MXPA97007852A - Homologo de fosfolipas - Google Patents

Abstract

The present invention provides nucleotide and amino acid sequences that identify and encode a phospholipase C homolog (plch and PLCH). The present invention also provides anti-sense molecules to the plch nucleotide sequences, expression vectors for the production of phospholipase C homologue purifying, antibodies that can specifically bind the phospholipase C homolog, hybridization probes or oligonucleotides to detect excess of nucleotide sequences encoding the homolog of phospholipase C, host cells genetically engineered for the expression of the homologue of phospholipase C, diagnostic tests for cells and / or tissues activated, inflamed, diseased, and resistant to hydroxyurea based on the sequences of nucleotide encoding the phospholipase C homologue or antibodies specific for the phospholipase homologue

Description

HOMOLOGO DE FOSFOLIPASA C TECHNICAL FIELD The present invention is in the field of molecular biology more particularly, the present invention describes the nucleic acid and the amino acid sequences of a novel phospholipase C homologue derived from a mast cell library.

PREVIOUS TECHNIQUE Phospholipase C Phospholipase C (PLC) belongs to a family of enzymes, also known as disulfide isomerases, which play a very important role in the transmembrane signal transduction. Many extracellular signaling molecules including hormones, growth factors, neurotransmitters, and immunoglobulins bind to their respective surface receptors in the cell and activate phospholipases C. The role of an activated phospholipase C is to catalyze the hydrolysis of phosphatidyl-inositol bisphosphate - 4, 5- (PIP2), a minor component of the plasma membrane, to produce diacylglycerol and 1, 4, 5-inositol triphosphate (IP3). In their respective biochemical trajectories, the 1, 4, 5-inositol triphosphate and diacylglycerol serve as secondary messengers and elicit a series of intracellular responses. 1, 4, 5-inositol triphosphate induces the release of Ca ++ from the internal cell store, and diacylglycerol activates protein kinase C (PKC). Both trajectories are part of the mechanism of transduction of the transmembrane signal which regulates cellular processes which include secretion, neural activity, metabolism, and proliferation. For example, signaling of the interleukin 4 receptor in human monocytes involves the activation of phospholipase C (Ho, JL et al. (1994) J Exper Med 180: 1457-69). Many different isoforms of phospholipase C have been identified and are categorized as phospholipase C-Beta, phospholipase C-Gama, and phospholipase C-Delta. Subtypes are designated by the addition of Arabic numbers after Greek letters, for example, phospholipase C-β-1. Phospholipases C have a molecular mass of 62-68 kDa, and their amino acid sequences show two regions of significant similarity. The first region designated X has approximately 170 amino acids, and the second region or Y contains approximately 260 amino acids.

The Mechanism of Protein G-Mediated Transmembrane Signaling Activates a particular phospholipase C via a regulatory protein binding with guanine nucleotide (G-protein) or by the intrinsic activity of tyrosine kinase of surface receptors. of the cell. Many plasma membrane binding receptors, including hormone receptors, activate the G proteins of cells. Each G protein can act as a molecular switch by activating one or more membrane binding effectors such as adenylate cyclase, ion channels, or phospholipase C. G proteins are heterotrimers with alpha, beta, and gamma subunits. The inactive G proteins have a guanine diphosphate (GDP) molecule tightly bound to its alpha subunit. When the G protein binding receptor binds an extracellular ligand, such as a hormone, the hormone receptor complex causes dissociation of guanine diphosphate from the alpha subunit. Immediately after this, the guanine diphosphate molecules fill the site, and the intrinsic ATPase activity of the alpha subunit causes the dissociation of the G protein from the hormone receptor complex. Simultaneously, guanine diphosphate binding reduces the affinity between the alpha, beta and gamma subunits and releases the beta-gamma complex. In some systems, the beta-gamma complex then activates phospholipase C beta-2 (Katz A et al. (1992) Nature 360: 686-9).

Isoforms of Phospholipase and its Cellular Activity The catalytic activities of the three isoforms of phospholipase C are Ca + + -dependent. It has been suggested that the binding sites for Ca + + in the phospholipases C are located in the Y region, one of the two conserved regions. Hydrolysis of phospholipids containing common inositol - phosphatidylinositol (Pl) 4-phosphatidylinositol monophosphate (PIP), and 4,5-phosphatidylinositol biphosphate (PIP2) by any of the isoforms produces cyclic and non-cyclic inositol phosphates. It is known that a large number of hormones and related molecules activate phospholipases.

Phospholipase C-beta Isoforms Both beta-1 and beta-2 isoforms of phospholipase C are activated by certain G protein subtypes and related G protein alpha subunits during signal transduction from the cell's surface receptors to the cell. phospholipase C. There may be two different types of G proteins, one sensitive to pertussis toxin and the other insensitive, which activate the beta-1 isoform. Katz A et al (supra) suggests that the beta subunit of the G protein can also activate phospholipase C beta-1. Activation of the phospholipases C is achieved by increasing its intrinsic activity rather than by reducing the Ca ++ requirement of the phospholipases C in the cytosol.

C-gamma Phospholipase Isoforms The phospholipase C gamma-1 isoform is phosphorylated and activated primarily by growth factor receptors that belong to the tyrosine kinase family. In addition, growth factor receptors are associated with the gamma-1 isoform before any tyrosine phosphorylation occurs. It appears that the major sites of tyrosine phosphorylation by the receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and nerve growth factor (NGF) are Tyr-771, Tyr-783 , and Tyr 1254 in the amino acid sequence of phospholipase C. The phosphorylation of Tyr-783 in the gamma-1 isoform by receptor tyrosine kinase is essential for the activation of the gamma-1 isoform. Other evidence suggests that the tyrosine kinase of the non-receptor protein can also phosphorylate and activate the gamma-1 isoform in response to certain surface receptors of the cell in leukocytes. For example, the T-cell antigen receptor complex can recognize antigens and transduce signals through the plasma membrane. Similarly, it seems that tyrosine kinases of non-receptor protein can activate the gamma-2 isoform. The evidence suggests that activation of the gamma-2 isoform is required for the stimulation of phospholipase D by platelet-derived growth factor (Yeo E-J et al. (1994) J Biol Chem 269 (45): 27823-27826). Other evidence suggests that the B cell surface antigen CD20 is associated with tyrosine and serine kinases and that it is involved in the tyrosine phosphorylation and activation of the gamma-1 and gamma-2 isoforms (Deans JP et al. (1993) ) J Immunol 151 (9): 4494-4504). It seems that the activation induced by the growth factor of phospholipases C is independent of the mediation by protein G. However, Marrero MB et al. (1994, J Biol Chem 269: 10935-39), reported an exception in the cells Aortic vessels of soft rat muscle where phospholipase C gamma-1 was activated by a G-protein coupled receptor.

C-delta phospholipase No receptors or transducers of the C-delta phospholipase isoforms have been identified.

Inhibition of Phospholipases C by Protein Kinase The evidence suggests that the activation of protein kinases can serve as negative feedback signals and attenuate the activity of receptor coupled phospholipase C including the magnitude and duration in certain cell types. For example, phosphorylation of Thr-654 at the epidermal growth factor receptors by the protein kinase prevents activation of the gamma-1 isoform by reducing the ability of the receptor tyrosine kinase to phosphorylate the gamma isoform. 1. In addition, activators of protein kinase C such as cAMP and 12-phorbol-13-phthalate acetate (PMA) attenuate the hydrolysis of phosphatidyl-inositol 4,5-bisphosphate induced by T cell antigen receptors. Similarly, it appears that the beta-1 isoform of phospholipase C is regulated by protein kinase C in certain cells.

Effects of the Second Messengers and Calcium Cations: Inositol Triphosphate and Diacylglycerol Once activated, phospholipases C catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce diacylglycerol and 1,4-inositol triphosphate, all of which serve as second messengers. Inositol triphosphate releases Ca ++ from the intracellular stores and increases the inward flow of Ca ++ from the extracellular fluid. Ca ++ directly regulates target enzymes and indirectly affects other enzymes by functioning as a second messenger and interacting with Ca ++ + fixative proteins, such as troponin C and calmodulin. The deactivation of the path of the inositol triphosphate is achieved through the active transport of Ca ++ inside the cells and the extrusion of ions through the fixation with plasma membrane, Ca ++ pumping ATPases. Similarly, sequential phosphorylation degrades inositol triphosphate. Diacylglycerol, a product of hydrolysis by phospholipases C, acts as a second messenger by activating protein kinase C. After protein kinase C binds to diacylglycerol, the Ca + + requirement of protein kinase C decreases to the level of free Ca ++ found in the cytosol. Activated kinase C of the protein phosphorylates a large number of intracellular proteins. The termination of the effect of diacylglycerol is achieved by enzymatic recycling to form phosphatidylinositol. Alternatively, a diacylglycerol lipase breaks diacylglycerol.

Phospholipase C and Disease Evidence indicates that a high percentage of primary human mammary carcinomas concomitantly show increased levels of epidermal growth factor receptor and phospholipase C-gamma-1. Similarly, studies in spontaneous hypertensive rats have suggested that one of the main causes of hypertension is an abnormal activation of phospholipase C-delta-1 that results from critical mutations in the X and Y regions of the amino acid sequence of Phospholipase C. The biology of phospholipase C is reviewed, inter alia, in Isselbacher KJ et al. (1994) Harrison's Principies of Internal Medicine, McGraw-Hill, New York NY; Kuruvilla A et al (1993) J Immun 151: 637-648; and Rhee SG and Choi KD (1994) J Biol Chem 267: 12393-12396. DESCRIPTION OF THE INVENTION The present invention provides a unique nucleotide sequence which encodes a novel human phospholipase C (PLCH) homologue. The cDNA, designated herein, was first found within the Incyte Clone Number 9118 from a human mast cell cDNA library. The invention also comprises the use of this homolog of phospholipase C or its variants to intercede in the conditions or diseases involving physiological or pathological situations which includes the steps of testing a sample or an extract with plch nucleic acids, fragments or oligomers of the same. Aspects of the invention include the anti-sense DNA of the plch; cloning or expression vectors containing plch, host cells or organisms transformed with expression vectors containing plch; a method for the production and recovery of the phospholipase C homologue from host cells; purified protein, homolog of phospholipase C, which can be used to generate antibodies for the diagnosis and therapy of activated and inflamed cells and / or tissues; and a diagnostic test for use in a biological sample obtained from a tumor for which hydroxyurea could be an appropriate therapy.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A and B visually display the nucleotide sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ IQ NO: 2) of the phospholipase C homologue from a DNA cDNA library. human mast cell. Alignment of the nucleic acid and the amino acid sequences took place using MacADNasis (Hitachi Software Engineering Co Ltd). Figure 2 shows the amino acid alignments of the consensus phospholipase C homolog (SEQ ID NO: 2, upper line) with MAP5PR0MR, family C of hamster protein disulfide isomerase / phosphoinositide-specific phospholipase Form I (Chaudhuri MM et al. (1992) Biocehm J. 281: 645-50). Figure 3 visually displays an analysis of the hydrophobicity of the phospholipase C homologue based on the sequence and composition of predicted amino acids. Figure 4 is a representation of the DNA sequences that were used to assemble the consensus sequence for the inventive phospholipase C homolog provided in SEQ ID NO: 1. The DNA sequences were armed using the INHERIT ™ 670 Sequence Analysis System given as SEQ ID NOS: 3-13.

MODES FOR CARRYING OUT THE INVENTION Definitions As used herein, PLCH (upper kit) refers to homologues of phospholipase C polypeptides, naturally occurring phospholipase C homologs and active fragments thereof, which are encoded by transcribed mRNAs. from the cDNA (lower kit, plch) of SEQ ID NO: 1. "Active" refers to those forms of homologue of phospholipase C which retains the biological and / or immunological activities of a phospholipase C homolog occurring naturally . "Naturally occurring phospholipase C homologue" refers to homologs of phospholipase C produced by human cells which have not been genetically engineered and specifically contemplates different phospholipase C homologs that originate from subsequent modifications to the translation of the phospholipase C. polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. "Derivative" refers to homologs of phospholipase C chemically modified by techniques such as ubiquitination, labeling (for example, with radionuclides, different enzymes, etc.), pegylation (derivatization with polyethylene glycol), and insertion or substitution by chemical synthesis of amino acids such as ornithine, which does not normally occur in human proteins. "Recombinant variant" refers to any polypeptide which differs from naturally occurring phospholipase C homologs by amino acid insertions, deletions, and substitutions, created using recombinant DNA techniques. A guide can be found to determine which amino acid residues can be replaced, added or deleted without eliminating the activities of interest, such as normal signal transduction, by comparing the sequence of the particular phospholipase C homologue with that of the homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology. Preferably, amino acid "substitutions" are the result of replacing an amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine; that is, conservative amino acid replacements. "Insertions" or "deletions" are typically in the range of about 1 to 15 amino acids. The permitted variation can be determined experimentally by systematically inserting, deleting or substituting amino acids in a phospholipase C homolog molecule using recombinant DNA techniques and testing the resulting recombinant variants to see their activity. When desirable, a "leader or signal sequence" can direct the polypeptide through the membrane of a cell. Such a sequence may be naturally present in the polypeptides of the present invention or may be provided from heterologous protein sources by recombinant DNA techniques. A "fragment", "portion", or "segment" of polypeptide is a stretch of amino acid residues of at least about 5 amino acids, often at least 7 amino acids, typically at least 13 amino acids, and, in different embodiments, at least 17 or more amino acids. To be active, any polypeptide of the phospholipase C homolog must have sufficient length to visually display the biological and / or immunological activity. A "fragment," "portion," or "segment" of "oligonucleotide" or polynucleotide is a stretch of nucleotide residues which is long enough to be used in the polymerase chain reaction (PCR) or different methods of Hybridization to amplify or simply reveal the related parts of the mRNA or DNA molecules. The present invention comprises the phospholipase C homologue polypeptide purified from natural or recombinant sources, cells transformed with recombinant nucleic acid molecules encoding the phospholipase C homolog. Different methods for the isolation of the phospholipase C homologue polypeptide can be achieved by methods well known in the art. For example, said polypeptide can be purified by immunoaffinity chromatography by using the antibodies provided by the present invention. The different protein purification methods well known in the art include those described by Deutscher M (1990) Methods in Enzymology, volume 182, Academic Press, San Diego, CA; and by Scopes R (1982) Protein Purification: Principles and Practice, Springer-Verlag, New York, NY both incorporated herein by reference. "Recombinant" can also refer to a polynucleotide that encodes the phospholipase C homolog and is prepared using recombinant DNA techniques. The DNA encoding the phospholipase C homolog may also include allelic or recombinant variants and mutants thereof. The "nucleic acid probes" are prepared based on the cDNA sequence which encodes the phospholipase C homolog provided by the present invention. The oligonucleotides comprise portions of the DNA sequence having at least 15 nucleotides, usually at least 20 nucleotides. Nucleic acid probes comprise portions of the sequence having fewer nucleotides of about 6 kb, usually less than about 1 kb. After appropriate tests to eliminate false positives, these probes can be used to determine whether the mRNAs encoding the phospholipase C homologue are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described. Walsh PS et al. (1992 PCR Methods Appl 1: 241-250). The probes can be derived from naturally occurring or recombinant nucleic acids of one or two chains or be chemically synthesized. They can be labeled by precise translation, Klenow filling reaction, polymerase chain reaction or other methods well known in the art. The probes of the present invention, their preparation and / or labeling are explained in Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY; or in Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, both incorporated herein by reference. "Activated" cells as used in this application are those that are occupied in the exchange of extracellular or intracellular membrane, including the export of neurosecretory or enzymatic molecules as part of a normal or disease process. Alternatively, recombinant variants encoding these same polypeptides or the like can be synthesized or selected by making use of "redundancy" in the genetic code. Different codon substitutions, such as silent changes which produce different restriction sites, can be introduced to optimize cloning within a plasmid or viral vector or expression in a normal prokaryotic or eukaryotic system. Mutations or domains of other aggregated peptides can also be introduced to modify the properties of any part of the polypeptide, to change affinities of ligand binding, interchain affinities, or rate of degradation / change.

Detailed Description of the Invention The present invention provides a unique nucleotide sequence that identifies a novel human phospholipase C homologue which was first identified in human mast cells. The first sequence identified in SEQ ID NO: 3 is shown. It was found to be homologous to the hamster P5 protein (Chaudhuri MM et al. (1992) Biochem J 281: 645-50), which was identified for the first time in cell lines with increased resistance to hydroxyurea, an antineoplastic agent. . Therefore, the expression of the phospholipase C homolog is extremely useful for diagnostic purposes. Because the phospholipase C homologue is specifically expressed in active cells in immunity, the nucleic acid (plch), the polypeptide (PLCH) and the phospholipase C homologue antibodies are useful in investigations and interventions in normal physiological and pathological processes and abnormal which comprise the role of the mast cell in immunity. Therefore, a diagnostic test for the upregulated expression of the phospholipase C homolog can accelerate the diagnosis and proper treatment of tumors, conditions, or diseases caused by systemic and local infections, traumatic and other types of tissue damage, hereditary diseases or environmental factors associated with hypertension, carcinomas, and other physiological / pathological problems associated with abnormal signal transduction. In addition, because the expression of the P5 protein in hydroxyurea-resistant hamster cells is increased, we propose a test for this gene to be used in the identification of drug resistance of hydroxyurea in human neoplastic cells such as those They can be found in chronic granulocytic leukemia. The nucleotide sequence encoding the phospholipase C homologue (or its complement) has numerous other applications in the techniques known to those skilled in the art of molecular biology. These techniques include the use of hybridization probes, the use of oligomers for the polymerase chain reaction, the use of chromosome and gene mapping, the use in the recombinant production of the phospholipase C homolog and the use in generation of antisense DNA or RNA, its chemical analogues and the like. The uses of the nucleotide encoding the phospholipase C homolog described herein are exemplary of the known techniques and are not intended to limit their use in any known technique to a person of ordinary skill in the art. On the other hand, the nucleotide sequences described herein can be used in molecular biology techniques that have not yet been developed, provided that the new techniques depend on the properties of the nucleotide sequences that are currently known, for example, the triple genetic code, specific base pair interactions, and so on. Those skilled in the art will appreciate that as a result of the degradation of the genetic code, a multitude of nucleotide sequences encoding the phospholipase C homolog may be produced, some bearing minimal homology to the nucleotide sequence of any known gene and that happen naturally. The invention specifically contemplates each and every possible variation of the nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triple genetic code as applied to the nucleotide sequence of the naturally occurring phospholipase C homologue, and all these variations must be considered as being specifically described. Although the nucleotide sequences encoding the phospholipase C homologue and its variants may preferably hybridize to the nucleotide sequence of the phospholipase C homolog occurring naturally under stringent conditions, it may be advantageous to produce nucleotide sequences encoding the homologue of phospholipase C or its derivatives possessing a substantially different codon usage. The codons can be selected to increase the rate at which the expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host according to the frequency with which the host uses the particular codons. Other reasons for substantially altering the nucleotide sequence of the phospholipase C homologue and its derivatives without altering the encoded amino acid sequence include the production of RNA transcripts having more desirable properties, such as longer half-life, than the transcripts produced from of the sequence that occurs naturally. The sequence of nucleotides encoding the phospholipase C homolog may be linked to a variety of other nucleotide sequences by well-established recombinant DNA techniques (cf Sambrook J et al., Supra). Useful nucleotide sequences for binding to the plch include a classification of cloning vectors, for example, plasmids, cosmids, lambda phage derivatives, phagemids, and the like, which are well known in the art. Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and the like. In general, the vectors of interest may contain a functional replication origin in at least one organism, suitable restriction endonuclease sensitive sites, and markers that can be selected for the host cell. Another aspect of the present invention is to provide plch-specific nucleic acid hybridization probes that can hybridize to nucleotide sequences encoding the naturally occurring phospholipase C homologue. Said probes can also be used for the detection of the phospholipase C receptor coding sequences and should preferably contain at least 50 percent of the nucleotide of this plch coding sequence. The hybridization probes of the present invention can be derived from the nucleotide sequence of SEQ ID NO: 1 or from the genomic sequence including promoter, enhancer and introns elements of the respective naturally occurring phospholipases C. Hybridization probes can be labeled by a variety of reporter groups, including radionuclides such as • P or • 3JS, or enzymatic labels such as alkaline phosphatase coupled to the probe by avidin / biotin coupling systems, and the like.

The polymerase chain reaction as described in U.S. Patent Nos. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based on the nucleotide sequences which encode the phospholipase C homolog. Said probes used in the polymerase chain reaction can be of recombinant origin, can be chemically synthesized, or can be a mixture of both and comprise a discrete nucleotide sequence for use in diagnosis or a degenerate combination of possible sequences for the identification of closely related genomic sequences. Another means for producing hybridization probes specific for plch DNAs includes the cloning of nucleic acid sequences encoding the phospholipase C homologue or phospholipase C homolog derivatives within vectors for the production of mRNA probes. Such vectors are known in the art and are commercially available and can be used to synthesize the RNA probes in vitro by the addition of the appropriate RNA polymerase such as T7 or SP6 RNA polymerase and the radioactively appropriate labeled nucleotides. It is now possible to produce a DNA sequence, or portions thereof, that encodes the phospholipase C homologue and its derivatives by synthetic chemistry alone, after which the gene can be inserted into the many available DNA vectors using reagents, vectors and cells that are known in the art at the time of submitting this application. Furthermore, synthetic chemistry can be used to introduce mutations within the plch sequences or any portion thereof. The nucleotide sequence can be used to construct an assay to detect activation, inflammation, or disease associated with abnormal levels of expression of the phospholipase C homologue. The nucleotide sequence can be labeled by methods known in the art and added to a sample of fluid or tissue of a patient under hybridization conditions. After an incubation period, the sample is washed with a compatible fluid which optionally contains an ink (or other label that requires a developer) if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed, the ink is quantified and compared to a standard. If the amount of ink is significantly elevated, the nucleotide sequence has hybridized to the sample, and the assay indicates the presence of inflammation and / or disease. The nucleotide sequence for the plch can be used to construct hybridization probes for gene mapping. The nucleotide sequence provided herein can be mapped to a chromosome and specific regions of a chromosome using well known genetic and / or chromosome mapping techniques. These techniques include in situ hybridization, binding analysis against known chromosomal markers, hybridization screening with libraries or chromosomal preparations selected by flux specific for known chromosomes, and the like. Among other places, the fluorescent in situ hybridization technique of chromosome diffusions has been described in Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, NY. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques can be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 1981f). The correlation enters the place of the plch on a map of physical chromosome and a specific disease (or predisposition to a specific disease) can help to delimit the region of DNA associated with that genetic disease. The nucleotide sequence of the present invention can be used to detect differences in gene sequence between normal and carrier or affected individuals. The nucleotide sequence encoding the phospholipase C homologue can be used to produce the one produced. In addition to recombinant production, fragments of the phospholipase C homolog may be produced by direct peptide synthesis using solid phase techniques (cf Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J Am Chem Soc 85: 2149-2154). Protein synthesis can be performed in vitro using manual techniques or by automation. Automated synthesis can be performed, for example, using Applied Biosystems 413A Peptide Synthesizer (ABI, Foster City, California) in accordance with the instructions provided by the manufacturer. They can be chemically synthesized and combine different fragments of the phospholipase C homolog separately using chemical methods to produce the full-length molecule. The homolog of phospholipase C by induction of antibody does not require biological activity; however, the protein must be immunogenic. The peptides used to induce specific antibodies can have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. These must mimic a portion of the amino acid sequence of the protein and can contain the complete amino acid sequence of a naturally occurring small molecule such as the phospholipase C homolog. Small stretches of purified phospholipase C homolog can be fused using well known methods of recombinant DNA technology. Among the many publications that teach methods for gene expression after they have been isolated, is Goedeel (1990) Gene Expression Technology, Methods and Enzymology, volume 185, Academic Press, San Diego, CA. The phospholipase C homolog can be expressed in a variety of host cells, either prokaryotic or eukaryotic. The host cells may be of the same species in which the plch nucleotide sequence is endogenous or of different species. The advantages of producing the phospholipase C homologue by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures. The transformed cells can be cultured with DNA encoding the phospholipase C homolog under suitable conditions for the expreration of foefolipaea C and the recovery of the protein from the cell culture. The homolog of foefolipaea C produced by a recombinant cell, ee may secrete or may be contained intracellularly, depending on the particular genetic construct used. In general, it is more convenient to prepare recombinant proteins in the form of secretion. The purification steps vary with the production process and the particular amino acid protein of the phospholipase C homologue with those of other proteins such as an orifice limpet hemocyanin and antibodies raised against the fusion protein. Antibodies specific for the homologue of foefolipaea C can be produced by vaccination of a suitable animal with the polypeptide or an antigenic fragment. An antibody is specific to the homologue of foefolipase C if it is produced against an epitope of the polypeptide and binds to at least one part of the natural or recombinant protein. The production of antibodies includes not only the stimulation of an immune response by injection in animals, but also analogous steps in the production of synthetic antibodies or other specific binding molecules such as the screening of recombinant immunoglobulin libraries (cf Orlandi R et al. (1989) PNAS 86: 3833-3837, or Huse WD and collaborator (1989) Science 256: 1275-1281) or the in vitro stimulation of lymphocyte populations. Current technology (Winter G and Milstein C (1991) Nature 349: 293-299) provides a number of highly specific binding reagents based on the principles of antibody formation. These techniques can be adapted to produce molecules that specifically bind the homologue of foefolipaea C. A further embodiment of the present invention is the use of antibodies, receptors or similaree specific to the homologue of phospholipase C, as a bioactive agent for treating infectious and localized infections, traumatisms and other tissue damage, hereditary or environmental diseases associated with hyperemission, carcinomae, and other physiological and / or pathological problems associated with abnormal signal transduction. Bioectivating compositions comprising agonists, antagonists, or receptors of the homologue of foefolipaea C in a suitable therapeutic method can be administered by any of many methods including clinical studies in mammalian specimens to determine the maximum tolerable dose in normal human subjects to determine the doeie. eegura Additionally, the bioactive agent can be complexed with a variety of well-established compounds or compositions which improve stability or pharmacological properties such as half-life. It is contemplated that a therapeutic, bioactive composition may be applied by intravenous infusion into the bloodstream or any other effective means that could be used for treatment. The following examples are provided to illustrate the present invention. These examples are provided by way of illustration and are not included for the purpose of limiting the invention.

INDUSTRIAL APPLICABILITY I mRNA Isolation and Construction of the cDNA Library The majority of the phospholipase C sequence of consensus was identified in Incyte Clone 9118 (SEQ ID NO: 3) among the sequences comprising the human mast cell library. SEQ ID NOs: 4-13 were found in the Incyte data bank which has been derived from a number of different libraries. Using these sequences, one skilled in the art has sufficient information to construct full-length cDNAs and use them to produce the homolog of phospholipase C. The cells used to prepare the human mast cell library of a cancer patient at the Mayo Clinic were obtained. The mast cell cDNA library was prepared by purification of the poly-A + mRNA and enzymatically synthesizing the double-stranded complementary DNA. The synthetic adapters were ligated to the blunt-ended cDNAs which were then ligated to the Uni-ZAPMR vector (Stratagene, La Jolla, CA) derived from lambda phage, which was used, in turn, to infect XLl-Blue ® ( Stratagene) of E. coli host chain. The quality of the library of CDNA using DNA probes and then phagemid pBluescript (Stratagene) was excised by the live m extirpation process. PBluescript is a smaller, single-stranded circular DNA molecule that includes all the necessary DNA sequences of the plasmid and the cDNA insert.

Isolation of cDNA Clones The phagemid was released from the cells, purified, and used to re-infect fresh bacterial host cells (SOLR, Stratagene), where the phagemid double-stranded DNA was produced. Because the phagemid carries the gene for ß-lactamaea, freshly transformed lae bacteriae were selected in medium containing ampicillin. The phagemid DNA was purified using the QIAWell-8 Plasmid Purification System or the DNA Purification System, QIAGEN® (QIAGEN Ine, Chatsworth, CA). This is a fast and reliable high-throughput method for causing lysis in bacterial cells and for straightening the highly purified phagemid DNA. The DNA was leached from the purification resin for DNA sequencing or other analytical manipulations.

III Sequencing of cDNA Clones The insertions of the cDNA were partially sequenced from the random isolates of the mast cell library. Methods for DNA sequencing are well known in the art. Conventional enzymatic methods employed a Klenow fragment of DNA polymerase, SEQUENASE (US Biochemical Corp., Cleveland, OH) or Taq polymerase to extend the DNA strands from an annealed primer or oligonucleotide to the DNA template of interest. Methods have been developed for the production of both single and double chain seedlings. The gelee urea-acrylamide chain termination reaction product was electrophoresed and detected either by autoradiography (for precursors labeled by radionuclides) or by fluorescence (for precureoree labeled with fluorescent). The recent improvements in the preparation of the mechanized reaction, the sequencing and the analysis of the fluorescent detection method, have allowed the expansion in the number of eequences that can be determined per day (using talee machines such as the Catalyst 800 and the sequencers of Applied Biosysteme 377 or 373 DNA).

IV Homology Search of cDNA Clones and Deduced Proteins Each eequence obtained in this way compared to the sequence in the GenBank using a search algorithm developed by Applied Biosystems and incorporated into the Sequence Analysis System INHERITNR 670. In this algorithm, used the Pattern Specification Language (developed by TRW Inc., Loe Angelee, CA) to determine the regions of homology. The tree parameters that determine how the sequence comparisons were made were the window size, the window desfaeamiento, and the error tolerance. Using a combination of these three parameters, the sequences containing regions of homology to the search sequence were searched in the DNA database, and the appropriate sequences were marked with an initial value. Subsequently, these homologous regions were examined using dot matrix homology diagrams to distinguish regions of homology from random couplings. Smith-Waterman alignments were used to visually display the results of the homology search. The homology of the peptide and protein sequence was determined using the INHERIT in a manner similar to that used in the homology of the DNA sequence. The Pattern Specification Language and parameter window were used to search the protein data bases for sequences that contained regions of homology that were marked with an initial value. The point matrix homology diagrams were examined to distinguish regions of significant homology from random couplings. Alternatively, BLAST, which means Basic Local Alignment Search Tool, was used to search for local sequence alignments (Altschul SF (1993) J Mol Evol 36: 290-300; Altechul, SF and collaboraree (1990) J Mol Biol 215: 403-10). The Báeico Local Alignment Search Tool produces alignments of both nucleotide and amino acid sequences to determine the similarity of the sequence. Due to the local nature of the alignments, the Basic Local Alignment Search Tool is especially useful for determining exact links or for identifying homologs. While that is ideal for links that do not contain intervals, it is inappropriate for searching for motif style. The fundamental unit of the Algorithm Output of the Basic Local Alignment Search Tool of the High Dial Segment Par (HSP). The High-Dial Segment Pair has two arbitrary but equal longitude sequence fragments whose alignment is locally maximum and for which alignment the dialing meets or exceeds a cut-off threshold set by the user. The approach of the Basic Local Alignment Search Tool is to look for the High Dial Segment Pairs between the inquiry sequence and a database sequence, to evaluate the eetective significance of any coupling encountered, and to report only those couplings that meet the threshold of significance selected by the user. Parameter E establishes the statistically significant threshold for reporting the couplings of the data base sequence. E is interpreted as the upper link of the expected frequency of the random occurrence of a High Dialing Segment Pair (or high Dialing Segment Pairs placement) within the context of the complete data base search. At the end of the program, any frequency of the data bank whose satiefaga coupling E is reported.

V Identification, Full-length Sequencing and Gene Translation. The analysis of INHERIT1 ^ results from the randomly selected and sequenced clonee portions of the identified maceil cell library Incyte Clone 9118 (SEQ ID NO: 3) as a homologue of the P5 gene of hameter, or protein dieulfide ieomeraea (Chaudhuri MM and collaborator, supra). The cDNA insert comprising Incyte 9118 was completely sequenced using the methods described above. The insert completely consisted of an open reading frame corresponding to nucleotides numbered about 130 on 950 (no data shown). Looking at the insertion of 9118, our database of clones (see SEQ ID NOe: 4-13), INHERIT ^ 11 and the hameter sequence as a guide, we assembled the sequence of the connection shown in Figure 1. We searched for the translation (SEQ ID NO: 2) shown in Figure 1 against the protein database such as SwiseProt and PIR and no exact coupling was found. Figure 2 shows the comparison of our etiology of the phospholipase C homologue with that of the hamster P5 protein. Figure 3 shows the hydrophobicity diagram for the phospholipase C homolog. Figure 4 is a proportional representation of the assembly of SEQ ID NOs: 3-13.

VI Anti-sense analysis Knowledge of the correct cDNA sequence, complete with the phospholipase C homologue, will allow euueus as a tool for antisense technology in the investigation of gene function. Oligonucleotides, either genomic or cDNA fragments comprising the antisense strand of the plch are used either in vitro or in vivo to inhibit mRNA expression. Such technology is now well known in the art, and probes are designated in different places along the nucleotide sequences. By treating cells or test animals complete with said antisense sequence, the gene of interest is effectively deviated. Frequently, the function of the gene can be ascertained by observing the behavior at the intracellular, cellular, tissue, or organic level (for example, mortality, loss of differentiated function, change in morphology, etc.). In addition to editing the sequences constructed to interrupt the tranecring of the open reading frame,. They obtain modifications of the gene expression by means of the designation of frequency and antieense to the region of the intron, promoter elements / enhancers, or haeta to trane-acting regulatory genes. In a similar way, inhibition is linked using the Hogeboom base pairing methodology, also known as "triple helix" base pairing.

VII Expression of the phospholipase C homologue Plch expression can be achieved by subcloning the cDNAs within appropriate expression vectors and transfecting the vectors within appropriate expression hosts. In this particular case, the cloning vector previously used for generation of the tissue library also provides direct expression of the plch sequences in E. coli. Upstream of the cloning site, this vector contains a promoter for the β-galactoeidase, followed by the sequence containing the amino-terminal Met and the subeequent 7 residues of β-galactoeidase. Immediately after this eight reeiduoe is a useful bacteriophage promoter useful for the artificial priming and tranecription and a number of unique restriction sites, including Eco Rl, for cloning. Induction of the isolated, bacterial strain transfected with IPTG using standard methods will yield a fusion protein corresponding to the first seven residues of β-galactosidase, approximately 15 residues of "linker", and the peptide encoded within the cDNA. Since cDNA clone insertions are • generated by an essentially random process, there is a possibility in three that the included cDNA will fall into the correct frame for the appropriate translation. If the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases by well-known methods including in vitro mutagenesis, exonuclease III digestion or mongo bean nuclease, or oligonucleotide linker incubation. The DNA plches from one side to another within other vectors of which it is known that they are useful for the expression of the specific host protein. Oligonucleotide amplimers containing cloning sites can be chemically synthesized as well as a DNA segment sufficient to hybridize to the stretches at both ends of the target cDNA (25 bases), by standard methods. These primers are then used to amplify the eegment of the desired gene by polymerase chain reaction. The new resulting gene segments are digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, similar gene segments are produced by digestion of the cDNA with the appropriate restriction enzymes and filling the missing gene segments with oligonucleotide and chemically synthesized. The segments of the coding sequence of more than one gene are ligated together and cloned into the appropriate vectors to optimize the expreration of the recombinant sequence. Suitable expression hosts for said chimeric molecules include but are not limited to mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae. . and bacteria such as E. coli. For each of these cellulatory systems, a useful expiration vector may also include a replication origin to allow propagation in the bacterium and a selectable marker such as the β-lactamase antibiotic resistance gene to allow selection in the bacteria. In addition, the vectors can include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells. The vectors for use in host cells of eukaryotic expression may require RNA processing elements such as 3 'polyadenylation sequences if the DNA is not part of the cDNA of interest. Additionally, the vector may contain promoters or enhancers, which increase the expreation of the gene. Said promotion of the host specific and include MMTV, SV40, and metallothionine promoters for Chinese haem ovary cells, - trp, lac, tac- and T7 promoters for bacterial hosts; and the alpha factor, oxidase of alcohol and promoter of PGH for the yeast. Transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, can be used in mammalian host cells. Once the homogeneous cultures of recombinant cells are obtained through the standard culture method, they recover a large amount of the homologue of foefolipase C produced recombinantly from the conditioned medium and analyzed using chromatography methods known in the art.

VIII Isolation of Recombinant Phospholipase C Homologue The phospholipase C homolog can be expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate purification of the protein. Said domains for facilitation of purification include, but are not limited to, metal chelation peptides such as hietidine / tryptophan modules that allow immobilized metalee purification, protein A domain that allows purification in immobilized immunoglobulin, and the domain used in the FLAGS exteneion / affinity purification system (Immunex Corp., Seattle WA). The inclusion of a dissociable linker sequence such as Factor XA or enterokinase (Invitrogen) between the purification domain and the plch sequence may be useful to facilitate the expression of the homolog of phospholipaea C.

IX Production of Specific Antibodies of the Homolog of Phospholipase C Two approaches are used to originate antibodies against the homolog of phospholipase C, and each approach is useful to generate antibodies either polyclonal or monoclonal. In one approach, the denatured protein of separation of the homologue of phospholipaea C from faee inverea is obtained in a quantity of 75 milligrams. Eeta denatured protein is used to immunize mice or rabbits using standard protocols; Approximately 100 micrograms are suitable for immunization of a mouse, whereas 1 milligram can be used to immunize a rabbit. To identify the mouse hybridomas, the denatured protein is radioiodized and tested to screen murine B-cell-enhanced hybridoma for those that produce antibodies. This procedure only requires small amounts of protein, so 20 milligrams would be enough to label and track several thousand clones. In the second approach, the amino acid sequence of the phospholipase C homologue, as deduced from the translation of the cDNA, is analyzed to determine the region of high immunogenicity. The oligopeptides comprising appropriate hydrophilic regions, as illustrated in Figure 3, are synthesized and used in suitable immunization protocols to elicit antibodies. In Ausubel FM et al (supra), the analysis is selected to select appropriate epitopes. The optimal amino acid sequence for immunization is usually in the C-terminus, the N-terminus and those involved, hydrophilic regions of the polypeptide that are likely to be exposed to the external environment when the protein is in its natural conformation. Typically, the selected peptides, approximately 15 residues in length, are synthesized using an Applied Biosyetems Model 431A Peptide Synthesizer using fmoc chemistry and coupled to orifice limpet hemocyanin (KLH, Sigma) by reaction with the M ester. -maleimidobenzoyl-N-hydroxysuccinimide (MBS; see Ausubel FM et al., supra). If necessary, a cysteine can be introduced at the N-terminus of the peptide to allow attachment to the hemocyanin of orifice limpete. Rabbits are immunized with the peptide-hemocyanin complex of orifice limpet in complete Freund's adjuvant. The resultant antisera are tested to see the antipeptide activity by fixing the peptide with plactic, blocking with 1% bovine serum albumin, reacting with antiserum, washing and reacting with labeled goat anti-rabbit IgG. (radioactive or fluoreactive), purified affinity, specific. Hybridomas can also be prepared and screened using standard techniques. Hybridomae of interest are detected by means of the stratification with the labeled homolog of foefolipase C to identify those fusionee that produce the monoclonal antibody with the desired specificity. In a typical protocol, the plate wells are covered (FAST; Becton-Dickinson, Palo Alto, CA) during incubation with rabbit-anti-mouse antibody (or suitable anti-species) with purified affinity, specific to 10 milligrams / milliliter. The wells are blocked with 1% bovine serum albumin, washed and incubated with supernatants of hybridomas. After washing, the wells are incubated with the homologue of foefolipase C labeled at 1 milligram / milliliter. The eobrenated with specific antibodies bind more phospholipase C homolog labeling than can be detected in the background. Afterwards, the clonee that produce specific antibodies is expanded and subjected to two cloning cycles at a limiting dilution (1 cell / 3 wells). The cloned hybridomas are injected into prietane-treated mice to produce aecitis, and the monoclonal antibody from mouse ascites fluid is purified by affinity chromatography on Protein A. Monoclonal antibodies with affinity at least 10 M, preferably from 10 to 10. Or stronger, will typically be made by standard procedures as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, New York, NY, both incorporated herein by reference.

X Diagnostic Test Using Specific Antibodies of Homolog of Phospholipase C. Loe antibodies of the homolog of fosfolipaea C particularee are useful for the investigation of the resistance of the tumor to hydroxyurea, signal transduction, and the diagnosis of infectious or hereditary conditions or diseases. which are characterized by differences in the amount or distribution of the phospholipase C homolog. Since the phospholipase C homologue was found in a human mast cell library, it seems to be over-regulated in cellular types involved primarily in protection or immune defense . Lae diagnostic tests for the phospholipase C homologue include methods that use the antibody and a label to detect the homolog of phospholipase C in human body fluids, membranes, cells, tissues or extracts thereof, polypeptides and antibodies can be used of the present invention with or without modification. Frequently, the polypeptides and antibodies will be labeled by means of binding them, either covalently or non-covalently, with a substance that provides a signal that can be detected. A wide variety of labeling and conjugation techniques are known and have been reported extensively in both patent and scientific literature. Suitable labels include radionuclides, enzymes, eutetrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Lae Patentists teaching the disclosure of labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins can be produced as shown in U.S. Patent No. 4,816,567, incorporated herein by reference. A variety of protocols for measuring the phospholipase C homolog or membrane-bound phospholipase C are known in the art, using either polyclonal or monoclonal antibodies specific for the protein. Examples include the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and selection of fluorescent activated cells (FACS). A two-site monoclonal-based immunoassay using monoclonal antibodies reactive to the two non-interfering epitopes on the phospholipase C homologue is preferred, but a competitive binding assay can be employed. These assays are described, inter alia, in Maddox, DE et al. (1983, J Exp Med 158: 1211).

XI Purification of Homologue of Native Phospholipase C Using Specific Antibodies The native or recombinant phospholipase C homologue is purified by immunoaffinity chromatography using antibodies specific for the homologue of foefolipaea C. In general, an immunoaffinity column is assembled by covalently coupling the anti-homologous antibody of foefolipaea C to an activated chromatographic reein. Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Similarly, monoclonal antibodies are prepared from mouse fluid ascites by precipitation of ammonium sulfate or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently bound to a chromatographic resin such as Sepharose activated by CnBr (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derived resin is washed in accordance with the manufacturer's instructions. Such immunoaffinity columns can be used in the purification of the phospholipase C homologue by preparing a fraction from cells containing the homologue of phospholipaea C in a soluble form. This preparation can be derived by the eolubilization of whole cell or an eubcellular fraction obtained by differential centrifugation (with or without the addition of detergent) or by any other method well known in the art. Alternatively, the homolog of soluble C-effecipaea containing a signal sequence in a useful amount within the medium in which the cells are grown can be determined. A preparation containing the homologue of soluble foefolipaea C is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential abeorbence of the homologue of foefolipaea C (for example, regulator of the pH of high ionic strength in the presence of Detergent) . The column is then leached under conditions that break the antibody / homolog bind of phospholipase C (eg, a pH regulator with a pH of 2-3 or an elevated concentration of a chaotrope such as urea or thiocinate ion) , and the phospholipaea C homolog is collected.

XII Drug Screening The invention is particularly useful for the screening of therapeutic compounds by the passage of the homologue of foefolipaea C and binding fragments thereof in any variety of drug screening techniques. The polypeptide or fragment inserted in said test may already be free in solution, fixed to a solid support, originated on a cellular surface or located intracellularly. A method of drug screening utilizes eukaryotic or prokaryotic huéeed cells which are transformed in an eetable manner with recombinant nucleic acids expressing the polypeptide or fragment. The drugs are screened against said cells transformed into competitive binding assays. Dichae cells are already in viable or fixed form, they are used for standard fixation assays. One can measure, for example, the formation of complexes between the homolog of phospholipase C and the agent being tested. Alternatively, one can examine the decrease in complex formation between the phospholipase C homologue and a receptor caused by the agent being tested. In this manner, the present invention provides methods of screening drugs or any other agents that affect signal transduction. This method comprises contacting said agent with a polypeptide of the homologue of foefolipase C or fragment thereof and making test (1) for the presence of a complex between the agent and the polypeptide of the homologue of foefolipaea C or fragment, or (ii) for the presence of a complex between the polypeptide of the homolog of foefolipaea C or fragment and the cell, by methods well known in the art. In such competitive binding assays, the polypeptide of the phospholipase C homologue or fragment is typically labeled. After a suitable incubation, the polypeptide of the homologue of free foefolipaea C or fragment of that preend in the bonding form, and the amount of free or uncomplexed tag, is a measure of the ability of the particular agent to fix the counterpart of phospholipase C or to interfere with the homologue of phospholipase C and the agent complex. Another technique for drug screening provides high throughput screening for compounds that have adequate binding affinity to the polypeptide of the homologue of foefolipase C and is described in detail in European Patent Application 84/03564, published on September 13. of 1984, incorporated herein by reference. Briefly stated, large amounts of different peptide test compounds are synthesized on a solid euettra, such as plastic pins or some other surface. The peptide test compounds are reacted with the homologous polypeptide of foefolipaea C and washed. Deepuée detects the phospholipase C homolog of the binding polypeptide by methods well known in the art. The purified phospholipase C homolog is coated directly on plates for use in the drug screening techniques mentioned above. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug screening assays in which the neutralizing antibodies that can bind the phospholipase C homologue, compete specifically with a test composition to fix the polypeptide of the homologue of foefolipase C or fragments thereof. In this manner, antibodies are used to detect the presence of any peptide that shares one or more antigenic determinants with the phospholipase C homologue.

XIII Rational Drug Design The purpose of the rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, for example, agonietas, antagonistae, or inhibidoree. Any of these examples are devised to devise drugs which are more active or stable forms of the polypeptide or which improve or interfere with the function of a polypeptide in vivo (cf Hodgson J (1991) Bio / Technology 9: 19-21, incorporated to the preend as reference). In one approach, the three-dimensional structure of a protein of interest, or of a protein inhibitor complex, is determined by x-ray crystallography, by computer design or, more typically, by a combination of the two approaches. Both the manner and charges of the polypeptide must be ascertained to elucidate the structure and to determine the active site (s) of the molecule. Less frequently, useful information could be gained with reference to the structure of a polypeptide by design based on the homologous protein structure. In all cases, relevant structural information is used to design efficient inhibitors. The examples of rational drug use may include molecules that have improved activity or stability as shown in Braxton S and Wells JA (1992 Biochemietry 31: 7796-7801) or that act as inhibitors, agonists, or antagonists of native peptides. as shown in Athauda SB et al. (1993 J Biochem 113: 742-746), incorporated herein by reference. It is also possible to isolate a specific target antibody, selected by functional assay, as described above, and then dissolve its crystal structure. This approach, in principle, produces a farmanúcleo on which can be based subeecuentes drug design. It is possible to deviate the protein crystallography completely by generating anti-idiotypic (anti-ide) antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, it is expected that the anti-idiotypic binding site is an analogue of the original receptor. Next, the anti-idiotypic is used to identify and isolate peptides from chemically or biologically produced peptide libraries. Afterwards, the peptide molecules act as the farmanucleus. By virtue of the present invention, a sufficient amount of polypeptide can be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the amino acid sequence of the phospholipase C homolog provided herein will provide guidance for those that they are using computer design techniques instead of or in addition to the X Ray X-ray.

XIV Identification of Other Members of the Signal Transduction Complex The homolog of inventive purified foefolipaea C is a search tool for the identification, characterization and purification of traneduction proteins of interaction or signal. Radioactivae labels are incorporated into the homologue of foefolipase C by different methods known in the art and used to capture molecules either soluble or fixed with membrane. A preferred method involves the labeling of the primary amino groups in the phospholipase C homolog with the 125 Bolton-Hunter reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133: 529). This reagent has been used to label different molecules without concomitant loss of biological activity (Hebert CA et al (1991) J Biol Chem 266: 18989; McColl S and collaborator (1993) J Immunol 150: 4550-4555). The membrane-bound molecules are incubated with the molecules of the labeled foefolipaea C homologue, washed and the unfixed molecules are removed, and the phospholipase C homolog complex is quantified. The data obtained is used using different concentrations of the phospholipase homologue. C to calculate the values for the number, affinity, and association of the homolog complex of foefolipaea C. The homologue of foefolipase C tagging is also useful as a reagent for the purification of the molecules with which the phospholipase C homologue interacts. an affinity purification mode, the phospholipase C homologue is covalently coupled to a chromatography column. The cells and their membranes are extracted, the homologue of foefolipaea C and ee paean is removed on the different column eubcomponentee free from the homologue of foefolipaea C. The molecules are fixed to the column by means of their affinity to the homologue of foefolipaea C. It recovers the complex of the foefolipaea C homolog of the column is dissociated and the recovered molecule is subjected to sequencing of N-terminus protein. This amino acid sequence is then used to identify the captured molecule or to degenerate eondee of degenerate oligonucleotide for cloning. gene from an appropriate cDNA library. In another alternative method, the antibodies are raised against the homologue of phospholipase C, specifically monoclonal antibodies. The monoclonal antibodies are identified to identify those that inhibit the binding of the labeled homologue of foefolipase C. Then, monoclonal antibodies are present in the affinity purification or the cloning of expression of associated molecules. Other soluble binding molecules are identified in a similar manner. The phospholipase C homolog labeling is incubated with extracts of other appropriate materials derived from mage cell and putative target cells. After incubation, complexes of the phospholipase C homologue (loe elee mae grandee) than the homologous molecule of foefolipaea C alone are identified by a size classification technique such as size exclusion chromatography or density gradient centrifugation. and they are purified by methods known in the art. The soluble binding protein (s) (e) is subjected to N-terminal sequencing to obtain sufficient information for the identification of the database, if the soluble protein is known, or for cloning, if the soluble protein is unknown.

XV Use and Administration of Antibodies, Inhibitors, Receptors or Antagonists of the Fospholipase C Homologue Antibodies, inhibitors, receptors or antagonists of the phospholipase C homolog (or other treatment to limit signal transduction, TST) provide different effects when administered in a therapeutic way. The TSTs will be formulated in a pharmaceutically acceptable inert non-toxic aqueous carrier medium preferably at a pH of about 5 to 8, preferably 6 to 8, although the pH may vary in accordance with the characteristics of the antibody, inhibitor, or antagonist that is being formulated and the condition that is going to be treated. Characteristics of TSTs include molecule solubility, half-life and antigenicity / immunogenicity; This and other characteristics can help to define an effective carrier. Native human proteins such as TSTs are preferred, but organic or einthetic molecules that re-emerge from drug screening in particular situations can also be equally effective. TSTs can be applied by known routes including but not limited to topical creams and gels, aerosols and aerobes of transmucosal; patches and bandages tranedérmicoe; injectable formulations, intravenous and organ washing; and orally administered orally formulated liquids and pills, particularly for re-acidizing stomachs and enzymes. The attending physician will determine the particular formulation, the exact doeie, and the route of administration and these will vary according to each specific situation. Such determinations are made by considering multiple variables such as the condition to be treated, the TST to be administered, and the pharmacokinetic profile of the particular TST. The additional factors that may be taken into account include the patient's disease status (eg, severity), age, weight, gender, diet, time and frequency of administration, drug combination, reactive responsiveness, and tolerance / response to the therapy. Long-acting TST formulations can be administered every 3 or 4 days, every week, or once every two weeks depending on the half-life and rate of removal of the particular TST. The normal dosage amounts may vary from 0.1 to 100,000 microgram, a total doe of about 1 gram, depending on the route of administration. In the literature, guidance is given as to the dosage and particularee method of application. See Patents of the United States of North America Nos. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art will employ different formulations for different TSTs. Administration to cells such as nerve cells requires application in a different manner than other cells such as vascular endothelial cells. It is contemplated that conditions or diseases that cause mast cell activity can precipitate damage that can be treated with TSTs. They can be diagnosed in a specific way, be conditioned or ill through the test described above, and they should make such a test in the cases in which they suspect of sietemic and local infections, trauma and other tissue damage, hereditary or environmental diseases associated with hypertension, carcinoma, and other physiological / pathological problems associated with abnormal signal transduction. All publications and patents mentioned in the above specification are incorporated in the preamble as a reference. The different modification and variation of the method and method of the invention will appear apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to the specific preferred modalities, it should be understood that the invention as claimed, should not be unduly limited to said modality of specificity. Actually, it is intended that the modification be modified as previously described to carry out the invention, which is obvious for those experts in the field of molecular biology or related fields, are within the scope of the following claim.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: INCYTE PHARMACEUTICALS, INC. (Ü) TITLE OF THE INVENTION: HOMOLOGO DE FOSFOLIPASA C (iii) NUMBER OF SEQUENCES: 13 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: INCYTE PHARMACEUTICALS, INC. (B) STREET: 3174 PORTER DRIVE (C) CITY: PALO ALTO (D) STATE: CA (E) COUNTRY: USA (F) POSTAL CODE: 94304 (v) LEGIBLE FORM BY COMPUTER: (A) TYPE OF MEANS: DÍ8CO of 5 1/4 (B) COMPUTER: IBM compatible with PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Releaee # 1.0, Vereion # 1.30 (vi) CURRENT REQUEST DATA: (A) ) PCT APPLICATION NUMBER: TO BE ASSIGNED (B) SUBMISSION DATE: April 10, 1996 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) SERIAL NUMBER OF APPLICATION: US 08 / 419,078 (B) SUBMISSION DATE: April 10, 1996 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: LUTHER, BARBARA J. (B) REGISTRATION NUMBER: 33954 (C) ATTORNEY / REFERENCE NUMBER: PF0030 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415 -? ? ? - 0572 INFORMATION FOR SE II 10: 1: (i) CHARACTERISTICS JT? THE SEQUENCE: (A) LENGTH: 1221 pairs of baeee (B) TYPE: acide r .-: le: co (C) TYPE OF CHAIN unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) SOURCE IMMEDIATE: (A) GENOTECA: None (B) CLON: 9118 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 ATGGCTCTCC TGGTGCTCGG TCTGGTGAGC TGTACCTTCT TTCTGGCAGT GAATGGTCTG TATTCCTCTA GTGATGATGT GATCGAATTA ACTCCATCAA ATTTCAACCG AGAAGTTATT CAGAGTGATA GTTTGTOGCT TGTAGAATTC TATGCTCCAT GGTGTGGTCA CTGTCAAAGA TT? ACACCAG AATGGAAGAA AGCAGCAACT GCATTAAAAG ATGTTGTCAA AGTTGGTGCA GTTGATGCAG ATAAGCATCA TTCCCTAGGA GGTCAGTATG GTGTTCAGGG ATTTCCTACC ATTAAGATTT TTGGATCCAA CAAAAACAGA CCAGAAGATT ACCAAGGTGG CAGAACTGGT GAAGCCATTG TAGATGCTGC GCTGAGTGCT CTGCGCCAGC TCGTGAAGGA TCGCCTCGGG GCACGGAGCG GAGGATACAG TTCTGGAAAA CAAGGCAGAA GTGATAGTTC AAGTAAGAAG GATGTGATTG AGCTGACAGA CGACAGCTTT GATAAGAATG TTCTGGACAG TGAAGATGTT TGGATGGTTG AGTTCTATGC TCCTTGGTGT GGACACTGCA AAAACCTAGA GCCAGAGTGG GCTGCCGCAG CTTCAGAAGT AAAAGAGCAG ACGAAAGGAA AAGTGAAACT GGCAGCTGTG GATGCTACAG TCAATCAGGT TCTGGCCTCC CGATACGGGA TTAGAGGATT TCCTACAATC AAGATATTTC AGAAAGGCGA GTCTCCTGTG GATTATGACG GTGGGCGGAC AAGATCCGAC ATCGTGTCCC GGGCCCTTGA TTTGTTTTCT GATAACGCCC CACCTCCTGA GCTGCTTGAG ATTATCAACG AGGACATTGC CAAGAGGACG TGTGAGGAGC ACCAGCTCTG TGTTGTGGCT GTCCTCCCCC ATATCCTTGA TACTGGAGCT GCAGGCAGAA ATTCTTATCT GGAAGTTCTT CAGACAAATA CAAAAAGAAA ATGTGGGGGT GGCTGTGGAC AGAAGCTGGA GCCCAGTCTG AACTTGAGAC CGCGTTGGGG ATTGGAGOGT TTGGGTACCC GCCATCAATG CACGCAAGAT GAAATTTGCT CTGCTAAAAG GCTCCTTCAG TGAGC AGGC ATCAACGAGT TTCTCAGGGA GCTCTCTTTT GGGCGTGGCT CCACGGCACC TGTAGGAGG: GGGGCTTTCC CTACCATCGT TGAGAGAGAG CCTTGTTACG GCAGGGATGG CGAGCTTCCC 126 GTGGAGGATG ACATTGACCT CAGTAATGTG GAGCTTTATG ACTTAGGGAA AGATGAGTTG 1J2C TA 1322 INFORMATION FOR S Q ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 566 amino acids (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: prcteína (vii) IMMEDIATE SOURCE: (A) GENOTECA: None (B) CLON: 9118 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Mee? Leu Leu Val Leu Gly Leu Val Ser Cys Thr Phe Phe Leu Wing 1 5 10 15 Val Asn Gly Leu Tyr Ser Ser Asp Asp Val lie Glu Leu Thr Pro 20 25 30 Ser Asn Phe Asn Arg Glu Val lie Gln Ser Asp Ser Leu Trp Leu Val 35 40 45 Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Gln Arg Leu Thr Pro Glu 50 55 60 Trp Lys Lys Wing Wing Thr Wing Leu Lys Asp Val Val Lys Val Gly Wing 65 70 75 SC Val Asp Ala Asp Lys His His Ser Leu Gly Gly Gln Tyr Gly Val Glr. 85 9C S5 Gly Phe Pro Thr He Lys He Phe Gly Ser Asn Lys Asn Arg Pro Gl IDO 5 :: c Asp Tyr Glr. Gly Gly Arg Thr Gly Glu Wing He Val Asp Ala Wing Leu 115 120 125 Ser Ala Leu Arg Gin Leu Val Lys Asp Arg Leu Gly Gly Arg Ser Gly 130 135 140 Gly Tyr Ser Ser Gly Lys Gln Gly Arg Ser Asp Ser Set Ser Lys Lvs 145 150 155 160 Asp Val He Glu Leu Thr Asp Asp Ser Phe Asp Lys Asn Val Leu Asp 165 170 175 Ser Glu Asp Val Trp Met Val Glu Phe Tyr Ala Pro Trp Cys Gly H¿s 180 185 190 Cys Lys Asn Leu Glu Pro Glu Trp Wing Wing Wing Wing Ser Glu Val Lys 195 200 205 Glu Gln Thr Lys Gly Lys Val Lys Leu Ala Wing Val Asp Wing Thr Val 210 215 220 Asn Gln Val Leu Wing Being Arg Tyr Gly He Arg Gly Phe Prc T .-. R He 225 23C 235 240 Lys He Phe Gln Lys Gly Glu Ser Pro Val Asp Tyr Asp Gly Gly Arg 245 250 255 Thr Arg Ser Asp He Val Ser Arg Ala Leu Asp Leu Phe Ser Asp As 260 265 270 Wing Pro Pro Pro Glu Leu Glu He He Asn Glu Asp He Wing Lys 275 280 285 Arg Thr Cys Glu Glu His Gln Leu Cys Val Val Wing Val Leu Pro His 290 295 300 He Leu Asp Thr Gly Ala Wing Gly Arg Asn Ser Tyr Leu Glu Val Leu 305 310 315 320 Leu Lys Leu? La? Sp Lys Tyr Lys Lys Lys Mßt Trp Gly Trp Leu Trp 325 330 335 Thr Glu? The Gly? The Gln Ser Glu Leu Glu Thr Wing Leu Giy He Gly 340 345 350 Gly Phß Gly Tyr Pro Wing Ala Wing Wing He Asn Wing Arg Lys Mee Lys 355 360 365 Phe Wing Leu Leu Lys Gly Ser Phe Ser Glu Gln Gly He Asn Glu Phe 370 375 380 Leu Arg Glu Leu Ser Phe Gly Arg Gly Be Thr Ala Pro Val Gly Gly 385 390 395 40C Gly Wing Phe Pro Thr He Val Glu Arg Glu Pro Cys Tyr Gly Arg Asp 405 410 415 Gly Glu Leu Pro Val Glu Asp Asp He Asp Leu As Asn Val Glu Leu 420 425 430 • yr Asp Leu Gly Lys Asp Glu Leu Xaa Trp Glx -eu Va: 435 440 445 Trp Gln Gln Glx Gln Asn Gly Xaa Trp Val Ala Leu Val Val Leu? Rc 450 455 460 Val Val Val Val Trp Gln Val Val Val Val Asn Asx Val Thr Val Ala 465 470 475 480 Leu Pro Asn Thr Glu Gln Asn Cys Lys Phe Trp H? S K? S Phe Gly H? S 465 490 495 His Asp Arg Asx His Leu Wing Ser Glu Arg Trp Arg He Thr Glu Arg 500 505 510 Asn Glu Trp Tyr Arg Lys Phe Ala Ala Pro His Pro Pro His Leu He 515 520 525 Pro Ala Ser Glu Cys Ala Met He Asn Ala Cys He Asp Ser Glu Glr. 530 535 540 Pro His He Leu His Wing Trp Lys He Asn Ser Pro His He Le-- Hi s 545 550 555 5 6 2 Wing Trp Lys He Asn Ser 565 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 898 base pairs (B) TI PO: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) ) IMMEDIATE SOURCE: (A) GENOTECA: Human Mast Cell (B) CLON: 9118 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TTCACCCGAG AAGTTATTC? GAGTGATAGT TTGTGGCTTG TAGAATTCTA TGCTCCATGG 62 TGTGGTCACT GTCAAAGATT AACACCAGAA TGGAAGAAAG CAGCAACTGC AT AAAAGAT 1: GTTGTCAAAG TTGGTGCAGT TGATGCAGAT AAGCATCATT CCCTAGGAGG TCAGTATGGT 1: 2 GTTCAGGGAT TTCCTACCAT TAAGATTrTT GGATCCAACA AAAACAGACC AGAAGATTAC 24: CAAGGTGGCA GAACTGGTGA AGCCATTGTA GATGCTGCGC TGAGTGCTCT GCGCCAGCTC 2- 22 GTGAAGGATC GCCTCGGGGG ACGGAGCGGA GGATACAGTT CTGGAAAACA AGGCAGAAGT 362 GATAGTTCAA GTAAGAAGGA TGTGATTGAG CTGACAGACG ACAGCTTTGA TAAGAATGTT 422 CTGGACAGTG AAGATGTTTG GATGGTTGAG TTCTATGCTC CTTGGTGTGG ACACTGCAAA 482 AACCTAGAGC CAGAGTGGGC TGCCGCAGCT TCAGAAGTAA AAGAGCAGAC GAAAGGAAAA: 42 GTGAAACTGG CAGCTGTGGA TGCTACAGTC AATCAGGTTC TGGCCTCCCG ATACGGGATT 622 AGAGGATTTC CTACAATCAA GATATTTCAG AAAGGCGAGT CTCCTGTGGA TTATGACGG? £ 62 GGGCGGACAA GATCCGACAT CGTGTCCCGG GCCCTTGATT TGTTTTCTGA TAACGCCCCA CCTCCTGAGC TGCTTGAGAT TATCAACGAG GACATTGCCA AGAGGACGTG TGA GA3CAC "82 CAGCTCTGTG TTGTGGCTGT CCTCCCCCAT ATCCTTGATA CTGGAGCTGC AGGCAGAAAT 842 TCTTATCTGG AAGTTCTTCT GAAGTTC-GCA GACAAAATCC AAAAAAAAAA AA? A? AAA £ 98 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30C pairs of baees (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Lymphoblast T / B Hybrid (B) CLON: 043866 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO:: CTGGAGCCCA GTTGAACTTG AGACCGCTTG GGGATTGGAG GGTTTGGGTA CCCCGCCATG 62 GCCGCCATCA ATGCACGCAA GATGAAATTT GCTCTGCTAA AAGGCTCCTT CAGTGAGCAA 120 GGCATCAACG AGTTTTCAGG GAGCTCTCTT TTGGGCGTGG CTCCACGGCA CCTGTAGGAG ISO GCGGGGCTTT CCCTACCATC GTTG? G? GAG AGCCTTGTAC GGCAGGGATG GCGAGCTTCC 240 CGTGGAGGAT GAC? TTG? CC TC? GTG? TGT GG? GCTTG? T G? CTT? GGG? AAGATGAAGT 300 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17C pairs of baees (B) TYPE: nucleic acid (C) TYPE OF CHAIN *, unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE; CDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Horny Stromal (B) CLON: 046611 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5 C? TC ACG? G TTTT? GGG? G CTCTCTTTTGGGCGTGGCTC C? CGGC? CCT GTAGGAGGCG GGGCTTTCCC TACC? TCGTT G? G? GAGAGC CTTGTTACGG C? GGG? TGGC G? GCTTCCCG TGG? GG? TG? C? TTG? CCTC? GT? TGTGGA GCTTTATG? C TT? GGG ??? G (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 170 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Fibroblast (B) CLON: 054216 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CATCAACGAG TTTTAGGGAG CTCTCTTTTG GGCGTGGCTC CACGGCACCT GTAGGAGGCG GGGCTTTCCC TACC? TCGTT GAGAGAGAGC CTTGTTACGG CAGGGATGGC GAGCTTCCCG TGGAGG? TGA CATTGACCTC AGTATGTGGA GCTTTATGAC TTAGGGAAAG < ) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 180 pairs of cells (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Human Fetal Endothelium - Stressed (B) CLON: 067172 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: AGAC ??? T? C AAAAAGAAAA TGTGGGGGTG GCTGTGGAC? G ?? GCTGG? G CCC? GTCTGA 60 ACTTGAG? CC GCTTGGGG? T TGG? GGGTTT GGGT? CCCCG CC? TGGCCGC C? TCAATGCA 12 C CGCAAG? TG? TTTGCTCT GCT? AA? GGC TCCTTC? GTG AGC ?? GGC? T C ?? CG? GTTT 180 i INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 191 pairs of baees (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Human Fetal Endothelium - Stressed (B) CLON: 067990 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8: GGGTACCCCG CCATGGCCCC ATCA? TGC? C GCAAGATG ?? ? TTTCTCTGC T ?? ? GGCTC 60 CTTC? GTGAG CAAGGCATC? ? CGAGTTTTT CAGGGAGCTC TCTTTTGGGC GTGGCTCCAC 122 GGCACCTGTA GGAGGCGGGG CTTTCCCTAC CATCGTTGAG AG? G? GCCTT GTTCGGAGGG 182 ATGGCGAGCT T 191 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 172 pairs of baeee (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Human Fetal Endothelium - Stressed (B) CLON: 082161 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 *.

CATGGCTCTC CTGGTGCTCG GCCTGGTGAG CTGT? CCTCT TTTGGCAGTG AATGGCTGTT 60 TCCTCTAGTG ATGATGTG? T CGATTTAACT CCTC ??? TTT CACCGAGAAG TTATTCAGAG 122 TGATAGTTTT GGCTTGTAG? ATTTATGCCC ATGGTGTGGT CACTGTCA ?? ?? 72 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 195 pairs of baeee (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Stimulated THP-l cells (B) CLON: 154746 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 1C GCGGTGGGGA CTGC? CGT? G CCCGGCGCTC GG? TGGCTCT CCTGGTGCTC GGTCTGGTGA sCTGTACCTT CTTTCTGGC? TG ?? TGGTCT GT? TTCCTCT AGTGATGATG TGATCGAAAT AACTCCATC? A? TTT? CCG? G? GTT? TT C? G? GTG? T? GTTTGTGGCT TGTAGAATTC 1 TATGCTCC? T GGTGT 19í (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 250 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: Unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Stimulated THP-l cells (B) CLON: 157482 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: GCGGCACGTG CAAGGGCTG? AGCGGCGGCG GCGGTGGGGA CTGCACGTAG CCCGGCGCTC GGCATGGCTC TCCTGGTGCT CGGTCTGGTG AGCTGTACCT TCTTTTGGCA GTGAATGGTC TGTATTCCTC TAGTGATGAT GTGATCGATT AACTCCATCA AATTTCAACC GAGAAGTTAT TCAGAGTGAT AGTTTGTGGC TTGTAGAATT CTATGCTCCC ATGGGTGTGG TCACTGTCAA AATTAACACC (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 206 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Inflamed Adenoid (B) CLON: 159363 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: G? GGGTTTGG GT? CCCGCC? TGGCCGCC? T C? ATGC? CGC ?? G? TG ??? T TTGCTCTGCT 60 AA ?? GGCTCC TTC? GTG? GC A? GGC? TC ?? CG? GTTTCTC? GGG? GCTCT CTTTTGGGCG 120 TGGCTCC? CG GC? CCTGT? G G? GGCGGGGC TTTCCCT? CC? TCGTTG? G? G? G? GCCTTG 1BC GG? CGGC? GG G? TGGCGAGC TTCCGT 06 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 212 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: Stomach (B) CLON: 219512 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13 G? GG? GCACC AGCTCTGTTT GTGGCTGTGC TGCCCCATTC CTTGAT? CTG G? GCTGCAGG CAG ??? TTCT T? TCTGG ?? G TTCTTCTGAA GTTGGCAGC? AATACAAAAA G? AAATTGGG GGTGGCTGTG GAC? G ?? GCT GGAGCCCAGT CTGAACTTGA G? CCGCGTTG GGGATTGGAG GGTTTGGGTA CCCCGCC? TG GCCGCCATCA AT

Claims (5)

1. A purified polynucleotide encoding the phospholipase C homologue polypeptide (PLCH) with an amino acid sequence that is shown in SEQ ID NO:

2. 2. The polynucleotide of Claim 2 wherein the nucleotide sequence connects of SEQ ID NO: 1, or its complement.

3. A diagnostic test for a tumor, condition or disease associated with an excess phospholipase C homologue in a biological sample comprising the steps of: a) combining the biological sample with the polynucleotide of Claim 1, or a fragment of the same, under conditions suitable for the formation of the hybridization complex; and b) detecting the hybridization complex, whereby the detection of the complex is the diagnosis of the tumor, condition or disease. . An expreration vector comprising the polynucleotide of Claim 1. 5. A huéeped cell trane-formed with the expreration vector of Claim

4. 6. A method for producing a polypeptide comprising the amino acid sequence shown in SEQ ID. NO: 2, said method comprising the steps of: a) culturing the host cell of Claim 5 under conditions suitable for expression of the polypeptide; and b) recovering the polypeptide from the culture of the host cell. 7. A purified polypeptide, also known as a phospholipase C homolog, comprising the amino acid sequence of SEQ ID NO: 2. 8. An anti-sense molecule comprising the nucleotide sequence complementary to a portion of the polynucleotide of claim 1. 9. A pharmaceutical composition comprising the anti-sense molecule of claim 8 and a pharmaceutically acceptable excipient. 10. A method for the treatment of a subject with a tumor, condition or disease aeociated with an excess phospholipase C homolog comprising the administration of an effective amount of the pharmaceutical composition of Claim 9 to the subject. 11. An antibody specific for the purified polypeptide of Claim 7. 12. A diagnostic test for a tumor, condition or disease wherein the excess homologue of phospholipase C ee cell characteristic resiates to the hydroxyurea in a biological sample comprising the paeoe of: a) combining the biological sample with the antibody of Claim 11, under appropriate conditions so that the antibody binds the homologue of foefolipase C and forms an antibody complex: phospholipase C homologue; and b) detecting the antibody complex: phospholipase C homolog, wherein the preeminence of the complex is in the diagnosis of the tumor, condition or disease. 13. A specific inhibitor for the purified polypeptide of Claim 7. 14. A pharmaceutical composition comprising the inhibitor of Claim 13 and a pharmaceutically acceptable excipient. 1

5. A method for the treatment of a subject with a tumor, condition or disease associated with an excess phospholipase C homolog comprising the administration of an effective amount of a pharmaceutical composition of the Claim 14 to the subject.