WO 99/55878 PCT/US99/07396 NOVEL NUCLEIC ACIDS AND POLYPEPTIDES RELATED TO A FARNESYL-DIRECTED CYSTEINE CARBOXYMETHYLTRANSFERASE BACKGROUND OF THE INVENTION 5 Farnesvl-directed cvsteine carboxvmethyltransferases are involved in the processing of various proteins, such as proteins involved in signaling pathways. fungal mating factors, and Ras polvpeptides. An activity of these methyltransferases is to perform methylesterification on prenylated proteins. See., e.g., Ashby et al., Yeast, 9:907-913, 1993; Khosravi-Far et al., Cell Growth and Differentiation, 3:461 469. 1992. 10 DESCRIPTION OF THE INVENTION The present invention concerns carboxvmethvltransferases ("MTase"), especially mammalian farnesvl-directed cvsteine MTases. such as human STE 14. MTases catalyze the transfer of a methyl group from a methyl donor to a methyl acceptor. There are at least seven different categories of MTases, distinguished by the type of methyl acceptor upon which the enzyme acts and the nature of the 15 chemical bond which is formed. See, e.g., Kagan and Clarke, Arch. Biochem. Biophys. 310: 417-427, 1994. An aspect of the invention relates to nucleic acids, polvpeptides, and fragments thereof, coding for carboxvmethvltransferases. especially a mammalian farnesyl-directed cysteine carboxvmethyltransferase, such as human STE14 and murine MTase. The invention further relates to 20 methods of using such nucleic acids and polvpeptides in therapeutics, diagnostics, and research. For example, the nucleic acids and polypeptides can be utilized in methods to identify modulators of MTase activity and to obtain ligands which bind to MTase. BRIEF DESCRIPTION OF THE DRAWINGS Fig. I shows a nucleotide and amino acid coding sequence for a human farnesyl-directed 25 cvsteine carboxvmethyltransferase. Fig. 2 shows a comparison between methyltransferase sequences from different species. Fig. 3 shows a nucleotide and amino acid coding sequence for a mouse farnesyl-directed cvsteine carboxvmethyltransferase DETAILED DESCRIPTION OF THE INVENTION 30 Novel nucleic acid and poiypeptide sequences have been identified which code for mammalian famesvl-directed cysteine carboxvmethyltransferases. These enzymes are involved in a variety of biological processes, including pathways which are associated with the maturation of signaling molecules, such as ras, rho, rab. rac, gamma-subunits of GTP-binding protein, related G-proteins, nuclear laminins, and fungal mating pheromones. A farnesyl-directed cysteine WO 99/55878 PCT/US99/07396 carboxvmethyltransferase has at least one of the following activities: ability to bind to, or attach to, a methyl donor substrate, e.g., S-adenosyl-L-methionine ("AdoMet"); ability to catalyze the transfer of a methyl group to a methyl acceptor ("methyltransferase activity"), where the methyl acceptor is preferably a prenylated cysteine. such as a S-farnesyl-cysteine: methylesterification of a newly exposed 5 alpha-carboxyl group, where the alpha-carboxyl group is a prenylated cysteine, such as a S-farnesyl cvsteine; ability to bind to, or attach to, a prenylated cysteine; promote protein/protein interactions; a transformation-modulating activity activity; or, an immunogenic activity, e.g., the polypeptide or polvnucleotide is capable of eliciting an immune response specific for an MTase of the present invention. 10 The above-mentioned activities of an MTase can be measured according to available assays or as described in the examples below. See, e.g., Imai et al.. Mol. Cell. Bio., 17:1543-1551, 1997: Hrvcyna et al.. Methods Enzvmol.. 250:251-266, 1995. Substrate binding is generally considered the first step in enzyme catalysis because the substrate, acting as a ligand. must first attach to the enzyme surface to enable the enzyme to carry out 15 its catalytic reactions. This enzyme surface can be referred to as the active site of the enzyme. Binding of the substrate to the enzyme surface can involve multiple interactions with the enzyme, e.g., chemical bonding with one or more amino acids and/or functional groups which comprise the enzyme. A methyl donor substrate binding activity as used herein means that a methyl donor substrate attaches to an MTase of the present invention. Attachment to the enzyme can be accomplished by one or more of the 20 interactions which hold its naturally-occurring substrate to it; however, a polypeptide can have a methyl donor substrate binding activity when it holds the substrate with less than the naturally-occurring number and quality of interactions. Methyl-donor substrate binding and catalytic activity can be dissociated from each other. Thus, an MTase polypeptide in accordance with the invention can possess substrate binding activity but not a 25 catalytic activity. A methyl donor substrate binding activity can optionally be effective: to achieve catalysis of the substrate, to competitively or noncompetitively bind to the active site, to irreversibly attach to the enzyme, to result in the loss of catalytic activity (e.g., where it is a suicide substrate), etc. Methyl donor substrate binding activity (e.g., binding of S-adenosyl-L-methionine) can be measured conventionally. For instance, a competition binding assay can be employed to identify 30 substrates which attach to a polypeptide. or derivative thereof, e.g., by combining under effective conditions, a substrate containing a detectable marker, an STE 14 polypeptide, or fragments thereof, and a compound which is to be tested for substrate binding activity. The assay can be accomplished in liquid phase. where bound and free substrate are separated by a membrane, or, it can be accomplished 2 WO 99/55878 PCTIUS99/07396 in solid phase. as desired. Solid-phase assays can be performed using high-throughput procedures, e.g., on chips, wafers, etc. A polvpeptide according to the present invention can also possess a catalytic activity, e.g., transfer of a methyl group to a methyl acceptor substrate. Generally. the catalysis results in 5 methylesterification of a newly exposed alpha-carboxyl group of an amino acid, especially a prenylated cvsteine. Thus, a catalytic activity in accordance with the present invention is a carboxylmethylation activity, especially of prenylated polvpeptides. This activity is described, e.g., in Hrvcyna et al., Methods Enzvmol., 250:251-266, 1995: Imai et al., Mol. Cell. Bio., 17:1543-1551, 1997. This activity can be measured in vitro or in vivo. 10 A polypeptide. or a nucleotide coding sequence thereof, can also possess a "transformation modulating activity". This can be an activity that modulates a transformed phenotype of cells, e.g., induces cell division, induces anchorage independent growth. increases ras activity. etc. The effect can be partial or incomplete. For example, expression of a STE 14 coding sequence in cells can cause a transformed phenotype, or it can enhance the phenotype of already transformed cells. A transformation 15 promoting activity can be enhanced by the presence of defects in other genes which contribute to transformation, such as ras, p53, Rb, cell-cycle regulatory genes, etc. A mammalian farnesvl-directed cysteine carboxvmethyltransferase (e.g., human STE14) is a mammalian polypeptide. or fragment thereof. having an amino acid sequence which is obtainable from a natural source. It therefore includes naturally-occurring, normal, mutant, polymorphic, etc., amino 20 acid sequences. Natural sources include, e.g., living cells, e.g., obtained from tissues or whole organisms. cultured cell lines, including primary and immortalized cell lines, biopsied tissues, etc. The present invention also relates to fragments of a full-length mammalian MTase, such as human STE 14 or murine MTase. The fragments are preferably biologically-active. By biologically-active, it is meant that the polypeptide fragment possesses an activity in a living system or with components of a living 25 system. Biological-activities include those mentioned, e.g., a methyltransferase activity, a methyl donor substrate binding activity, a transformation-modulating activity, and/or an immunogenic activity. Fragments can be prepared according to any desired method, including, chemical synthesis, genetic engineering, cleavage products, etc. See, below. The present invention also relates to a human farnesyl-directed cysteine 30 carboxvmethvltransferase having an amino acid sequence of amino acids 1 to 284. See, Fig. 1. The 284 amino acid polypeptide has a predicted molecular weight of 31.9 kilodaltons. In addition to the human STE 14 sequence, an MTase from another mammalian species. mouse, has been cloned and identified. A sequence of this is identified in Fig. 2 and 3. A full-length nucleic acid containing, e.g., a complete coding sequence for a mouse MTase. its promoter. and/or enhancer 3 WO 99/55878 PCT/US99/07396 region, etc., can be routinely identified and obtained, e.g., by using the above-mentioned fragments as probes for a cDNA or genomic library, by PCR, etc. Other homologs from mammalian and non-mammalian can be obtained according to various methods. For example, hybridization with an oligonucleotide (see below) selective for a mammalian 5 famesyl-directed cysteine carboxvmethyltransferases can be employed to select such homologs, e.g., as described in Sambrook et al., Molecular Cloning, 1989, Chapter 11. Such homologs can have varying amounts of nucleotide and amino acid sequence identity and similarity to farnsyl-directed carboxvmethyltransferase. Non-mammalian organisms include, e.g., vertebrates, invertebrates, zebra fish, chicken. Drosophila, C. elegans, roundworms, prokarvotes, plants, Arabidopsis, viruses, etc. 10 The invention also relates to famesyl-directed cvsteine carboxvmethyl-transferase specific amino acid sequences, e.g., a defined amino acid sequence which is found in the particular human or mouse sequences of Figs. I and 3 but not in another amino acid sequence, preferably not in Xenopus Xmam4, S. pombe mam4, S. cerevisiae STE 14, or mouse MTase. See. Imai et al.. Mol. Cell.. Bio., 17:1543-1551, 1997; Sapperstein et al.. Mol. Cell. Bio.. 14:1438-1449, 1994. 15 A specific amino acid sequence can be found routinely, e.g., by searching a gene/protein database using the BLAST set of computer programs. Mammalian specific sequences can be selected from about the first 65 amino acids of the MTase, e.g., CAARAPP, etc. A human specific amino acid sequence is. for instance, ICGVSYALTV. A famesyl-directed cysteine carboxvmethyltransferases specific amino acid sequence can be useful to produce peptides as antigens to generate an immune 20 response specific for it. Antibodies obtained by such immunization can be used as a specific probe for a mammalian famesyl-directed cysteine carboxvmethvltransferases protein for diagnostic or research purposes. A polypeptide of the invention, e.g., having a polypeptide sequence as shown in Fig. I or Fig. 3, can by analyzed by available methods to identify structural and/or functional domains in the 25 polvpeptide. For example, when the polvpeptide coding sequence set forth in Fig. 1 is analyzed by hydropathy and hydrophilicity analysis (e.g., Kyte and Doolittle, J. Mol. Bio., 157:105, 1982) putative membrane spanning regions are identified at: L16 to T34: L44 to Y59; 168 to F85; 1156 to L173, and V225 to W241. A putative catalytic region is VI 10 to L284. Various other programs can be used to analyze its structure and routinely predict functional domains, including, EMBL Protein Predict; Rost 30 and Sander, Proteins, 19:55-72, 1994. As mentioned polypeptides of the present invention can comprise a complete coding sequence for a mammalian famesyl-directed cysteine carboxymethyltransfer-ase, or fragments thereof. For example, an N-terminal region of a mammalian famesyl-directed cysteine carboxymethyltransferase can modulate its enzymatic activity, e.g., by enhancing its activity or stabilizing it. Thus, useful fragments 4 WO 99/55878 PCT/US99/07396 include, about amino acids 1-65 of the murine or human sequences in Fig. I and Fig. 2. These fragments can be used to modulate, stabilize, or enhance activity of other MTases, or other polypeptides by joining them in reading-frame with the polypeptide of interest. One or more fragments can be used, e.g., a human or murine STE 14 can comprise two or more N-terminal regions which possess a 5 modulatory activity. A fragment of a farnesyl-directed cysteine carboxvmethyltransferases polypeptide can be selected to have a specific biological activity, e.g., a methyl donor binding activity, a methylesterification activity, a methyltransferase activity, a transformation-modulatory activity, an immunogenic activity, etc. A useful fragment can be identified routinely by testing such fragments for a desired activity. The measurement of these activities is described below and in the examples. These 10 peptides can also be identified and prepared as described in EP 496 162. A polypeptide of the present invention can also have 100% or less amino acid sequence identity to the amino acid sequence set forth in Fig. 1 or 3. For the purposes of the following discussion: Sequence identity means that the same nucleotide or amino acid which is found in the sequence set forth in Fig 1 or Fig. 3 is found at the corresponding position of the compared sequence(s), e.g., Fig. 2. A 15 polypeptide having less than 100% sequence identify to the amino acid sequence set forth in Fig. 1 or 3 can contain various substitutions from the naturally-occurring sequence, including homologous amino acid substitutions. See below for examples of homologous amino acid substitution. The sum of the identical and homologous residues divided by the total number of residues in the sequence over which the farnesvl-directed cysteine carboxvmethvltransferases polvpeptide is compared is equal to the percent 20 sequence similarity. For purposes of calculating sequence identity and similarity, the compared sequences can be aligned and calculated according to any desired method, algorithm, computer program, etc., including, e.g., FASTA, BLASTA. A polypeptide having less than 100% amino acid sequence identity to the amino acid sequence of Fig. 1 can comprise e.g., about 99%, 97%, 95%, preferably about greater than 71% homology, such as 75% or more, with the proviso that the sequence is not 25 Xenopus Xmam4, S. pombe mam4, S. cerevisiae STE14, or mouse MTase. See, Imai et al., Mol. Cell., Bio., 17:1543-1551, 1997; Sapperstein et al., Mol. Cell. Bio., 14:1438-1449. The fragment of Fig 3 can also be excluded. A mammalian farnesyl-directed cysteine carboxymethyltransferases polypeptide, fragment, or substituted polypeptide can also comprise various modifications, where such modifications include lipid 30 modification. methylation, phosphorylation. glycosylation. covalent modifications (e.g., of an R-group of an amino acid), amino acid substitution, amino acid deletion, or amino acid addition. Modifications to the polypeptide can be accomplished according to various methods, including recombinant, synthetic, chemical, etc. 5 WO 99/55878 PCT/US99/07396 Polvpeptides of the present invention (e.g., human STE 14 or mouse MTase, fragments thereof, mutations thereof) can be used in various ways, e.g., in assays, as immunogens for antibodies as described below, as biologically-active agents (e.g., having one or more of the activities associated with STE14). 5 A polypeptide coding for a farnesyl-directed cysteine carboxvmethvltransferase, a derivative thereof, or a fragment thereof, can be combined with one or more structural domains, functional domains, detectable domains, antigenic domains, and/or a desired polypeptides of interest, in an arrangement which does not occur in nature, i.e., not naturally-occurring, e.g., as in a human or murine STE 14 gene. a genomic fragment prepared from the genome of a living organism, e.g., an animal. 10 preferably a mammal, such as human, mouse, or cell lines thereof. A polypeptide comprising such features is a chimeric or fusion polypeptide. Such a chimeric polypeptide can be prepared according to various methods, including, chemical. synthetic. quasi-synthetic, and/or recombinant methods. A chimeric nucleic acid coding for a chimeric polypeptide can contain the various domains or desired polypeptides in a continuous (e.g., with multiple N-terminal domains to stabilize or enhance activity) or 15 interrupted open reading frame, e.g., containing introns, splice sites, enhancers, etc. The chimeric nucleic acid can be produced according to various methods. See, e.g., U.S. Pat. No. 5,439,819. A domain or desired polypeptide can possess any desired property, including, a biological function such as catalytic, signalling, growth promoting, cellular targeting (e.g., signal sequence, targeting sequence, such as to endosomes, lysosomes. ER, nuncleus), etc., a structural function such as hydrophobic. hydro 20 philic, membrane-spanning, etc., receptor-ligand functions, and/or detectable functions, e.g., combined with enzyme. fluorescent polypeptide. green fluorescent protein, (Chalfie et al., 1994, Science, 263:802; Cheng et al.. 1996. Nature Biotechnology, 14:606: Levy et al., 1996, Nature Biotechnology, 14:610, etc. In addition, a polypeptide, or a part of it, can be used as selectable marker when introduced into a host cell. For example, a nucleic acid coding for an amino acid sequence according to the present 25 invention can be fused in frame to a desired coding sequence and act as a tag for purification, selection, or marking purposes. The region of fusion can encode a cleavage site to facilitate expression, isolation, purification, etc. A polypeptide according to the present invention can be produced in an expression system, e.g., in vivo, in vitro, cell-free, recombinant, cell fusion, etc., according to the present invention. 30 Modifications to the polypeptide imparted by such system include, glycosylation, amino acid substitution (e.g., by differing codon usage), polypeptide processing such as digestion, cleavage, endopeptidase or exopeptidase activity, attachment of chemical moieties, including lipids and phosphates, etc. 6 WO 99/55878 PCT/US99/07396 A polypeptide according to the present invention can be recovered from natural sources, transformed host cells (culture medium or cells) according to the usual methods, including, detergent extraction (e.g., CHAPS, octylglucoside), ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction 5 chromatography. hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. A mammalian farnesyl-directed cysteine carboxvmethyltransferase nucleic acid, or fragment thereof, is a nucleic acid having a nucleotide sequence obtainable from a natural source. or comprising a 10 coding sequence coding for a mammalian famesyl-directed cysteine carboxymethyltransferase. See, above. It therefore includes naturally-occurring, normal, mutant, polymorphic, degenerate sequences, etc., alleles. Natural sources include, e.g., living cells obtained from tissues and whole organisms, cultured cell lines, including primary and immortalized cell lines. Expression of human STE 14 is relatively ubiquitous, e.g., it is expressed in e.g., heart, brain, placenta. lung, liver, skeletal muscle, 15 kidney, pancrease, spleen, thvmus, prostate, testis, over, small intestine, colon, and peripheral blood leucocvtes. It is also expressed in various cancer cells, including, HL-60, Hela cell S3. chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW 480, lung carcinoma A549. and melanoma G361. The approximate size of the transcripts are about 2 kb, 3.5 kb. and 5 kb. 20 A nucleic acid sequence of a human allele of a mammalian famesyl-directed cysteine carboxvmethyltransferase. STE 14, is shown in Fig. 1. It contains open-reading frame of 284 amino acids at nucleotide positions 90 to 944. It contains 5' untranslated sequences at 1 to 89 and 3' untranslated sequences at 945 to 2556. A nucleic sequence of the invention can contain the complete coding sequence from amino acid 1 to amino acid 284 (i.e., full-length, having a start codon and a 25 termination codon), degenerate sequences thereof, and fragments thereof. A nucleic acid according to the present invention can also comprise a nucleotide sequence which is 100% complementary, e.g., an anti-sense, to any nucleotide sequence mentioned above and below. The present invention also relates to mouse nucleotide sequence coding for all or part of a MTase, e.g., as shown in Fig. 3. As for the human allele, the invention relates to degenerate sequences 30 thereof. and anti-sense fragments thereof. A nucleic acid according to the present invention can be obtained from a variety of different sources. It can be obtained from DNA or RNA, such as polyadenylated mRNA, e.g., isolated from tissues, cells, or whole organism. The nucleic acid can be obtained directly from DNA or RNA, or from 7 WO 99/55878 PCT/US99/07396 a cDNA library. The nucleic acid can be obtained from a cell at a particular stage of development. having a desired genotype, phenotype (e.g., an oncogenicallv transformed cell or a cancerous cell), etc. As for the polypeptides mentioned above, a nucleic acid comprising a nucleotide sequence coding for a polypeptide according to the present invention can include only coding sequence: a coding 5 sequence and additional coding sequence (e.g., sequences coding for leader, secretary. targeting, enzymatic, fluorescent or other diagnostic peptides), coding sequences and non-coding sequences, e.g., untranslated sequences at either a 5' or 3' end, or dispersed in the coding sequence, e.g., introns. A nucleic acid comprising a nucleotide sequence coding without interruption for a polypeptide means that the nucleotide sequence contains an amino acid coding sequence for a farnesyl 10 directed cysteine carboxvmethyltransferase. with no non-coding nucleotides interrupting or intervening in the coding sequence. e.g., absent intron(s). Such a nucleotide sequence can also be described as contiguous. A genomic DNA coding for a human or mouse MTase, etc., can be obtained routinely. A nucleic acid according to the present invention also can comprise an expression control sequence operably linked to a nucleic acid as described above. The phrase "expression control 15 sequence" means a nucleic acid sequence which regulates expression of a polypeptide coded for by a nucleic acid to which it is operably linked. Expression can be regulated at the level of the mRNA or polypeptide. Thus, the expression control sequence includes mRNA-related elements and protein related elements. Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators. etc. An expression control sequence is operably linked to a 20 nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence. For example, when a promoter is operably linked 5' to a coding sequence, expression of the coding sequence is driven by the promoter. Expression control sequences can be heterologous or endogenous to the normal gene. A nucleic acid in accordance with the present invention can be selected on the basis of nucleic 25 acid hybridization. The ability of two single-stranded nucleic acid preparations to hybridize together is a measure of their nucleotide sequence complementarity, e.g., base-pairing between nucleotides, such as A-T, G-C, etc. The invention thus also relates to nucleic acids which hybridize to a nucleic acid comprising a nucleotide sequence as set forth in Fig. 1 or Fig. 3, preferably. Fig. 1. A nucleotide sequence hybridizing to the latter sequence will have a complementary nucleic acid strand. or act as a 30 template for one in the presence of a polvmerase (i.e., an appropriate nucleic acid synthesizing enzyme). The present invention includes both strands of nucleic acid, e.g., a sense strand and an anti-sense strand. Hybridization conditions can be chosen to select nucleic acids which have a desired amount of nucleotide complementarity with the nucleotide sequence set forth in Fig. 1. A nucleic acid capable of hybridizing to such sequence, preferably, possesses about 95%, more preferably, 97%, etc., 8 WO 99/55878 PCT/US99/07396 complementarity. between the sequences. The present invention particularly relates to DNA sequences which hybridize to the nucleotide sequence set forth in Fig. 1. or its complement, under stringent conditions. As used here, stringent conditions means, for example: 50% formamide, 6X SSC or 6X SSPE, and optionally, a blocking agent (s)s (e.g., Denhardt's reagent; BLOTTO, heparin. denatured, 5 fragmented salmon sperm DNA) at 42 C (or 68 C if the formamide is omitted). Washing and hybridization can be performed as described in Sambrook et al., Molecular Cloning, 1989, Chapter 9. Hybridization can also be based a calculation of melting temperature (Tm) of the hybrid formed between the probe and its target, as described in Sambrook et al. Such stringent conditions can select sequences which have, e.g., at least about 95%, preferably 97%. nucleotide complementarity between 10 the nucleic acids, with the proviso that such nucleic acid is not Xenopus Xmam4, S. pombe mam4, S. cerevisiae STE14. or mouse MTase. See, Imai et al., Mol. Cell.. Bio., 17:1543-155 1, 1997: Sapperstein et al., Mol. Cell. Bio., 14:1438-1449, 1994. According to the present invention, a nucleic acid or polypeptide can comprise one or more differences in the nucleotide or amino acid sequence set forth in Fig. 1 or Fig. 3. Changes or 15 modifications to the nucleotide and/or amino acid sequence can be accomplished by any method available, including directed or random mutagenesis. A nucleic acid coding for a human or mouse MTase according to the invention can comprise nucleotides which occur in a naturally-occurring MTase gene e.g., naturally-occurring polymorphisms, normal or mutant alleles (nucleotide or amino acid), mutations which are discovered in a natural 20 population of mammals, such as humans, monkeys, pigs, mice, rats, or rabbits. By the term naturally occurring, it is meant that the nucleic acid is obtainable from a natural source, e.g., animal tissue and cells, body fluids, tissue culture cells, forensic samples. Naturally-occurring mutations can include deletions (e.g., a truncated amino- or carboxy-terminus), substitutions, or additions of nucleotide sequence. These genes can be detected and isolated by nucleic acid hybridization according to methods 25 which one skilled in the art would know. It is recognized that, in analogy to other oncogenes. naturally occurring variants include deletions, substitutions, and additions which produce pathological conditions in the host cell and organism. A nucleotide sequence coding for a polypeptide of the invention can contain codons found in a naturally-occurring gene, transcript, or cDNA, for example, e.g., as set forth in Fig. 1, or it can contain 30 degenerate codons coding for the same amino acid sequences. Modifications to a sequence of the invention, e.g., mutations, can also be prepared based on homology searching from gene data banks, e.g., Genbank, EMBL. Sequence homology searching can be accomplished using various methods, including algorithms described in the BLAST family of computer programs, the Smith-Waterman algorithm, etc. For example, conserved amino acids can be 9 WO 99/55878 PCT/US99/07396 identified between various sequences. See, e.g., Fig. 2. A mutation(s) can then be introduced into a sequence by identifying and aligning amino acids conserved between the polypeptides and then modifying an amino acid in a conserved or non-conserved position. A nucleic acid and corresponding polypeptide of the present invention include sequences which 5 differ from the nucleotide sequence of Fig. 1 (or less preferably Fig. 3) but which are phenotypically silent. These sequence modifications include, e.g., nucleotide substitution which do not affect the amino acid sequence (e.g., different codons for the same amino acid or degenerate sequences), replacing naturally-occurring amino acids with homologous amino acids, e.g., (based on the size of the side chain and degree of polarization) small nonpolar: cysteine, proline, alanine, threonine: small polar: seine, 10 glycine, aspartate, asparagine; large polar: glutamate, glutamine, lysine, arginine: intermediate polarity: tyrosine, histidine, tryptophan; large nonpolar: phenylalanine, methionine, leucine, isoleucine, valine. Homologous acids can also be grouped as follows: uncharged polar R groups, glycine. serine. threonine, cysteine. tyrosine, asparagine, glutamine: acidic amino acids (negatively charged), aspartic acid and glutamic acid; basic amino acids (positively charged), lysine. arginine, histidine. Homologous 15 substitutions also include those described by Dayhoff in the Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8. 779-785 (1989). Muteins in accordance with the present invention include amino acid sequences where a residue in the human sequence is replaced by a residue from a corresponding domain in the Xenopus Xmam4, S. pombe mam4, S. cerevisiae STE 14. or mouse MTase.at a corresponding position. 20 A nucleic acid can comprise a nucleotide sequence coding for a polvpeptide having an amino acid sequence as set forth in Fig. 1 or Fig. 3, except where one or more positions are substituted by homologous amino acids; or a nucleotide sequence coding for a polypeptide having an amino acid sequence as set forth in Fig. 1 (less preferably Fig. 3). except having 1, 5, 10, 15, or 20 substitutions. e.g., wherein the substitutions are conservative amino acids. The invention also relates to polypeptides 25 coded for by such nucleic acids. In addition, it may be desired to change the codons in the sequence to optimize the sequence for expression in a desired host. A nucleic acid according to the present invention can comprise, e.g., DNA, RNA, synthetic nucleic acid, peptide nucleic acid, modified nucleotides, or mixtures. A DNA can be double- or single stranded. Nucleotides comprising a nucleic acid can be joined via various known linkages, e.g., ester, 30 sulfamate, sulfamide, phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc., depending on the desired purpose, e.g., resistance to nucleases, such as RNase H, improved in vivo stability, etc. See, e.g., U.S. Pat. Nos. 5,378,825. Various modifications can be made to the nucleic acids, such as attaching detectable markers (avidin, biotin, radioactive elements), moieties which improve hybridization, detection, or stability. The 10 WO 99/55878 PCT/US99/07396 nucleic acids can also be attached to solid supports. e.g., nitrocellulose, magnetic or paramagnetic microspheres (e.g., as described in USP 5,411,863; USP 5.543,289; for instance, comprising ferromagnetic, supermagnetic, paramagnetic, superparamagnetic, iron oxide and polysaccharide), nylon, agarose, diazotized cellulose, latex solid microspheres. polvacrylamides. etc., according to a desired 5 method. See, e.g., U.S. Pat. Nos. 5,470,967, 5,476,925, 5,478,893. Another aspect of the present invention relates to oligonucleotides and nucleic acid probes. Such oligonucleotides or nucleic acid probes can be used, e.g., to detect, quantitate, or isolate a human or mouse MTase, such as STE 14, nucleic acid in a test sample. Detection can be desirable for a variety of different purposes, including research, diagnostic, and forensic. For diagnostic purposes. it may be 10 desirable to identify the presence or quantity of a such a nucleic acid sequence in a sample, where the sample is obtained from tissue, cells, body fluids, etc. In a preferred method, the present invention relates to a method of detecting a nucleic acid comprising, contacting a target nucleic acid in a test sample with an oligonucleotide under conditions effective to achieve hybridization between the target and oligonucleotide; and detecting hybridization. An oligonucleotide in accordance with the invention 15 can also be used in synthetic nucleic acid amplification such as PCR (e.g., Saiki et al., 1988, Science, 241:53: U.S. Pat. No. 4,683,202; PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, New York, 1990) or differential display (See, e.g., Liang et al., Nucl. Acid. Res., 21:3269-3275, 1993; USP 5,599.672: W097/18454). Useful oligonucleotides include, e.g., 5'-CAGATAGCCATCCGAGCTTGT-3' (282-302 nucleotide position); 20 5-CTCCTGAATCACAGCCTGGAGTA-3' (462-484 nucleotide position); 5'-CCTGGAGTATACAGTAGCTGCT'-3' (476-497 nucleotide position). Such detection can be accomplished in combination with oligonucleotides for other genes. such as ras, p53, Rb, cell-cycle regulatatory genes, etc. For methods and probes, e.g., USP 5,591,582. Another aspect of the present invention is a nucleotide sequence which is unique to human 25 STE 14 or mouse MTase. By a unique sequence to famsyl-directed carboxvmethvltransferase. it is meant a defined order of nucleotides which occurs in human or mouse STE 14, e.g., in the nucleotide sequence of Fig. 1, but rarely or infrequently in other nucleic acids, especially not in an animal nucleic acid, preferably mammal, such as human, rat, mouse, etc. Both sense and antisense nucleotide sequences are included. A unique nucleic acid according to the present invention can be determined 30 routinely. A nucleic acid comprising such a unique sequence can be used as a hybridization probe to identify the presence of, e.g., human or mouse STE 14, in a sample comprising a mixture of nucleic acids, e.g., on a Northern blot. Hybridization can be performed under stringent conditions to select nucleic acids having at least 95% identity (i.e., complementarity) to the probe, but less stringent conditions can also be used. A unique famsyl-directed carboxymethyltransferase nucleotide sequence 11 WO 99/55878 PCT/US99/07396 can also be fused in-frame, at either its 5' or 3' end, to various nucleotide sequences as mentioned throughout the patent, including coding sequences for other parts of STE 14, enzymes, GFP, etc, expression control sequences, etc. See. e.g. Hrvcyna et al.. Methods Enzymol.. 250:251-266, 1995. Hybridization can be performed under different conditions, depending on the desired selectivity, 5 e.g., as described in Sambrook et al., Molecular Cloning, 1989. For example, to specifically detect human or mouse MTase, an oligonucleotide can be hybridized to a target nucleic acid under conditions in which the oligonucleotide only hybridizes to it, e.g., where the oligonucleotide is 100% complementary to the target. Different conditions can be used if it is desired to select target nucleic acids which have less than 100% nucleotide complementarity, at least about, e.g., 99%. 97%. 95%, 10 90%, 70%, 67%. with the proviso that the sequence is not Xenopus Xmam4, S. pombe mam4, S. cerevisiae STE14, See. Imai et al., Mol. Cell., Bio., 17:1543-1551, 1997; Sapperstein et al., Mol. Cell. Bio.. 14:1438-1449, 1994. Since a mutation in a human or mouse MTase of the present invention can cause or enhance diseases or pathological conditions, e.g., cancer, benign tumors, an oligonucleotide according to the present invention can be used diagnostically. For example, a patient having symptoms 15 of a cancer or other condition associated with the Ras signaling pathway (see below) can be diagnosed with the disease by using an oligonucleotide according to the present invention, in polymerase chain reaction followed by DNA sequencing to identify whether the sequence is normal, in combination with other oligonucleotides to oncogenes or genes in the ras signalling pathway, etc., e.g., GRB2. H-, K- and N-ras, c-Raf, MAP kinases. p42, p 44 . Ser/Thr kinases, Elk-i, c-myc, c-Jun, G-proteins, Ftase, PPSEP, 20 PPSMT. etc. In a preferred method, the present invention relates to a method of diagnosing a cancer comprising contacting a sample comprising a target nucleic acid with an oligonucleotide under conditions effective to permit hybridization between the target and oligonucleotide: detecting hybridization. wherein the oligonucleotide comprises a sequence of a human or mouse MTase, preferably a unique sequence of; and determining the nucleotide sequence of the target nucleic acid to 25 which the oligonucleotide is hybridized. The sequence can be determined according to various methods, including isolating the target nucleic acid, or a cDNA thereof, and determining its sequence according to a desired method. Oligonucleotides of the present invention can comprise any continuous nucleotide sequence of Fig. 1. These oligonucleotides (nucleic acid) according to the present invention can be of any desired 30 size, e.g., about 10-200 nucleotides, 12-100, preferably 12-50, 12-25, 14-16, at least about 15, at least about 20, etc. The oligonucleotides can have non-naturally-occurring nucleotides, e.g., inosine. The oligonucleotides have 100% identity or complementarity to a sequence of Fig. 1 or Fig. 3, or it can have mismatches or nucleotide substitutions, e.g., 1, 2, 3, 4, or 5 substitutions. In accordance with the present invention, the oligonucleotide can comprise a kit. where the kit includes a desired buffer (e.g., 12 WO 99/55878 PCT/US99/07396 phosphate, tris, etc.), detection compositions, etc. The oligonucleotide can be labeled or unlabeled, with radioactive or non-radioactive labels as known in the art. Anti-sense nucleic acid can also be prepared from a nucleic acid according to the present, preferably an anti-sense to a coding sequence of Fig. 1, less preferably Fig. 3. Antisense nucleic acid 5 can be used in various ways, such as to regulate or modulate expression of farnsyl-directed carboxymethyltransferase, e.g., inhibit it. to detect its expression, or for in situ hybridization. These oligonucleotides can be used analogously to USP 5,576,208 describing inhibition of ras. For the purposes of regulating or modulating expression of famsyl-directed carboxymethyltransferase, an anti sense oligonucleotide can be operably linked to an expression control sequence. 10 The nucleic acid according to the present invention can be labelled according to any desired method. The nucleic acid can be labeled using radioactive tracers such as 32 P, "S, '251, 3 H, or 4 C, to mention only the most commonly used tracers. The radioactive labelling can be carried out according to any method such as, for example, terminal labeling at the 3' or 5' end using a radiolabeled nucleotide, polynucleotide kinase (with or without dephosphorvlation with a phosphatase) or a ligase (depending on 15 the end to be labelled). A non-radioactive labeling can also be used, combining a nucleic acid of the present invention with residues having immunological properties (antigens. haptens), a specific affinity for certain reagents (ligands), properties enabling detectable enzyme reactions to be completed (enzymes or coenzymes. enzyme substrates, or other substances involved in an enzymatic reaction), or characteristic physical properties, such as fluorescence or the emission or absorption of light at a 20 desired wavelength, etc. A nucleic acid according to the present invention, including oligonucleotides, anti-sense nucleic acid, etc., can be used to detect expression of farnsyl-directed carboxymethyltransferase in whole organs, tissues, cells, etc., by various techniques, including Northern blot, PCR. in situ hybridization, etc. Such nucleic acids can be particularly useful to detect disturbed expression, e.g., cell-specific 25 and/or subcellular alterations. of farnsvl-directed carboxvmethyltransferase. The levels of famsvl directed carboxymethyltransferase can be determined alone or in combination with other genes products (e.g., Ras-H, -N, -K4A, -K4B, p53, Rb, RCE1, etc.). A nucleic acid according to the present invention can be expressed in a variety of different systems, in vitro and in vivo, according to the desired purpose. For example, a nucleic acid can be 30 inserted into an expression vector, introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for the nucleic acid. Effective conditions includes any culture conditions which are suitable for achieving production of the polypeptide by the host cell, including effective temperatures, pH, medias, additives to the media in which the host cell is cultured (e.g., additives which amplify or induce expression such as butvrate. or methotrexate if the coding 13 WO 99/55878 PCTIUS99/07396 nucleic acid is adjacent to a dhfr gene), cvclohexamide, cell densities, culture dishes, etc. A nucleic acid can be introduced into the cell by any effective method including, e.g., naked DNA, calcium phosphate precipitation, electroporation, injection, DEAE-Dextran mediated transfection, fusion with liposomes, associated with agents which enhance its uptake into cells. viral transfection. A cell into which a 5 nucleic acid of the present invention has been introduced is a transformed host cell. The nucleic acid can be extrachromosomal or integrated into a chromosome(s) of the host cell. It can be stable or transient. An expression vector is selected for its compatibility with the host cell. Host cells include, mammalian cells, e.g., COS-7, CHO, HeLa, LTK, NIH 3T3, HEK 293, yeast, insect cells, such as Sf9 (S. frugipeda) and Drosophila, bacteria, such as E. coli, Streptococcus. bacillus. yeast, fungal cells, 10 plants, embryonic stem cells (e.g., mammalian, such as mouse or human), cancer or tumor cells. Sf9 expression can be accomplished in analogy to Graziani et al., Oncogene, 7:229-235. When the human gene was expressed in Sf9 cells, its activity was highest in the membrane fraction. Expression control sequences are similarly selected for host compatibility and a desired purpose, e.g., high copy number, high amounts, induction, amplification, controlled expression. Other sequences 15 which can be employed include enhancers such as from SV40, CMV, inducible promoters, cell-type specific elements, or sequences which allow selective or specific cell expression. Promoters that can be used to drive its expression, include, e.g., the endogenous promoter, SV40 etc. Another gene of interest can be introduced into the same host for purposes of, e.g., modulating expression famsyl-directed carboxymethyltransferase. elucidating farnsyl-directed 20 carboxymethyltransferase function or that of the gene of interest. Genes of interest include other oncogenes, genes involved in the cell cycle, such as p53, Rb, etc. Such genes can be the normal gene, or a variation. e.g., a mutation, chimera, polymorphism. etc. A nucleic acid or polypeptide of the present invention can be used as a size marker in nucleic acid or protein electrophoresis, chromatography, etc. Defined restriction fragments can be determined 25 by scanning the sequence for restriction sites, calculating the size, and performing the corresponding restriction digest. The human farnsyl-directed carboxymethyltransferase cDNA can also be used as a 2.5 kb molecular weight marker on a gel. Another aspect of the present invention relates to the regulation of biological pathways in which an MTase gene is involved, particularly pathological conditions. For example: cell proliferation (e.g., 30 cancer), growth control, apoptosis, differentiation, morphogenesis, mating type, G-protein signalling, cell adhesion, etc. For example, a mammalian MTase of the present invention is involved in the processing of various proteins, e.g., polvpeptides which are lipid-modified (for instance, containing a steroid intermediate such as faransyl or geranylgemanyl, or other prenylated species) and/or which contain an N-terminal C. CC, CCXX, CXC, See, e.g., Cox and Der, Biochim. Biopkys. Acta 1333, 14 WO 99/55878 PCTIUS99/07396 F5 1 -F71, 1997. Of particular interest, is the role that a mammalian farnesyl-directed cysteine carboxvmethyltransferase plays in processing ras polypeptides since the latter is involved in carcinogenesis and transformation. Ras and other polypetides containing the above-mentioned motifs are processed through one or more steps involving, farnsylation. endoproteolysis, and methylation. See, 5 e.g., Gelb, Science, 275, 1751-1751, 1997. Over-expression of ras (wild-type, mutated, constitutive, etc., ras), optionally in combination with aberrant expression of other genes, leads to oncogenic activity. One approach to treating ras over-expression is inhibiting the ras maturation pathway so that incompletely processed and/or inactive ras accumulates, eliminating or reducing its oncogenic effect. In accordance with the present invention. the ras maturation pathway can be inhibited by blocking 10 mammalian famesyl-directed cvsteine carboxvmethvltransferase, such as STE 14, Blocking can be accomplished in various ways, including by administering: STE14 antibodies, or other STE14 ligands, STE 14 peptides (especially, those that bind to a methyl donor but lack methyltransferase or methyltransesterification activity), inhibitors of STE 14 catalytic activity (e.g., methyl-donor mimics or competitors or antagonists), inhibitors of STE 14 gene expression (e.g., anti-sense or double-stranded 15 RNA, such as, Fire et al., Nature, 391:806-811, 1998). Blocking agents can be identified according to the methods described herein or those available in the art. One aspect of the invention relates to identifying compounds which modulate farnesyl-directed cvsteine carboxvmethyltransferase activity. The activity can be modulated by increasing, reducing, antagonizing, promoting, stabilizing, etc. its activity. Thus, one object of the invention is to facilitate 20 screening for compounds which modulate the incorporation of a methyl group into a methyl-acceptor substrate of the methyltransferase enzyme. Accordingly, the present invention relates to identifying compounds that modulate a mammalian farnesvl directed cysteine carboxvmethyltransferase, especially a human or mouse MTase of Fig. 1 or Fig. 3. comprising: reacting, in the presence of a test compound. a methyl-donor substrate, a methyl 25 acceptor substrate, and a mammalian famesyl-directed cysteine carboxvmethyltransferase, or a fragment thereof having methyltransferase activity, under conditions effective for the mammalian carboxvmethvltransferase, or said fragment, to methylate said methyl-acceptor substrate; detecting the methylation of said methyl-acceptor substrate; and identifying whether the test compound modulates methyltransferase activity by comparing the amount of 30 methylation in the presence and absence of the test compound. Any functional methyl-donor substrate is acceptable, including detectably-labeled S-adenosyl methionine, especially S-adenosyl-L-[methyl- 14 C]methionine or S-adenosyl-L-[methyl- 3 H]methionine. Likewise, any functional methyl-acceptor is satisfactory, including polypeptides which comprise a terminal farnsylated cysteine, polypeptides which terminate in CC, CCXX, or CXC. and/or are 15 WO 99/55878 PCT/US99/07396 cysteine-prenylated with, for instance, a farnsyl or geranylgeranyl. Functional methyl-acceptor substrates include, e.g., Ras-H. -N, -K4A, -K4B, Lamins A and B. RhoB, RhoE. others, Rap2, Rheb, Phosphorvlase kinase and , Rhodopsin kinase, Transducin , cGMP phosphodiesterasae , IFN induced guanylate-binding protein 1, IP 3 5-phosphatase. PxF. PRL-1/PTPCAAX 1 and 2. biotin-Lys 5 Lys-Ser-Lys-Thr-Lys-(Famesvl)Cys, etc. Other substrates include, hepatitis delta virus (Otto et al., J. Biol. Chem., 271:4569-72, 1996). In general, any substrate is suitable if it can be acted upon in the methyltransferase reaction. Thus, a substrate can comprise other atoms, such as additional amino acid residues linked by peptide or other bonds. and can be modified in any desirable way. For example. a substrate can be affixed to a solid 10 suport, e.g., comprising, latex, sepharose, silica, agarose, sephadex, cellulose, polysaccharides, glass, polymers, etc. A substrate can also be detectably labeled, e.g., with antibody, avidin, biotin, radioactive labels, aptamers, fluorescent labels, nucleic acid, etc. The substrates can also comprise phosphates, methyl groups, sugars, or lipids. The test compound is preferably reacted with substrates in a milieu in which methylation of the 15 acceptor-substrate is accomplished. Such a milieu can be referred to as effective conditions. These conditions can be determined in the absence of the test compound to establish a baseline activity, e.g., as in a control. The effective reaction conditions can be routinely selected, e.g., using salts, buffers, reducing and/or oxidizing agents, pH's, etc. For instance, when utilizing a methyl-acceptor substrate acceptor comprising a farnsylated C-terminal cysteine. effective methylation results in its methylation. 20 After the step of reacting the substrates, test compound, and MTase, under conditions in which methylation can be achieved, the next step is to determine the effect of the test compound on methylation. Detecting methylation, can be optimized in the absence of the test compound to establish a baseline activity for MTase. Generally, detecting methylation involves measuring the incorporation of a labeled methyl group (e.g., 3 H or 14 C) into an acceptor-substrate of the MTase. In a preferred 25 embodiment, the methyl-acceptor substrate is biotin-Lys-Lys-Ser-Lys-Thr-Lvs-(Famesyl)Cys and the methyl-donor substrate is detectably-labeled S-adenosyl methionine (for instance. S-adenosyl-L [methyl- 14 C]methionine or S-adenosvl-L-[methyl- 3 H]methionine) and the methylation detecting is accomplished by capturing the methyl-acceptor substrate using an streptavidin-coated bead; and measuring the amount of labeled methyl incorporated into said methyl-acceptor substrate. However, the 30 capture can be accomplished using any available means, depending upon the anchor (such as biotin in the example) incorporated into the methyl-acceptor substrate, e.g., SPA beads coated with protein A can be bound to anti-Ras antibody which can be used to capture full-length protein from cells. If this cellular RAS is unmethylated, recombinant MTase can be used to incorporate label from 3 H-S-adenosyl methionine. 16 WO 99/55878 PCT/US99/07396 In general assays according to the invention involve capture of the methyl-substrate acceptor and distinguishing whether the substrate has been modified, i.e., by the addition of a methyl group. This can be accomplished by any effective means, including antibodies (for instance, an antibody that recognizes the methylated substrate but not the unmethylated, or vice-versa): mass spectroscopy; 5 electrophoretic mobility shifts; chromatography; electrophoresis; etc. The MTase component can be added to the reaction mixture in a variety forms, e.g., substantially purified, as a component of cell membranes (such as, endoplasmic reticulum), or as a soluble extract. In each case, the MTase polypeptide can be obtained from a natural source, a recombinant source (e.g., a human STE14 expressed in an insect cell line or bacteria as a fusion or non 10 fusion protein, for instance as described in Hrvcyna et al., Methods Enzymol., 250:251-266, 1995), or it can be produced synthetically (produced chemically or enzymatically, e.g., cleavage of a full-length MTase. Preferably, the MTase is expressed in a cell line transformed with an MTase coding sequence (e.g., a cDNA, a gene, a genomic fragment, etc.). In the latter case. the MTase is present as a 15 heterologous component of the cell; by heterologous, it is meant that the MTase is not only expressed in a cell line of a different species, but it is also coded for by a coding sequence that has been introduced into the cell, e.g., by transfection, transformation, etc. Preferably, the MTase is expressed at high levels in the cell (bacterial, yeast, mammalian, etc.). A human farnesyl-directed cysteine carboxvmethyltransferase. or a fragment thereof, is a preferred coding sequence. See, e.g., Fig. 1. A 20 useful fragment of the human sequence comprises a methvltransferase activity and methyl-donor substrate binding activity. In a preferred aspect of the invention, the MTase is provided as a cell lysate, e.g., cells transformed with human STE 14 are lysed and the resulting lysate is used directly in the assay, i.e., a crude lysate. The crude lysate comprising the recombinant human STE 14 can optionally be refined or 25 enriched for human STE 14. For instance, e.g, a membrane fraction can be isolated, etc., as described in Hrycyna et al., Methods Enzvmol.. 250:251-266, 1995 . A purpose of the assay is select and identify compounds which modulate MTase activity. Thus, methylation is typically performed in the presence and absence of the test compound. Whether a compound modulates RCE can be determined routinely, e.g., by determining whether more or less 30 methylation has occurred in the presence of the test compound. The assav can also be conducted in whole cells. For instance, a modulator of famesyl-directed cvsteine carboxvmethyltransferase activity can be added to a desired host cell line and then its effect on the cell line can be observed. As mentioned, the MTases methylate a variety of different proteins in cells. See, above. Thus, the effect of an inhibitor on a whole cell (or extract, etc.) can be measured by 17 WO 99/55878 PCT/US99/07396 identifying whether the substrate has been methylated. For example, in cell lines that express ras, a whole cell assay can comprise: administering a test compound to a cell; determining or detecting whether the test compound modulates the processing of ras, e.g., by using an antibody or electrophoretic shift to detect whether a ras intermediate has accumulated in the cell. Cell lines can be engineered to 5 express the methyltransferase substrate, overexpress it, etc. An in vivo method of assaying for farnesyl directed cysteine carboxymethyltransferase further involves modifying test compounds to gain entry into the cell, e.g., derivatizing compounds, encapsulating compounds in liposome, and other means to enhance delivery to the cell, e.g., to stimulate phagocytosis. Agents can also be administered to such cells and tested for their ability to inhibit transformation, e.g., by monitoring cell morphology, etc. See, 10 e.g., USP 5,688,655. Assays can also be carried out as described in USP 5,710,171; 5,703,241; 5,585,359; 5,557,729; 5,532,359; 5,470,832; 5,420, 245; 5,185,248. Cell lines useful for the in vitro (e.g., as a source of membranes) and in vivo assays can express one or more heterologous genes, including FTase, RCEl, Rb, rac, p53, Ras-H, -N, -K4A, -K4B, Lamins A and B, RhoB, RhoE. Rap2, Rheb, Phosphorylase kinase and , Rhodopsin kinase, 15 Transducin . cGMP phosphodiesterasae . IFN-induced guanylate-binding protein 1, IP 3 5-phosphatase, PxF, PRL-1/PTPCAAX1 and 2. Compounds identified in this or other manners can be useful to modulate famesyl-directed cysteine carboxvmethyltransferase activity in a cell, a tissue, a whole organism, in situ, in vitro (test tube, a solid support, etc.), in vivo, or in any desired environment. In general, a compound having such 20 an in vitro activity will be useful in vivo to modulate a biological pathway associated with farnesyl directed cysteine carboxymethyltransferase, e.g., to treat a pathological condition associated with the biological and cellular activities mentioned above. The present invention thus also relates to the treatment and prevention of diseases and pathological conditions associated with ras, G-protein, etc. mediated signal transduction, e.g., cancer, diseases associated with abnormal cell proliferation. For 25 example, the invention relates to a method of treating cancer comprising administering, to a subject in need of treatment, an amount of a compound effective to treat the disease, where the compound is a regulator of farnesyl-directed cysteine carboxymethyltransferase gene or polypeptide expression. Treating the disease can mean, delaying its onset, delaying the progression of the disease, improving or delaying clinical and pathological signs of disease. A regulator compound, or mixture of compounds, 30 can be synthetic, naturally-occuring, or a combination. A regulator compound can comprise amino acids, nucleotides, hydrocarbons, lipids, polysaccharides, etc. A regulator compound is preferably a regulator of farnesyl-directed cysteine carboxvmethyltransferase, e.g., inhibiting or increasing its mRNA, protein expression, or processing. Expression can be regulated using different agents, e.g., an anti-sense nucleic acid, a ribozyme, an aptamer, a synthetic compound, or a naturally-occurring 18 WO 99/55878 PCT/US99/07396 compound. To treat the disease, the compound, or mixture, can be formulated into pharmaceutical composition comprising a pharmaceutically acceptable carrier and other excipients as apparent to the skilled worker. See, e.g., Remington's Pharmaceutical Sciences, Eighteenth Edition, Mack Publishing Company, 1990. Such composition can additionally contain effective amounts of other compounds. 5 especially for treatment of cancer. The present invention also relates to antibodies which specifically recognize farnesyl-directed cvsteine carboxvmethyltransferase. Antibodies. e.g., polyclonal, monoclonal. recombinant, chimeric, can be prepared according to any desired method. For example, for the production of monoclonal antibodies, a polypeptide according to Fig. I (a specific fragment thereof) can be administered to mice, 10 goats, or rabbit subcutaneously and/or intraperitoneally, with or without adjuvant, in an amount effective to elicit an immune response. The antibodies can also be single chain or FAb. The antibodies can be IgG, subtypes, IgG2a, IgG1, etc. Antibodies can also be generated by administering naked DNA See, e.g., USP 5,703,055; 5,589,466; 5,580,859. An antibody specific for farnesyl-directed cysteine carboxymethyltransferase means that the 15 antibody recognizes a defined sequence of amino acids within or including a famesyl-directed cysteine carboxvmethyltransferase, e.g., the human and murine sequences of Fig. 1 and Fig. 3. Thus, a specific antibody will bind with higher affinity to an amino acid sequence, i.e., an epitope, found in Fig. 1 than to a different epitope(s), e.g., as detected and/or measured by an immunoblot assay. Thus, an antibody which is specific for an epitope of human STE 14 is useful to detect the presence of the epitope in a 20 sample, e.g., a sample of tissue containing human STE14 gene product, distinguishing it from samples in which the epitope is absent. Such antibodies are useful as described in Santa Cruz Biotechnology, Inc., Research Product Catalog, can be formulated accordingly, e.g., 100 pg/ml. In addition. ligands which bind to a farnesyl-directed cysteine carboxymethyltransferase polypeptide according to the present invention, or a derivative thereof, can also be prepared, e.g., using 25 synthetic peptide libraries or aptamers (e.g., Pitrung et al., U.S. Pat. No. 5,143.854: Gevsen et al., 1987, J. Immunol. Methods, 102:259-274; Scott et al., 1990, Science, 249:386; Blackwell et al., 1990, Science, 250:1104; Tuerk et al., 1990, Science, 249: 505. Antibodies and other ligands which bind famesyl-directed cysteine carboxvmethyltransferase can be used in various ways, including as therapeutic, diagnostic, and commercial research tools, e.g, to 30 quantitate the levels of farnesyl-directed cysteine carboxymethyltransferase polypeptide in animals, tissues, cells, etc., to identify the cellular localization and/or distribution of it. to purify it, or a polypeptide comprising a part of it, to modulate the function of it, etc. Antibodies to it, or a derivative thereof, can be used in Western blots, ELIZA, immunoprecipitation, RIA, etc. The present invention relates to such assays, compositions and kits for performing them, etc. 19 WO 99/55878 PCTIUS99/07396 An antibody according to the present invention can be used to detect famesyl-directed cysteine carboxvmethvltransferase polypeptide or fragments thereof in various samples, including tissue, cells, body fluid, blood, urine, cerebrospinal fluid. A method of the present invention comprises, e.g., (a) contacting a ligand which binds to a peptide of Fig. 1 under conditions effective, as known in the art. to 5 achieve binding, and (b) detecting specific binding between the ligand and peptide. By specific binding, it is meant that the ligand attaches to a defined sequence of amino acids, e.g., within or including the amino acid sequence of Fig. 1 or derivatives thereof. The antibodies or derivatives thereof can also be used to inhibit expression of famsyl-directed carboxymethyltransferase or a fragment thereof. The levels of famesyl-directed cysteine carboxymethvltransferase polvpeptide can be determined alone or in 10 combination with other gene products. In particular, the amount (e.g., its expression level) of famesyl directed cvsteine carboxvmethvltransferase polypeptide can be compared (e.g., as a ratio) to the amounts of other polypeptides in the same or different sample, e.g., ras. FTase, endoprotease. etc. A ligand for famesyl-directed cysteine carboxvmethyltransferase can be used in combination with other antibodies, e.g., antibodies that recognize oncological markers of cancer, including, ras, etc. In general, 15 reagents which are specific for famesyl-directed cysteine carboxvmethyltransferase can be used in diagnostic and/or forensic studies according to any desired method, e.g., as U.S. Pat. Nos. 5,397,712; 5,434,050; 5,429,947. The present invention also relates to a labeled famsyl-directed carboxymethyltransferase polypeptide. prepared according to a desired method, e.g., as disclosed in U.S. Pat. No. 5,434,050. A 20 labelled polvpeptide can be used, e.g., in binding assays, such as to identify substances that bind or attach to famsyl-directed carboxvmethyltransferase, to track the movement of famsyl-directed carboxvmethvltransferase in a cell, in an in vitro. in vivo. or in situ system. etc. A nucleic acid, polypeptide, antibody, famsyl-directed carboxvmethyltransferase ligand etc.. according to the present invention can be isolated. The term "isolated" means that the material is in a 25 form in which it is not found in its original environment, e.g., more concentrated, more purified, separated from component, etc. An isolated nucleic acid includes, e.g., a nucleic acid having the sequence of famsyl-directed carboxymethyltransferase separated from the chromosomal DNA found in a living animal. This nucleic acid can be part of a vector or inserted into a chromosome (by specific gene-targeting or by random integration at a position other than its normal position) and still be isolated 30 in that it is not in a form which it is found in its natural environment. A nucleic acid or polypeptide of the present invention can also be substantially purified. By substantially purified, it is meant that nucleic acid or polypeptide is separated and is essentially free from other nucleic acids or polypeptides, i.e., the nucleic acid or polypeptide is the primary and active constituent. 20 WO 99/55878 PCTIUS99/07396 The present invention also relates to a transgenic animal. e.g., a non-human-mammal, such as a mouse, comprising a famsyl-directed carboxymethyltransferase nucleic acid. Transgenic animals can be prepared according to known methods, including, e.g., by pronuclear injection of recombinant genes into pronuclei of 1-cell embryos, incorporating an artificial yeast chromosome into embryonic stem 5 cells, gene targeting methods. embryonic stem cell methodology. See, e.g., U.S. Patent Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad Sci., 77:7380-7384 (1980): Palmiter et al., Cell, 41:343-345 (1985); Palmiter et al., Ann. Rev. Genet., 20:465-499 (1986); Askew et al., Mol. Cell. Bio., 13:4115-4124, 1993; Games et al. Nature. 373:523-527, 1995; Valancius and Smithies, Mol. Cell. Bio., 11:1402-1408, 1991; Stacey 10 et al., Mol. Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995; Rubinstein et al., Nucl. Acid Res., 21:2613-2617,1993. A nucleic acid according to the present invention can be introduced into any non-human mammal. including a mouse (Hogan et al., 1986, in Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring Harbor, New York), pig (Hammer et al., Nature. 315:343-345, 1985), sheep (Hammer et al., Nature. 315:343-345, 15 1985), cattle, rat, or primate. See also, e.g., Church, 1987, Trends in Biotech. 5:13-19; Clark et al., 1987, Trends in Biotech. 5:20-24; and DePamphilis et al., 1988, BioTechniques, 6:662-680. In addition, e.g., custom transgenic rat and mouse production is commercially available. These transgenic animals are useful as a cancer model, e.g., to test drugs, as food for a snake, as genetic markers to detect strain origin, etc. Such transgenic animals can further comprise other transgenes genes. e.g., Rb, 20 p53, RCE 1. FTase, rho, rab, rac, gamma-subunits of GTP-binding protein, and any of the above mentioned genes throughout this disclosure. Generally, the nucleic acids, polvpeptides, antibodies, etc. of the present invention can be prepared and used as described in, U.S. Pat. Nos. 5.501,969, 5,506.133, 5,441,870 WO 90/00607; WO 91/15582: 25 For other aspects of the nucleic acids, polypeptides. antibodies, etc., reference is made to standard textbooks of molecular biology, protein science, and immunology. See, e.g., Davis et al. (1986), Basic Methods in Molecular Biologv, Elsevir Sciences Publishing, Inc., New York; Hames et al. (1985), Nucleic Acid Hybridization, IL Press, Molecular Cloning, Sambrook et al.; Current Protocols in Molecular Biologv. Edited by F.M. Ausubel et al., John Wiley & Sons, Inc; Current 30 Protocols in Human Genetics, Edited by Nicholas C. Dracopoli et al., John Wiley & Sons, Inc.; Current Protocols in Protein Science; Edited by John E. Coligan et al., John Wiley & Sons, Inc.; Current Protocols in Immunologv; Edited by John E. Coligan et al., John Wiley & Sons, Inc. 21 WO 99/55878 PCT/US99/07396 EXAMPLE We have devised our own version of this assay which utilizes a biotinylated, prenylated peptide substrate (Biotin-Lvs-Lys-Ser-Lys-Thr-Lys-(Famesvl)Cvs; based on the C-terminal sequence of K-Ras 4B, but lacking the final three residues). In short, the human STE 14-expressing bacterial or insect cell 5 membranes utilize the.co-substrate 3 H-S-adenosyl methionine to methylate the (Famesvl)Cvs-carboxvl group. The resulting label incorporated into the substrate peptide is quantified using streptavidin-coated SPA beads. The methylase is cloned into the pRSET (Invitrogen) and pFastBac (Gibco BRL) bacterial and insect cell expression vectors, respectively. A standard assay is performed in 96-well sample plates (Wallac Part NO. 1450-40 1) with a 10 total assay volume of 100 1 which generally contains: 50 1 compound, 25 1 membranes and 25 1 3
H
SAM/substrate added in that order. Final concentration of HEPES pH 7.4 is 100 mM. A volume of 25 1 of membranes in 100 mM HEPES pH 7.4 is added to each well, followed by 25 1 diluted substrate (methylase substrates is stored at -20*C in 100% DMSO but is diluted in 10% DMSO to the required working concentration immediately before use). To this is added the label, i.e. 15 3 H-SAM (-85Ci.mmol; ImCi/ml; 12 M): 0.125 1 per well made up to 25 1 with 100 mM HEPES pH 7.4. The plate is then sealed and incubated at room temperature for 60 mins. The reaction is stopped by adding 150 1 Stop Mix which contains SPA beads (250 g) in PBS pH 7.1 + 5 mM EDTA + 0.1% Tween-20. The plate is sealed again the beads are left to settle 20 overnight before reading on a scintillation counter. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. 25 The entire disclosure of all applications, patents and publications, cited above and in the figures are hereby incorporated by reference. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. 22