAA CTT TTT GGC TA - #C AGC TGG - # 273 Val His Asn Leu Pro Gln Gln Leu Phe Gly Ty - #r Ser Trp 55 - # 60 - # 65 - - TAC AAA GGG GAA AGA GTG GAT GGC AAC CGT CA - #A ATT GTA - # 312 Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gl - #n Ile Val 70 - # 75 - # 80 - - GGA TAT GCA ATA GGA ACT CAA CAA GCT ACC CC - #A GGG CCC - # 351 Gly Tyr Ala Ile Gly Thr Gln Gln Ala Thr Pr - #o Gly Pro 85 - # 90 - - GCA AAC AGC GGT CGA GAG ACA ATA TAC CCC AA - #T GCA TCC - # 390 Ala Asn Ser Gly Arg Glu Thr Ile Tyr Pro As - #n Ala Ser 95 - # 100 - # 105 - - CTG CTG ATC CAG AAC GTC ACC CAG AAT GAC AC - #A GGA TTC - # 429 Leu Leu Ile Gln Asn Val Thr Gln Asn Asp Th - #r Gly Phe 110 - # 115 - - TAC ACC CTA CAA GTC ATA AAG TCA GAT CTT GT - #G AAT GAA - # 468 Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Va - #l Asn Glu 120 1 - #25 1 - #30 - - GAA GCA ACT GGA CAG TTC CAT GTA TAC CCG GA - #G CTG CCC - # 507 Glu Ala Thr Gly Gln Phe His Val Tyr Pro Gl - #u Leu Pro 135 - # 140 - # 145 - - AAG CCC TCC ATC TCC AGC AAC AAC TCC ACC CC - #T GTG GAG - # 546 Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pr - #o Val Glu 150 - # 155 - - GAC AAG GAT GCT GTG GCC TTC ACC TGT GAA CC - #T GAG ACT - # 585 Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pr - #o Glu Thr 160 - # 165 - # 170 - - CAG GAC ACA ACC TAC CTG TGG TGG ATA AAC AA - #T CAG AGC - # 624 Gln Asp Thr Thr Tyr Leu Trp Trp Ile Asn As - #n Gln Ser 175 - # 180 - - CTC CCG GTC AGT CCC AGG CTG CAG CTG TCC AA - #T GGC AAC - # 663 Leu Pro Val Ser Pro Arg Leu Gln Leu Ser As - #n Gly Asn 185 1 - #90 1 - #95 - - AGG ACC CTC ACT CTA CTC AGT GTC ACA AGG AA - #T GAC ACA - # 702 Arg Thr Leu Thr Leu Leu Ser Val Thr Arg As - #n Asp Thr 200 - # 205 - # 210 - - GGA CCC TAT GAG TGT GAA ATA CAG AAC CCA GT - #G AGT GCG - # 741 Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Va - #l Ser Ala 215 - # 220 - - AAC CGC AGT GAC CCA GTC ACC TTG AAT GTC AC - #C TAT GGC - # 780 Asn Arg Ser Asp Pro Val Thr Leu Asn Val Th - #r Tyr Gly 225 - # 230 - # 235 - - CCG GAC ACC CCC ACC ATT TCC CCT TCA GAC AC - #C TAT TAC - # 819 Pro Asp Thr Pro Thr Ile Ser Pro Ser Asp Th - #r Tyr Tyr 240 - # 245 - - CGT CCA GGG GCA AAC CTC AGC CTC TCC TGC TA - #T GCA GCC - # 858 Arg Pro Gly Ala Asn Leu Ser Leu Ser Cys Ty - #r Ala Ala 250 2 - #55 2 - #60 - - TCT AAC CCA CCT GCA CAG TAC TCC TGG CTT AT - #C AAT GGA - # 897 Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Il - #e Asn Gly 265 - # 270 - # 275 - - ACA TTC CAG CAA AGC ACA CAA GAG CTC TTT AT - #C CCT AAC - # 936 Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Il - #e Pro Asn 280 - # 285 - - ATC ACT GTG AAT AAT AGT GGA TCC TAT ACC TG - #C CAC GCC - # 975 Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cy - #s His Ala 290 - # 295 - # 300 - - AAT AAC TCA GTC ACT GGC TGC AAC AGG ACC AC - #A GTC AAG - # 1014 Asn Asn Ser Val Thr Gly Cys Asn Arg Thr Th - #r Val Lys 305 - # 310 - - ACG ATC ATA GTC ACT GAG CTA AGT CCA GTA GT - #A GCA AAG - # 1053 Thr Ile Ile Val Thr Glu Leu Ser Pro Val Va - #l Ala Lys 315 3 - #20 3 - #25 - - CCC CAA ATC AAA GCC AGC AAG ACC ACA GTC AC - #A GGA GAT - # 1092 Pro Gln Ile Lys Ala Ser Lys Thr Thr Val Th - #r Gly Asp 330 - # 335 - # 340 - - AAG GAC TCT GTG AAC CTG ACC TGC TCC ACA AA - #T GAC ACT - # 1131 Lys Asp Ser Val Asn Leu Thr Cys Ser Thr As - #n Asp Thr 345 - # 350 - - GGA ATC TCC ATC CGT TGG TTC TTC AAA AAC CA - #G AGT CTC - # 1170 Gly Ile Ser Ile Arg Trp Phe Phe Lys Asn Gl - #n Ser Leu 355 - # 360 - # 365 - - CCG TCC TCG GAG AGG ATG AAG CTG TCC CAG GG - #C AAC ACC - # 1209 Pro Ser Ser Glu Arg Met Lys Leu Ser Gln Gl - #y Asn Thr 370 - # 375 - - ACC CTC AGC ATA AAC CCT GTC AAG AGG GAG GA - #T GCT GGG - # 1248 Thr Leu Ser Ile Asn Pro Val Lys Arg Glu As - #p Ala Gly 380 3 - #85 3 - #90 - - ACG TAT TGG TGT GAG GTC TTC AAC CCA ATC AG - #T AAG AAC - # 1287 Thr Tyr Trp Cys Glu Val Phe Asn Pro Ile Se - #r Lys Asn 395 - # 400 - # 405 - - CAA AGC GAC CCC ATC ATG CTG AAC GTA AAC TA - #T AAT GCT - # 1326 Gln Ser Asp Pro Ile Met Leu Asn Val Asn Ty - #r Asn Ala 410 - # 415 - - CTA CCA CAA GAA AAT GGC CTC TCA CCT GGG GC - #C ATT GCT - # 1365 Leu Pro Gln Glu Asn Gly Leu Ser Pro Gly Al - #a Ile Ala 420 - # 425 - # 430 - - GGC ATT GTG ATT GGA GTA GTG GCC CTG GTT GC - #T CTG ATA - # 1404 Gly Ile Val Ile Gly Val Val Ala Leu Val Al - #a Leu Ile 435 - # 440 - - GCA GTA GCC CTG GCA TGT TTT CTG CAT TTC GG - #G AAG ACC - # 1443 Ala Val Ala Leu Ala Cys Phe Leu His Phe Gl - #y Lys Thr 445 4 - #50 4 - #55 - - GGC AGG GCA AGC GAC CAG CGT GAT CTC ACA GA - #G CAC AAA - # 1482 Gly Arg Ala Ser Asp Gln Arg Asp Leu Thr Gl - #u His Lys 460 - # 465 - # 470 - - CCC TCA GTC TCC AAC CAC ACT CAG GAC CAC TC - #C AAT GAC - # 1521 Pro Ser Val Ser Asn His Thr Gln Asp His Se - #r Asn Asp 475 - # 480 - - CCA CCT AAC AAG ATG AAT GAA GTT ACT TAT TC - #T ACC CTG - # 1560 Pro Pro Asn Lys Met Asn Glu Val Thr Tyr Se - #r Thr Leu 485 - # 490 - # 495 - - AAC TTT GAA GCC CAG CAA CCC ACA CAA CCA AC - #T TCA GCC - # 1599 Asn Phe Glu Ala Gln Gln Pro Thr Gln Pro Th - #r Ser Ala 500 - # 505 - - TCC CCA TCC CTA ACA GCC ACA GAA ATA ATT TA - #T TCA GAA - # 1638 Ser Pro Ser Leu Thr Ala Thr Glu Ile Ile Ty - #r Ser Glu 510 5 - #15 5 - #20 - - GTA AAA AAG CAG TAATGAAACC TGTCCTGCTC ACTGCAGTGC - # - #1680 Val Lys Lys Gln 525 - - TGATGTATTT CAAGTCTCTC ACCCTCATCA CTAGGAGATT - # - # 1720 - - CCTTTCCCCT GTAGGGTAGA GGGGTGGGGA CAGAAACAAC - # - # 1760 - - TTTCTCCTAC TCTTCCTTCC TAATAGGCAT CTCCAGGCTG - # - # 1800 - - CCTGGTCACT GCCCCTCTCT CAGTGTCAAT AGATGAAAGT - # - # 1840 - - ACATTGGGAG TCTGTAGGAA ACCCAACCTT CTTGTCATTG - # - # 1880 - - AAATTTGGCA AAGCTGACTT TGGGAAAGAG GGACCAGAAC - # - # 1920 - - TTCCCCTCCC TTCCCCTTTT CCCAACCTGG ACTTGTTTTA - # - # 1960 - - AACTTGCCTG TTCAGAGCAC TCATTCCTTC CCACCCCCAG - # - # 2000 - - TCCTGTCCTA TCACTCTAAT TCGGATTTGC CATAGCCTTG - # - # 2040 - - AGGTAATGTC CTTTTCCATT AAGTACATGT GCCAGGAAAC - # - # 2080 - - AGCGAGAGAG AGAAAGTAAA CGGCAGTAAT GCTTCTCCTA - # - # 2120 - - TTTCTCCAAA GCCTTGTGTG AACTAGCAAA GAGAAGAAAA - # - # 2160 - - TCAAATATAT AACCAATAGT GAAATGCCAC AGGTTTGTCC - # - # 2200 - - ACTGTCAGGG TTGTCTACCT GTAGGATCAG GGTCTAAGCA - # - # 2240 - - CCTTGGTGCT TAGCTAGAAT ACCACCTAAT CCTTCTGGCA - # - # 2280 - - AGCCTGTCTT CAGAGAACCC ACTAGAAGCA ACTAGGAAAA - # - # 2320 - - ATCACTTGCC AAAATCCAAG GCAATTCCTG ATGGAAAATG - # - # 2360 - - CAAAAGCACA TATATGTTTT AATATCTTTA TGGGCTCTGT - # - # 2400 - - TCAAGGCAGT GCTGAGAGGG AGGGGTTATA GCTTCAGGAG - # - # 2440 - - GGAACCAGCT TCTGATAAAC ACAATCTGCT AGGAACTTGG - # - # 2480 - - GAAAGGAAT CAGAGAGCTGC CCTTCAGCGA TTATTTAAAT - # - # 2520 - - TGTTAAAGAA TACACAATTT GGGGTATTGG GATTTTTCTC - # - # 2560 - - CTTTTCTCTG AGACATTCCA CCATTTTAAT TTTTGTAACT - # - # 2600 - - GCTTATTTAT GTGAAAAGGG TTATTTTTAC TTAGCTTAGC - # - # 2640 - - TATGTCAGCC AATCCGATTG CCTTAGGTGA AAGAAACCAC - # - # 2680 - - CGAAATCCCT CAGGTCCCTT GGTCAGGAGC CTCTCAAGAT - # - # 2720 - - TTTTTTTGTC AGAGGCTCCA AATAGAAAAT AAGAAAAGGT - # - # 2760 - - TTTCTTCATT CATGGCTAGA GCTAGATTTA ACTCAGTTTC - # - # 2800 - - TAGGCACCTC AGACCAATCA TCAACTACCA TTCTATTCCA - # - # 2840 - - TGTTTGCACC TGTGCATTTT CTGTTTGCCC CCATTCACTT - # - # 2880 - - TGTCAGGAAA CCTTGGCCTC TGCTAAGGTG TATTTGGTCC - # - # 2920 - - TTGAGAAGTG GGAGCACCCT ACAGGGACAC TATCACTCAT - # - # 2960 - - GCTGGTGGCA TTGTTTACAG CTAGAAAGCT GCACTGGTGC - # - # 3000 - - TAATGCCCCT TGGGAAATGG GGCTGTGAGG AGGAGGATTA - # - # 3040 - - TAACTTAGGC CTAGCCTCTT TTAACAGCCT CTGAAATTTA - # - # 3080 - - TCTTTTCTTC TATGGGGTCT ATAAATGTAT CTTATAATAA - # - # 3120 - - AAAGGAAGGA CAGGAGGAAG ACAGGCAAAT GTACTTCTCA - # - # 3160 - - CCCAGTCTTC TACACAGATG GAATCTCTTT GGGGCTAAGA - # - # 3200 - - GAAAGGTTTT ATTCTATATT GCTTACCTGA TCTCATGTTA - # - # 3240 - - GGCCTAAGAG GCTTTCTCCA GGAGGATTAG CTTGGAGTTC - # - # 3280 - - TCTATACTCA GGTACCTCTT TCAGGGTTTT CTAACCCTGA - # - # 3320 - - CACGGACTGT GCATACTTTC CCTCATCCAT GCTGTGCTGT - # - # 3360 - - GTTATTTAAT TTTTCCTGGC TAAGATCATG TCTGAATTAT - # - # 3400 - - GTATGAAAAT TATTCTATGT TTTTATAATA AAAATAATAT - # - # 3440 - - ATCAGACATC GAAAAAAAAA A - # - # 3461 - - - - (2) INFORMATION FOR SEQ ID NO: 7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1964 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 7: - - CCGGGGGACA CGCAGGGCCA ACAGTCACAG CAGCCCTGAC - # - # 40 - - CAGAGCATTC CTGGAGCTCA AGCTCTCTAC AAAGAGGTGG - # - # 80 - - ACAGAGAAGA CAGCAGAGAC C ATG GGA CCC CCC TCA GCC - # - # 119 - # Met Gly Pro Pro Ser Ala - # - # -30 - - CCT CCC TGC AGA TTG CAT GTC CCC TGG AAG GA - #G GTC CTG - # 158 Pro Pro Cys Arg Leu His Val Pro Trp Lys Gl - #u Val Leu -25 - # -20 - - CTC ACA GCC TCA CTT CTA ACC TTC TGG AAC CC - #A CCC ACC - # 197 Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pr - #o Pro Thr15 - - #10 - #-5 - - ACT GCC AAG CTC ACT ATT GAA TCC ACG CCA TT - #C AAT GTC - # 236 Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Ph - #e Asn Val 1 - # 5 - # 10 - - GCA GAG GGG AAG GAG GTT CTT CTA CTC GCC CA - #C AAC CTG - # 275 Ala Glu Gly Lys Glu Val Leu Leu Leu Ala Hi - #s Asn Leu 15 - # 20 - - CCC CAG AAT CGT ATT GGT TAC AGC TGG TAC AA - #A GGC GAA - # 314 Pro Gln Asn Arg Ile Gly Tyr Ser Trp Tyr Ly - #s Gly Glu 25 - # 30 - # 35 - - AGA GTG GAT GGC AAC AGT CTA ATT GTA GGA TA - #T GTA ATA - # 353 Arg Val Asp Gly Asn Ser Leu Ile Val Gly Ty - #r Val Ile 40 - # 45 - # 50 - - GGA ACT CAA CAA GCT ACC CCA GGG CCC GCA TA - #C AGT GGT - # 392 Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Ty - #r Ser Gly 55 - # 60 - - CGA GAG ACA ATA TAC CCC AAT GCA TCC CTG CT - #G ATC CAG - # 431 Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Le - #u Ile Gln 65 - # 70 - # 75 - - AAC GTC ACC CAG AAT GAC ACA GGA TTC TAC AC - #C CTA CAA - # 470 Asn Val Thr Gln Asn Asp Thr Gly Phe Tyr Th - #r Leu Gln 80 - # 85 - - GTC ATA AAG TCA GAT CTT GTG AAT GAA GAA GC - #A ACC GGA - # 509 Val Ile Lys Ser Asp Leu Val Asn Glu Glu Al - #a Thr Gly 90 - # 95 - #100 - - CAG TTC CAT GTA TAC CCG GAG CTG CCC AAG CC - #C TCC ATC - # 548 Gln Phe His Val Tyr Pro Glu Leu Pro Lys Pr - #o Ser Ile 105 - # 110 - # 115 - - TCC AGC AAC AAC TCC AAC CCC GTG GAG GAC AA - #G GAT GCT - # 587 Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Ly - #s Asp Ala 120 - # 125 - - GTG GCC TTC ACC TGT GAA CCT GAG GTT CAG AA - #C ACA ACC - # 626 Val Ala Phe Thr Cys Glu Pro Glu Val Gln As - #n Thr Thr 130 - # 135 - # 140 - - TAC CTG TGG TGG GTA AAT GGT CAG AGC CTC CC - #G GTC AGT - # 665 Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pr - #o Val Ser 145 - # 150 - - CCC AGG CTG CAG CTG TCC AAT GGC AAC AGG AC - #C CTC ACT - # 704 Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Th - #r Leu Thr 155 1 - #60 1 - #65 - - CTA CTC AGC GTC AAA AGG AAC GAT GCA GGA TC - #G TAT GAA - # 743 Leu Leu Ser Val Lys Arg Asn Asp Ala Gly Se - #r Tyr Glu 170 - # 175 - # 180 - - TGT GAA ATA CAG AAC CCA GCG AGT GCC AAC CG - #C AGT GAC - # 782 Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn Ar - #g Ser Asp 185 - # 190 - - CCA GTC ACC CTG AAT GTC CTC TAT GGC CCA GA - #T GGC CCC - # 821 Pro Val Thr Leu Asn Val Leu Tyr Gly Pro As - #p Gly Pro 195 - # 200 - # 205 - - ACC ATT TCC CCC TCA AAG GCC AAT TAC CGT CC - #A GGG GAA - # 860 Thr Ile Ser Pro Ser Lys Ala Asn Tyr Arg Pr - #o Gly Glu 210 - # 215 - - AAT CTG AAC CTC TCC TGC CAC GCA GCC TCT AA - #C CCA CCT - # 899 Asn Leu Asn Leu Ser Cys His Ala Ala Ser As - #n Pro Pro 220 2 - #25 2 - #30 - - GCA CAG TAC TCT TGG TTT ATC AAT GGG ACG TT - #C CAG CAA - # 938 Ala Gln Tyr Ser Trp Phe Ile Asn Gly Thr Ph - #e Gln Gln 235 - # 240 - # 245 - - TCC ACA CAA GAG CTC TTT ATC CCC AAC ATC AC - #T GTG AAT - # 977 Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Th - #r Val Asn 250 - # 255 - - AAT AGC GGA TCC TAT ATG TGC CAA GCC CAT AA - #C TCA GCC - # 1016 Asn Ser Gly Ser Tyr Met Cys Gln Ala His As - #n Ser Ala 260 - # 265 - # 270 - - ACT GGC CTC AAT AGG ACC ACA GTC ACG ATG AT - #C ACA GTC - # 1055 Thr Gly Leu Asn Arg Thr Thr Val Thr Met Il - #e Thr Val 275 - # 280 - - TCT GGA AGT GCT CCT GTC CTC TCA GCT GTG GC - #C ACC GTC - # 1094 Ser Gly Ser Ala Pro Val Leu Ser Ala Val Al - #a Thr Val 285 2 - #90 2 - #95 - - GGC ATC ACG ATT GGA GTG CTG GCC AGG GTG GC - #T CTG ATA - # 1133 Gly Ile Thr Ile Gly Val Leu Ala Arg Val Al - #a Leu Ile 300 - # 305 - # 310 - - TAGCAGCCCT GGTGTATTTT CGATATTTCA GGAAGACTGG - # - # 1173 - - CAGATTGGAC CAGACCCTGA ATTCTTCTAG CTCCTCCAAT - # - # 1213 - - CCCATTTTAT CCCATGGAAC CACTAAAAAC AAGGTCTGCT - # - # 1253 - - CTGCTCCTGA AGCCCTATAT GCTGGAGATG GACAACTCAA - # - # 1293 - - TGAAAATTTA AAGGAAAAAC CCTCAGCCCT GAGGTGTGTG - # - # 1333 - - CCACTCAGAG ACTTCACCTA ACTAGAGACA GGCAAACTGC - # - # 1373 - - AAACCANNCC TCTTTCGCTT GGCAGGATGA TGGTGTCATT - # - # 1413 - - AGTATTTCAC AAGAAGTAGC TTCAGAGGGT AACTTAACAG - # - # 1453 - - AGTATCAGAT CTATCTTGTC AATCCCAACG TTTTACATAA - # - # 1493 - - AATAAGCGAT CCTTTAGTGC ACCCAGTGAG TGACATTAGC - # - # 1533 - - AGCATCTTTA ACACAGCCGT GTGTTCAAGT GTACAGTGGT - # - # 1573 - - CCTTTTCAGA GTTGGNNNTA CTCCAACTGA AATGTTAAGG - # - # 1613 - - AAGAAGATAG ATCCAATTAA AAAAAATTAA AACCAATTTA - # - # 1653 - - AAAAAAAAAA AGAACACAGG AGATTCCAGT CTACTTGAGT - # - # 1693 - - TAGCATAATA CAGAAGTCCC CTCTACTTTA ACTTTTACAA - # - # 1733 - - AAAAGTAACC TGAACTAATC TGATGTTAAC CAATGTATTT - # - # 1773 - - ATTTGTCTGG TTCTGTTTCC TTGTTCCAAT TTGACAAAAC - # - # 1813 - - CCACTGTTCT TGTATTGTAT TGCCCAGGGG GAGCTATCAC - # - # 1853 - - TGTACTTGTA GAGTGGTGCT GCTTTAAGTT CATAAATCAC - # - # 1893 - - AAATAAAAGC CAATTAGCTC TATAACTAAA AAAAAAAAAA - # - # 1933 - - AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A - # - # 1964 - - - - (2) INFORMATION FOR SEQ ID NO: 8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2009 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 8: - - ACAGCACAGC TGACAGCCGT ACTCAGGAAG CTTCTGGATC - # - # 40 - - CTAGGCTTAT CTCCACAGAG GAGAACACAC AAGCAGCAGA - # - # 80 - - GACC ATG GGG CCC CTC TCA GCC CCT CCC TGC A - #CA -# 114 Met Gly Pro Leu Ser Ala Pro Pro - # Cys Thr - # 5 - # 10 - - CAC CTC ATC ACT TGG AAG GGG GTC CTG CTC AC - #A GCA TCA - # 153 His Leu Ile Thr Trp Lys Gly Val Leu Leu Th - #r Ala Ser 15 - # 20 - - CTT TTA AAC TTC TGG AAT CCG CCC ACA ACT GC - #C CAA GTC - # 192 Leu Leu Asn Phe Trp Asn Pro Pro Thr Thr Al - #a Gln Val 25 - # 30 - # 35 - - ACG ATT GAA GCC CAG CCA CCC AAA GTT TCT GA - #G GGG AAG - # 231 Thr Ile Glu Ala Gln Pro Pro Lys Val Ser Gl - #u Gly Lys 40 - # 45 - - GAT GTT CTT CTA CTT GTC CAC AAT TTG CCC CA - #G AAT CTT - # 270 Asp Val Leu Leu Leu Val His Asn Leu Pro Gl - #n Asn Leu 50 - # 55 - # 60 - - GCT GGC TAC ATT TGG TAC AAA GGG CAA ATG AC - #A TAC GTC - # 309 Ala Gly Tyr Ile Trp Tyr Lys Gly Gln Met Th - #r Tyr Val 65 - # 70 - # 75 - - TAC CAT TAC ATT ACA TCA TAT GTA GTA GAC GG - #T CAA AGA - # 348 Tyr His Tyr Ile Thr Ser Tyr Val Val Asp Gl - #y Gln Arg 80 - # 85 - - ATT ATA TAT GGG CCT GCA TAC AGT GGA AGA GA - #A AGA GTA - # 387 Ile Ile Tyr Gly Pro Ala Tyr Ser Gly Arg Gl - #u Arg Val 90 - # 95 - # 100 - - TAT TCC AAT GCA TCC CTG CTG ATC CAG AAT GT - #C ACG CAG - # 426 Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Va - #l Thr Gln 105 - # 110 - - GAG GAT GCA GGA TCC TAC ACC TTA CAC ATC AT - #A AAG CGA - # 465 Glu Asp Ala Gly Ser Tyr Thr Leu His Ile Il - #e Lys Arg 115 1 - #20 1 - #25 - - CGC GAT GGG ACT GGA GGA GTA ACT GGA CAT TT - #C ACC TTC - # 504 Arg Asp Gly Thr Gly Gly Val Thr Gly His Ph - #e Thr Phe 130 - # 135 - # 140 - - ACC TTA CAC CTG GAG ACT CCC AAG CCC TCC AT - #C TCC AGC - # 543 Thr Leu His Leu Glu Thr Pro Lys Pro Ser Il - #e Ser Ser 145 - # 150 - - AGC AAC TTA AAT CCC AGG GAG GCC ATG GAG GC - #T GTG ATC - # 582 Ser Asn Leu Asn Pro Arg Glu Ala Met Glu Al - #a Val Ile 155 - # 160 - # 165 - - TTA ACC TGT GAT CCT GCG ACT CCA GCC GCA AG - #C TAC CAG - # 621 Leu Thr Cys Asp Pro Ala Thr Pro Ala Ala Se - #r Tyr Gln 170 - # 175 - - TGG TGG ATG AAT GGT CAG AGC CTC CCT ATG AC - #T CAC AGG - # 660 Trp Trp Met Asn Gly Gln Ser Leu Pro Met Th - #r His Arg 180 1 - #85 1 - #90 - - TTG CAG CTG TCC AAA ACC AAC AGG ACC CTC TT - #T ATA TTT - # 699 Leu Gln Leu Ser Lys Thr Asn Arg Thr Leu Ph - #e Ile Phe 195 - # 200 - # 205 - - GGT GTC ACA AAG TAT ATT GCA GGA CCC TAT GA - #A TGT GAA - # 738 Gly Val Thr Lys Tyr Ile Ala Gly Pro Tyr Gl - #u Cys Glu 210 - # 215 - - ATA CGG AAC CCA GTG AGT GCC AGC CGC AGT GA - #C CCA GTC - # 777 Ile Arg Asn Pro Val Ser Ala Ser Arg Ser As - #p Pro Val 220 - # 225 - # 230 - - ACC CTG AAT CTC CTC CCA AAG CTG TCC AAG CC - #C TAC ATC - # 816 Thr Leu Asn Leu Leu Pro Lys Leu Ser Lys Pr - #o Tyr Ile 235 - # 240 - - ACA ATC AAC AAC TTA AAC CCC AGA GAG AAT AA - #G GAT GTC - # 855 Thr Ile Asn Asn Leu Asn Pro Arg Glu Asn Ly - #s Asp Val 245 2 - #50 2 - #55 - - TTA ACC TTC ACC TGT GAA CCT AAG AGT GAG AA - #C TAC ACC - # 894 Leu Thr Phe Thr Cys Glu Pro Lys Ser Glu As - #n Tyr Thr 260 - # 265 - # 270 - - TAC ATT TGG TGG CTA AAT GGT CAG AGC CTC CC - #T GTC AGT - # 933 Tyr Ile Trp Trp Leu Asn Gly Gln Ser Leu Pr - #o Val Ser 275 - # 280 - - CCC AGG GTA AAG CGA CCC ATT GAA AAC AGG AT - #C CTC ATT - # 972 Pro Arg Val Lys Arg Pro Ile Glu Asn Arg Il - #e Leu Ile 285 - # 290 - # 295 - - CTA CCC AAT GTC ACG AGA AAT GAA ACA GGA CC - #T TAT CAA - # 1011 Leu Pro Asn Val Thr Arg Asn Glu Thr Gly Pr - #o Tyr Gln 300 - # 305 - - TGT GAA ATA CGG GAC CGA TAT GGT GGC ATC CG - #C AGT GAC - # 1050 Cys Glu Ile Arg Asp Arg Tyr Gly Gly Ile Ar - #g Ser Asp 310 3 - #15 3 - #20 - - CCA GTC ACC CTG AAT GTC CTC TAT GGT CCA GA - #C CTC CCC - # 1089 Pro Val Thr Leu Asn Val Leu Tyr Gly Pro As - #p Leu Pro 325 - # 330 - # 335 - - AGC ATT TAC CCT TCA TTC ACC TAT TAC CGT TC - #A GGA GAA - # 1128 Ser Ile Tyr Pro Ser Phe Thr Tyr Tyr Arg Se - #r Gly Glu 340 - # 345 - - AAC CTC TAC TTT TCC TGC TTC GGT GAG TCT AA - #C CCA CGG - # 1167 Asn Leu Tyr Phe Ser Cys Phe Gly Glu Ser As - #n Pro Arg 350 - # 355 - # 360 - - GCA CAA TAT TCT TGG ACA ATT AAT GGG AAG TT - #T CAG CTA - # 1206 Ala Gln Tyr Ser Trp Thr Ile Asn Gly Lys Ph - #e Gln Leu 365 - # 370 - - TCA GGA CAA AAG CTC TCT ATC CCC CAA ATA AC - #T ACA AAG - # 1245 Ser Gly Gln Lys Leu Ser Ile Pro Gln Ile Th - #r Thr Lys 375 3 - #80 3 - #85 - - CAT AGT GGG CTC TAT GCT TGC TCT GTT CGT AA - #C TCA GCC - # 1284 His Ser Gly Leu Tyr Ala Cys Ser Val Arg As - #n Ser Ala 390 - # 395 - # 400 - - ACT GGC AAG GAA AGC TCC AAA TCC ATC ACA GT - #C AAA GTC - # 1323 Thr Gly Lys Glu Ser Ser Lys Ser Ile Thr Va - #l Lys Val 405 - # 410 - - TCT GAC TGG ATA TTA CCC TGAATTCTAC TAGTTCCTCC - #- # 1361 Ser Asp Trp Ile Leu Pro 415 - - AATTCCATTT TCTCCCATGG AATCACGAAG AGCAAGACCC - # - # 1401 - - ACTCTGTTCC AGAAGCCCTA TAATCTGGAG GTGGACAACT - # - # 1441 - - CGATGTAAAT TTCATGGGAA AACCCTTGTA CCTGACATGT - # - # 1481 - - GAGCCACTCA GAACTCACCA AAATGTTCGA CACCATAACA - # - # 1521 - - ACAGCTACTC AAACTGTAAA CCAGGATAAG AAGTTGATGA - # - # 1561 - - CTTCACACTG TGGACAGTTT TTCAAAGATG TCATAACAAG - # - # 1601 - - ACTCCCCATC ATGACAAGGC TCCACCCTCT ACTGTCTGCT - # - # 1641 - - CATGCCTGCC TCTTTCACTT GGCAGGATAA TGCAGTCATT - # - # 1681 - - AGAATTTCAC ATGTAGTAGC TTCTGAGGGT AACAACAGAG - # - # 1721 - - TGTCAGATAT GTCATCTCAA CCTCAAACTT TTACGTAACA - # - # 1761 - - TCTCAGGGAA ATGTGGCTCT CTCCATCTTG CATACAGGGC - # - # 1801 - - TCCCAATAGA AATGAACACA GAGATATTGC CTGTGTGTTT - # - # 1841 - - GCAGAGAAGA TGGTTTCTAT AAAGAGTAGG AAAGCTGAAA - # - # 1881 - - TTATAGTAGA GTCTCCTTTA AATGCACATT GTGTGGATGG - # - # 1921 - - CTCTCACCAT TTCCTAAGAG ATACAGTGTA AAAACGTGAC - # - # 1961 - - AGTAATACTG ATTCTAGCAG AATAAACATG TACCACATTT - # - # 2001 - - GCAAAAAA - # - # -# 2009 - - - - (2) INFORMATION FOR SEQ ID NO: 9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1591 nucleo - #tides (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 9: - - GGGTGGATCC TAGGCTCATC TCCATAGGGG AGAACACACA - # - # 40 - - TACAGCAGAG ACC ATG GGA CCC CTC TCA GCC CCT CC - #C TGC - # 80 Met Gly - #Pro Leu Ser Ala Pro Pro Cys - # 5 - - ACT CAG CAC ATC ACC TGG AAG GGG CTC CTG CT - #C ACA GCA - # 119 Thr Gln His Ile Thr Trp Lys Gly Leu Leu Le - #u Thr Ala 10 - # 15 - # 20 - - TCA CTT TTA AAC TTC TGG AAC CTG CCC ACC AC - #T GCC CAA - # 158 Ser Leu Leu Asn Phe Trp Asn Leu Pro Thr Th - #r Ala Gln 25 - # 30 - # 35 - - GTA ATA ATT GAA GCC CAG CCA CCC AAA GTT TC - #T GAG GGG - # 197 Val Ile Ile Glu Ala Gln Pro Pro Lys Val Se - #r Glu Gly 40 - # 45 - - AAG GAT GTT CTT CTA CTT GTC CAC AAT TTG CC - #C CAG AAT - # 236 Lys Asp Val Leu Leu Leu Val His Asn Leu Pr - #o Gln Asn 50 - # 55 - # 60 - - CTT ACT GGC TAC ATC TTG TAC AAA GGG CAA AT - #G ACG GAC - # 275 Leu Thr Gly Tyr Ile Trp Tyr Lys Gly Gln Me - #t Thr Asp 65 - # 70 - - CTC TAC CAT TAC ATT ACA TCA TAT GTA GTA GA - #C GGT CAA - # 314 Leu Tyr His Tyr Ile Thr Ser Tyr Val Val As - #p Gly Gln 75 - # 80 - # 85 - - ATT ATA TAT GGG CCT GCC TAC AGT GGA CGA GA - #A ACA GTA - # 353 Ile Ile Tyr Gly Pro Ala Tyr Ser Gly Arg Gl - #u Thr Val 90 - # 95 - # 100 - - TAT TCC AAT GCA TCC CTG CTG ATC CAG AAT GT - #C ACA CAG - # 392 Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Va - #l Thr Gln 105 - # 110 - - GAG GAT GCA GGA TCC TAC ACC TTA CAC ATC AT - #A AAG CGA - # 431 Glu Asp Ala Gly Ser Tyr Thr Leu His Ile Il - #e Lys Arg 115 - # 120 - # 125 - - GGC GAT GGG ACT GGA GGA GTA ACT GGA TAT TT - #C ACT GTC - # 470 Gly Asp Gly Thr Gly Gly Val Thr Gly Tyr Ph - #e Thr Val 130 - # 135 - - ACC TTA TAC TCG GAG ACT CCC AAG CGC TCC AT - #C TCC AGC - # 509 Thr Leu Tyr Ser Glu Thr Pro Lys Arg Ser Il - #e Ser Ser 140 1 - #45 1 - #50 - - AGC AAC TTA AAC CCC AGG GAG GTC ATG GAG GC - #T GTG CGC - # 548 Ser Asn Leu Asn Pro Arg Glu Val Met Glu Al - #a Val Arg 155 - # 160 - # 165 - - TTA ATC TGT GAT CCT GAG ACT CCG GAT GCA AG - #C TAC CTG - # 587 Leu Ile Cys Asp Pro Glu Thr Pro Asp Ala Se - #r Tyr Leu 170 - # 175 - - TGG TTG CTG AAT GGT CAG AAC CTC CCT ATG AC - #T CAC AGG - # 626 Trp Leu Leu Asn Gly Gln Asn Leu Pro Met Th - #r His Arg 180 - # 185 - # 190 - - TTG CAG CTG TCC AAA ACC AAC AGG ACC CTC TA - #T CTA TTT - # 665 Leu Gln Leu Ser Lys Thr Asn Arg Thr Leu Ty - #r Leu Phe 195 - # 200 - - GGT GTC ACA AAG TAT ATT GCA GGG CCC TAT GA - #A TGT GAA - # 704 Gly Val Thr Lys Tyr Ile Ala Gly Pro Tyr Gl - #u Cys Glu 205 2 - #10 2 - #15 - - ATA CGG AGG GGA GTG AGT GCC AGC CGC AGT GA - #C CCA GTC - # 743 Ile Arg Arg Gly Val Ser Ala Ser Arg Ser As - #p Pro Val 220 - # 225 - # 230 - - ACC CTG AAT CTC CTC CCG AAG CTG CCC ATG CC - #T TAC ATC - # 782 Thr Leu Asn Leu Leu Pro Lys Leu Pro Met Pr - #o Tyr Ile 235 - # 240 - - ACC ATC AAC AAC TTA AAC CCC AGG GAG AAG AA - #G GAT GTG - # 821 Thr Ile Asn Asn Leu Asn Pro Arg Glu Lys Ly - #s Asp Val 245 - # 250 - # 255 - - TTA GCC TTC ACC TGT GAA CCT AAG AGT CGG AA - #C TAC ACC - # 860 Leu Ala Phe Thr Cys Glu Pro Lys Ser Arg As - #n Tyr Thr 260 - # 265 - - TAC ATT TGG TGG CTA AAT GGT CAG AGC CTC CC - #G GTC AGT - # 899 Tyr Ile Trp Trp Leu Asn Gly Gln Ser Leu Pr - #o Val Ser 270 2 - #75 2 - #80 - - CCG AGG GTA AAG CGA CCC ATT GAA AAC AGG AT - #A CTC ATT - # 938 Pro Arg Val Lys Arg Pro Ile Glu Asn Arg Il - #e Leu Ile 285 - # 290 - # 295 - - CTA CCC AGT GTC ACG AGA AAT GAA ACA GGA CC - #C TAT CAA - # 977 Leu Pro Ser Val Thr Arg Asn Glu Thr Gly Pr - #o Tyr Gln 300 - # 305 - - TGT GAA ATA CGG GAC CGA TAT GGT GGC ATC CG - #C AGT AAC - # 1016 Cys Glu Ile Arg Asp Arg Tyr Gly Gly Ile Ar - #g Ser Asn 310 - # 315 - # 320 - - CCA GTC ACC CTG AAT GTC CTC TAT GGT CCA GA - #C CTC CCC - # 1055 Pro Val Thr Leu Asn Val Leu Tyr Gly Pro As - #p Leu Pro 325 - # 330 - - AGA ATT TAC CCT TAC TTC ACC TAT TAC CGT TC - #A GGA GAA - # 1094 Arg Ile Tyr Pro Tyr Phe Thr Tyr Tyr Arg Se - #r Gly Glu 335 3 - #40 3 - #45 - - AAC CTC GAC TTG TCC TGC TTT GCG GAC TCT AA - #C CCA CCG - # 1133 Asn Leu Asp Leu Ser Cys Phe Ala Asp Ser As - #n Pro Pro 350 - # 355 - # 360 - - GCA GAG TAT TTT TGG ACA ATT AAT GGG AAG TT - #T CAG CTA - # 1172 Ala Glu Tyr Phe Trp Thr Ile Asn Gly Lys Ph - #e Gln Leu 365 - # 370 - - TCA GGA CAA AAG CTC TTT ATC CCC CAA ATT AC - #T ACA AAT - # 1211 Ser Gly Gln Lys Leu Phe Ile Pro Gln Ile Th - #r Thr Asn 375 - # 380 - # 385 - - CAT AGC GGG CTC TAT GCT TGC TCT GTT CGT AA - #C TCA GCC - # 1250 His Ser Gly Leu Tyr Ala Cys Ser Val Arg As - #n Ser Ala 390 - # 395 - - ACT GGC AAG GAA ATC TCC AAA TCC ATG ATA GT - #C AAA GTC - # 1289 Thr Gly Lys Glu Ile Ser Lys Ser Met Ile Va - #l Lys Val 400 4 - #05 4 - #10 - - TCT GGT CCC TGC CAT GGA AAC CAG ACA GAG TC - #T CAT- # 1325 Ser Gly Pro Cys His Gly Asn Gln Thr Glu Se - #r His 415 - # 420 - - TAATGGCTGC CACAATAGAG ACACTGAGAA AAAGAACAGG - # - # 1365 - - TTGATACCTT CATGAAATTC AAGACAAAGA AGAAAAAGGC - # - # 1405 - - TCAATGTTAT TGGACTAAAT AATCAAAAGG ATAATGTTTT - # - # 1445 - - CATAATTTTT ATTGGAAAAT GTGCTGATTC TTGGAATGTT - # - # 1485 - - TTATTCTCCA GATTTATGAA CTTTTTTTCT TCAGCAATTG - # - # 1525 - - GTAAAGTATA CTTTTGTAAA CAAAAATTGA AACATTTGCT - # - # 1565 - - TTTGCTCTCT ATCTGAGTGC CCCCCC - # - # 1591 - - - - (2) INFORMATION FOR SEQ ID NO: 10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 nucleoti - #des (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 10: - - GCTCCTTGTA AG - # - # - # 12 - - - - (2) INFORMATION FOR SEQ ID NO: 11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 nucleoti - #des (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 11: - - GCT GGT TGT AAG - # - # - # 12 Ala Gly Cys Lys - - - - (2) INFORMATION FOR SEQ ID NO: 12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 nucleoti - #des (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 12: - - G CTG GTT GTA AG - # - # - # 12 Leu Val Val - - (2) INFORMATION FOR SEQ ID NO: 13: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 nucleoti - #des (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 13: - - GC TGG TTG TAAG - # - # - # 12 Trp Leu - - (2) INFORMATION FOR SEQ ID NO: 14: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 nucleoti - #des (B) TYPE: nucleic a - #cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 14: - - GGGCCCTTAA - # - #- # 10__________________________________________________________________________

Number	Name	Date
3663684	Orkin et al.	May 1972
3697638	Hansen	Oct 1972
3852415	Vandervoorde	Dec 1974
3867363	Hansen	Feb 1975
3927193	Hansen et al.	Dec 1975
3956258	Hansen	May 1976
4086217	Hansen	Apr 1978
4140753	Edington et al.	Feb 1979
4145336	Edington et al.	Mar 1979
4180499	Hansen	Dec 1979
4228236	Jakstys et al.	Oct 1980
4272504	Kim et al.	Jun 1981
4299815	Hansen et al.	Nov 1981
4349528	Koprowski et al.	Sep 1982
4467031	Gallati et al.	Aug 1984
4489167	Ochi et al.	Dec 1984
4578349	Schaffel	Mar 1986

Number	Date	Country
92223	Apr 1983	EPX
92225	Oct 1983	EPX
83303759	Jan 1984	EPX
102008	Mar 1984	EPX
97373	Apr 1984	EPX
113072	Jul 1984	EPX
8402983	Aug 1984	WOX

	Number	Date
Parent	027974	Mar 1993
Parent	760031	Sep 1992
Parent	274107	Nov 1988

	Number	Date
Parent	207678	Jun 1988
Parent	060031	Jun 1987
Parent	016683	Feb 1987
Parent	896361	Aug 1986

cDNAs coding for members of the carcinoembryonic antigen family

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US Referenced Citations (17)

Foreign Referenced Citations (7)

Non-Patent Literature Citations (34)

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Continuation in Parts (4)

Entry
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