WO2002097073A2

WO2002097073A2 - Regulation of human phospholipase c delta-like enzyme

Info

Publication number: WO2002097073A2
Application number: PCT/EP2002/005814
Authority: WO
Inventors: Yonghong Xiao
Original assignee: Bayer Aktiengesellschaft
Priority date: 2001-05-29
Filing date: 2002-05-28
Publication date: 2002-12-05
Also published as: WO2002097073A3; AU2002321039A1

Abstract

Reagents that regulate human phospholipase C delta-like enzyme and reagents which bind to human phospholipase C delta-like enzyme gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, Central Nervous System (CNS Disorders), cancer, COPD, diabetes, and asthma.

Description

REGULATION OF HUMAN PHOSPHOLIPASE C DELTA-LIKE ENZYME

This application incorporates by reference co-pending provisional application Serial No. 60/293,517 filed May 29, 2001.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the regulation of human phospholipase C delta-like enzyme.

BACKGROUND OF THE INVENTION

Phospholipase C (PLC) is an enzyme that hydrolyzes glycerophospholipid and sphingophospholipid. See U.S. Patent 6,060,302. PLC is present in the spleen, the tunica mucosa interstini tenuis, and the placenta, of mammals and plays an important role in mammalian metabolism. For example, phosphatidylinositol-specific

I I phospholipase C (PI-PLC) hydrolyzes phosphatidylinositol 4,5-diphosphate to generate 1,2-diacylglycerol and inositol 1,4,5-triphosphate (Rhee et al, Science 244, 546-50, 1989). Various PLC isozymes have been described (e.g., Bennett et al, Nature 334, 268-70, 1988; Emori et al, J. Biol. Chem. 264, 21885-90, 1989; Katan et al, Cell 54, 171-77, 1988); Ohta et al, FEBS Lett. 242, 31-35, 1988; Stahl et al, Nature 332, 269-72, 1988; Suh et al, Cell 54, 161-69, 1988; and Kritz et al, CIBA Found. Symp. 150, 112-27, 1990). Because of the importance of PLC in mammalian metabolism, there is a need in the art to identify additional PLC-like enzymes, which can be regulated to provide therapeutic effects.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods of regulating a human phospholipase C delta-like enzyme. This and other objects of the invention are provided by one or more of the embodiments described below. One embodiment of the invention is a phospholipase C delta-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 72% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.

Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a phospholipase C delta-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

Binding between the test compound and the phospholipase C delta-like enzyme polypeptide is detected. A test compound which binds to the phospholipase C delta- like enzyme polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the activity of the phospholipase C delta-like enzyme.

Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a polynucleotide encoding a phospholipase C delta-like enzyme polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.

Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the phospholipase C delta-like enzyme through interacting with the phospholipase C delta-like enzyme mRNA.

Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a phospholipase C delta-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 72% identical to the amino acid sequence shown in SEQ LD NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.

A phospholipase C delta-like enzyme activity of the polypeptide is detected. A test compound which increases phospholipase C delta-like enzyme activity of the polypeptide relative to phospholipase C delta-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation. A test compound which decreases phospholipase C delta-like enzyme activity of the polypeptide relative to phospholipase C delta-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a phospholipase C delta-like enzyme product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.

Binding of the test compound to the phospholipase C delta-like enzyme product is detected. A test compound which binds to the phospholipase C delta-like enzyme product is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a phospholipase C delta-like enzyme polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ TD NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.

Phospholipase C delta-like enzyme activity in the cell is thereby decreased.

The invention thus provides a human phospholipase C delta-like enzyme that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site. Human phospholipase C delta-like enzyme and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the DNA-sequence encoding a phospholipase C delta-like enzyme

Polypeptide (SEQ ID NO: 1). Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.1 (SEQ ID NO: 2).

Fig. 3 shows the amino acid sequence of the protein identified by swissnew|P10688|PIDl_RAT (SEQ LD NO: 3).

Fig. 4 shows the DNA-sequence encoding a phospholipase C delta-like enzyme Polypeptide (SEQ ID NO: 4).

Fig. 5 shows the BLASTP - alignment of 556_protein (SEQ ID NO: 2) against swissnew|P10688|PIDl_RAT (SEQ ID NO: 3).

Fig. 6 shows the BLOCKS search results.

Fig. 7 shows the HMMPFAM - alignment of 556_protein (SEQ ID NO: 2) against pfam|hmm|PI-PLC-X.

Fig. 8 shows theHMMPFAM - alignment of 556_ρrotein (SEQ ID NO: 2) against pfam|hmm|PI-PLC-Y.

Fig. 9 shows the HMMPFAM - alignment of 556_protein (SEQ ID NO: 2) against ρfam|hmm|C

Fig. 10 shows the HMMPFAM - alignment of 556_protein (SEQ ID NO: 2) against ρfam|hmm|PH.

Fig. 11 shows the HMMPFAM - alignment of 556 protein (SEQ ID NO: 2) against pfam|hmm|efhand. Fig. 12 shows the BLASTP - alignment of 556_protein (SEQ ID NO: 2) against pdb|lDJG|lDJG-B.

Fig. 13 shows the Expression of human phospholipase C delta-like enzyme in various tissues.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide from the group consisting of:

a) a polynucleotide encoding a phospholipase C delta-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 72% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2;

b) a polynucleotide comprising the sequence of SEQ ID NO: 1 ;

c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a phospholipase C delta-like enzyme polypeptide;

d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a phospholipase C delta-like enzyme polypeptide; and

e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a phospholipase C delta-like enzyme polypeptide. Furthermore, it has been discovered by the present applicant that a novel phospholipase C delta-like enzyme, particularly a human phospholipase C delta-like enzyme, can be used in therapeutic methods to treat cancer, COPD, diabetes, and asthma. Human phospholipase C delta-like enzyme comprises the amino acid sequence shown in SEQ ID NO: 2. A coding sequence for human phospholipase C delta-like enzyme is shown in SEQ ID NO: 1. This sequence is contained within the longer sequence shown in SEQ ID NO: 4, which is located on chromosome 2, at 2q35. Related ESTs (BF310672); (BE313093); (AI366170); (BF206866) (BE046870); (AU152354); (AU152354); (AI914254); (W22094); (BE671129) (AA128575); (AA359453); (AA359449); (AL589403); (AW594023); (BF747275)

(AW176574); (AW166377); (AA777524) are expressed in brain neuroblastoma, NT2 neuronal precursor cells after 2-weeks retinoic acid (RA) induction of terato- carcinoma cells, brain oligodendroglioma, tongue squamous cell carcinoma, lung carcinoid, lung carcinoma, adult retina, 19-week fetal lung, adult spinal chord, cervix, pooled germ cell tumors, adult breast, adult colon, and 20-week fetal liver and spleen.

Human phospholipase C delta-like enzyme is 47% identical over 752 amino acids to swissnew|P10688|PIDl_RAT (SEQ ID NO: 3) (FIG. 1) and 49% identical over 606 amino acids to pdb|lDJG|lDJG-B (FIG. 8). the protein has clear homologies to a variety of phospholipase C delta enzymes, ranging from 47%) to 71% identity over its entire length. It also has prosite profiles for PTPLC_X_ OMAIN and PΓPLC_Y_DOMALN, as well as EFJHAND calcium binding region and C2 domain. HMMpFAM hits for all above domains are well beyond the threshold level. Alignments are shown in FIGS. 1 and 3-8.

Human phospholipase C delta-like enzyme of the invention is expected to be useful for the same purposes as previously identified human phospholipase C delta enzymes. Human phospholipase C delta-like enzyme is believed to be useful in therapeutic methods to treat disorders such as cancer, COPD, diabetes, and asthma. Human phospholipase C delta-like enzyme also can be used to screen for human phospholipase C delta-like enzyme activators and inhibitors.

Polypeptides

Human phospholipase C delta-like enzyme polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, or 762 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 2 or a biologically active variant thereof, as defined below. A human phospholipase C delta-like enzyme polypeptide of the invention therefore can be a portion of a human phospholipase C delta-like enzyme protein, a full-length human phospholipase C delta-like enzyme protein, or a fusion protein comprising all or a portion of a human phospholipase C delta-like enzyme protein.

Biologically Active Variants

Human phospholipase C delta-like enzyme polypeptide variants which are biologically active, e.g., retain enzymatic activity, also are human phospholipase C delta-like enzyme polypeptides. Preferably, naturally or non-naturally occurring human phospholipase C delta-like enzyme polypeptide variants have amino acid sequences which are at least about 72, preferably about 75, 80, 75, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof. Percent identity between a putative human phospholipase C delta-like enzyme polypeptide variant and an amino acid sequence of SEQ ID NO: 2 is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA <°P:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff & Henikoff, 1992. Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson & Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant. The FASTA algorithm is described by Pearson

& Lipman, Proc. Nat'l Acad. Sci. USA 55:2444(1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman- Wunsch- Sellers algorithm (Needleman & Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM

J. Appl. Math.26:7 l (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRLX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63

(1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default. Nariations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a human phospholipase C delta-like enzyme polypeptide can be found using computer programs well known in the art, such as DΝASTAR software.

The invention additionally, encompasses phospholipase C delta-like enzyme polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, N8 protease, ΝaBH , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The phospholipase C delta-like enzyme polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

The invention also provides chemically modified derivatives of phospholipase C delta-like enzyme polypeptides that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The polypeptides can be modified at random or predetermined positions within the molecule and can include one, two, three, or more attached chemical moieties.

Whether an amino acid change or a polypeptide modification results in a biologically active phospholipase C delta-like enzyme polypeptide can readily be determined by assaying for phospholipase c activity, as described for example, in Mullinax et al, J. Biomol Screen. 4, 151-55, 1999, or Litosch, Biochemistry 39, 7736-43, 2000).

Fusion Proteins

Fusion proteins are useful for generating antibodies against human phospholipase C delta-like enzyme polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a human phospholipase C delta-like enzyme polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A human phospholipase C delta-like enzyme polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, or 762 contiguous amino acids of SEQ ID NO: 2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length human phospholipase C delta-like enzyme protein.

The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horse- radish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSN) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the human phospholipase C delta-like enzyme polypeptide-encoding sequence and the heterologous protein sequence, so that the human phospholipase C delta-like enzyme polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DΝA methods can be used to prepare fusion proteins, for example, by making a DΝA construct which comprises coding sequences selected from SEQ ID NO: 1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown,

MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS). Identification of Species Homologs

Species homologs of human phospholipase C delta-like enzyme polypeptide can be obtained using human phospholipase C delta-like enzyme polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of human phospholipase C delta-like enzyme polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides

A human phospholipase C delta-like enzyme polynucleotide can be single- or double- stranded and comprises a coding sequence or the complement of a coding sequence for a human phospholipase C delta-like enzyme polypeptide. A coding sequence for human phospholipase C delta-like enzyme is shown in SEQ ID NO: 1.

Degenerate nucleotide sequences encoding human phospholipase C delta-like enzyme polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 1 or its complement also are human phospholipase C delta-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.

Complementary DNA (cDNA) molecules, species homologs, and variants of human phospholipase C delta-like enzyme polynucleotides that encode biologically active human phospholipase C delta-like enzyme polypeptides also are human phospholipase C delta-like enzyme polynucleotides. Polynucleotide fragments com- prising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ TD NO:

1 or its complement also are human phospholipase C delta-like enzyme poly- nucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.

Identification of Polynucleotide Variants and Homologs

Variants and homologs of the human phospholipase C delta-like enzyme polynucleotides described above also are human phospholipase C delta-like enzyme polynucleotides. Typically, homologous human phospholipase C delta-like enzyme polynucleotide sequences can be identified by hybridization of candidate poly- nucleotides to known human phospholipase C delta-like enzyme polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions-2X SSC (0.3 M NaCI, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50°C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each— homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15%) basepair mismatches.

Species homologs of the human phospholipase C delta-like enzyme polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of human phospholipase C delta-like enzyme polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_m of a double-stranded DNA decreases by 1-1.5°C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973). Variants of human phospholipase C delta-like enzyme polynucleotides or human phospholipase

C delta-like enzyme polynucleotides of other species can therefore be identified by hybridizing a putative homologous human phospholipase C delta-like enzyme polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising poly- nucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to human phospholipase C delta-like enzyme polynucleotides or their complements following stringent hybridization and/or wash conditions also are human phospholipase C delta-like enzyme polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20°C below the calculated T_m of the hybrid under study. The T_m of a hybrid between a human phospholipase C delta-like enzyme polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_m = 81.5°C - 16.6(log₁₀[Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 600/1), where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65°C, or 50%> formamide, 4X SSC at 42°C, or 0.5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.

Preparation of Polynucleotides

A human phospholipase C delta-like enzyme polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.

Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated human phospholipase C delta-like enzyme polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise human phospholipase C delta-like enzyme nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.

Human phospholipase C delta-like enzyme cDNA molecules can be made with standard molecular biology techniques, using human phospholipase C delta-like enzyme mRNA as a template. Human phospholipase C delta-like enzyme cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize human phospholipase C delta-like enzyme polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a human phospholipase C delta-like enzyme polypeptide having, for example, an amino acid sequence shown in SEQ ID NO: 2 or a biologically active variant thereof.

Extending Polynucleotides

Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFΓNDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.

Obtaining Polypeptides

Human phospholipase C delta-like enzyme polypeptides can be obtained, for example, by purification from human cells, by expression of human phospholipase C delta-like enzyme polynucleotides, or by direct chemical synthesis.

Protein Purification

Human phospholipase C delta-like enzyme polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with human phospholipase C delta-like enzyme expression constructs. A purified human phospholipase C delta-like enzyme polypeptide is separated from other compounds that normally associate with the human phospholipase C delta-like enzyme polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified human phospholipase C delta-like enzyme polypeptides is at least 80%) pure; preferably, the preparations are 90%>, 95%>, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS- polyacrylamide gel electrophoresis.

Expression of Polynucleotides

To express a human phospholipase C delta-like enzyme polynucleotide, the poly- nucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding human phospholipase C delta-like enzyme polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding a human phospholipase C delta-like enzyme polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions ~ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the

BLUESCRTPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a human phospholipase C delta-like enzyme polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the human phospholipase C delta-like enzyme polypeptide. For example, when a large quantity of a human phospholipase C delta-like enzyme polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRTPT (Stratagene). In a BLUESCRTPT vector, a sequence encoding the human phospholipase C delta-like enzyme polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pIN vectors (Van

Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol. 153, 516-544, 1987.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding human phospholipase C delta-like enzyme polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of

CaMV can be used alone or in combination with the omega leader sequence from

TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express a human phospholipase C delta-like enzyme polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding human phospholipase C delta-like enzyme polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of human phospholipase C delta-like enzyme polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which human phospholipase C delta-like enzyme polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be used to express human phospholipase C delta-like enzyme polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding human phospholipase C delta-like enzyme polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a human phospholipase C delta-like enzyme polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding human phospholipase C delta-like enzyme polypeptides. Such signals include the ATG initiation codon and adjacent sequences, hi cases where sequences encoding a human phospholipase C delta-like enzyme polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl Cell Differ. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed human phospholipase C delta-like enzyme polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phos- phorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express human phospholipase C deltalike enzyme polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced human phospholipase C delta-like enzyme sequences. Resistant clones of stably trans- formed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.

Any number of selection systems can be used to recover transformed cell lines.

These include, but are not limited to, the herpes simplex virus thymidine kinase

(Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tk^~ or apr cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine

(Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).

Detecting Expression

Although the presence of marker gene expression suggests that the human phospholipase C delta-like enzyme polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a human phospholipase C delta-like enzyme polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a human phospholipase C delta-like enzyme polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a human phospholipase C delta-like enzyme polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the human phospholipase C delta-like enzyme polynucleotide.

Alternatively, host cells which contain a human phospholipase C delta-like enzyme polynucleotide and which express a human phospholipase C delta-like enzyme polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a human phospholipase C delta-like enzyme polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a human phospholipase C delta- like enzyme polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a human phospholipase C deltalike enzyme polypeptide to detect transformants that contain a human phospholipase C delta-like enzyme polynucleotide.

A variety of protocols for detecting and measuring the expression of a human phospholipase C delta-like enzyme polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immuno- assay using monoclonal antibodies reactive to two non-interfering epitopes on a human phospholipase C delta-like enzyme polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding human phospholipase C delta-like enzyme polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a human phospholipase C delta-like enzyme polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia

Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding a human phospholipase C delta-like enzyme polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode human phospholipase C delta-like enzyme polypeptides can be designed to contain signal sequences which direct secretion of soluble human phospholipase C delta-like enzyme polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound human phospholipase C delta-like enzyme polypeptide.

As discussed above, other constructions can be used to join a sequence encoding a human phospholipase C delta-like enzyme polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system

(Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the human phospholipase C delta-like enzyme polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a human phospholipase C delta-like enzyme polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by BVIAC (immobilized metal ion affinity chromatography, as described in Porath et al, Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the human phospholipase C delta-like enzyme polypeptide from the fusion protein.

Vectors that contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.

Chemical Synthesis

Sequences encoding a human phospholipase C delta-like enzyme polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a human phospholipase C delta-like enzyme polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of human phospholipase C delta-like enzyme polypeptides can be separately synthesized and combined using chemical methods to produce a full- length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND

MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic human phospholipase C delta-like enzyme polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the human phospholipase C delta-like enzyme polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce human phospholipase C delta-like enzyme polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter human phospholipase C delta-like enzyme polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of a human phospholipase C delta-like enzyme polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of a human phospholipase C delta-like enzyme polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a human phospholipase C delta-like enzyme polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immuno- histochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immuno- radiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.

Typically, an antibody which specifically binds to a human phospholipase C delta- like enzyme polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immuno- chemical assay. Preferably, antibodies which specifically bind to human phospholipase C delta-like enzyme polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a human phospholipase C delta-like enzyme polypeptide from solution.

Human phospholipase C delta-like enzyme polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a human phospholipase C delta-like enzyme polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyro globulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitro- phenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and

Corynebacterium parvum are especially useful.

Monoclonal antibodies that specifically bind to a human phospholipase C delta-like enzyme polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984).

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608,

1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies that specifically bind to a human phospholipase C delta- like enzyme polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to human phospholipase C delta-like enzyme polypeptides.

Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Ewr. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in

Mallender & Voss, 1994, J Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DΝA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol. Meth. 165, 81-91).

Antibodies which specifically bind to human phospholipase C delta-like enzyme polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are mulfivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a human phospholipase C delta-like enzyme polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of human phospholipase C delta-like enzyme gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol.

Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.

Modifications of human phospholipase C delta-like enzyme gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the human phospholipase C delta-like enzyme gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in

Huber & Carr, MOLECULAR AND iMMUNOLOGic APPROACHES, Futura Publishing Co.,

Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a human phospholipase C delta-like enzyme polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a human phospholipase C delta-like enzyme polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent human phospholipase C delta-like enzyme nucleotides, can provide sufficient targeting specificity for human phospholipase C delta-like enzyme mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular human phospholipase C delta-like enzyme polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a human phospholipase C delta-like enzyme polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozvmes

Ribozymes are RNA molecules with catalytic activity. See, e.g. , Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a human phospholipase C delta-like enzyme polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the human phospholipase C delta-like enzyme polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within a human phospholipase C delta-like enzyme RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate human phospholipase C delta-like enzyme RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target. Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease human phospholipase C delta-like enzyme expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whose products interact with human phospholipase C delta-like enzyme. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, cancer, COPD, diabetes, and asthma. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human phospholipase C delta-like enzyme gene or gene product may itself be tested for differential expression. The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science -257, 967-71, 1992; U.S. Patent 5,262,311).

The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human phospholipase C delta-like enzyme. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human phospholipase C delta-like enzyme. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human phospholipase C delta-like enzyme gene or gene product are up-regulated or down-regulated.

Screening Methods

The invention provides assays for screening test compounds that bind to or modulate the activity of a human phospholipase C delta-like enzyme polypeptide or a human phospholipase C delta-like enzyme polynucleotide. A test compound preferably binds to a human phospholipase C delta- like enzyme polypeptide or polynucleotide.

More preferably, a test compound decreases or increases enzymatic activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

T st Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced re- combinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997. Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J.

Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin,

Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to human phospholipase C delta-like enzyme polypeptides or polynucleotides or to affect human phospholipase C delta-like enzyme activity or human phospholipase C delta-like enzyme gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the micro titer plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent

5,976,813. h this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding Assays

For binding assays, the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the human phospholipase C delta-like enzyme polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the human phospholipase C delta-like enzyme polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the human phospholipase C delta-like enzyme polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a human phospholipase C delta-like enzyme polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a human phospholipase C delta-like enzyme polypeptide. A microphysiometer

(e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a human phospholipase C delta-like enzyme poly- peptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a human phospholipase C delta-like enzyme polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705,

1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. fri yet another aspect of the invention, a human phospholipase C delta-like enzyme polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent

W0 94/10300), to identify other proteins which bind to or interact with the human phospholipase C delta-like enzyme polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a human phospholipase C delta-like enzyme polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the human phospholipase C delta-like enzyme polypeptide.

It may be desirable to immobilize either the human phospholipase C delta-like enzyme polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the human phospholipase C delta-like enzyme polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a human phospholipase C delta-like enzyme polypeptide (or poly- nucleotide) can be accomplished in any vessel suitable for containing the reactants.

Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the human phospholipase C delta-like enzyme polypeptide is a fusion protein comprising a domain that allows the human phospholipase C delta-like enzyme polypeptide to be bound to a solid support. For example, glutathione-S- transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma

Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed human phospholipase C delta-like enzyme polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a human phospholipase C delta-like enzyme polypeptide (or polynucleotide) or a test com- pound can be immobilized utilizing conjugation of biotin and streptavidin.

Biotinylated human phospholipase C delta-like enzyme polypeptides (or poly- nucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a human phospholipase C delta-like enzyme polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the human phospholipase C delta-like enzyme polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the human phospholipase C delta-like enzyme polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the human phospholipase C delta-like enzyme polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a human phospholipase C delta-like enzyme polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a human phospholipase C delta-like enzyme polypeptide or polynucleotide can be used in a cell-based assay system. A human phospholipase C delta-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a human phospholipase C delta-like enzyme polypeptide or polynucleotide is determined as described above.

Enzyme Assays

Test compounds can be tested for the ability to increase or decrease the enzymatic activity of a human phospholipase C delta-like enzyme polypeptide. Enzymatic activity can be measured, for example, as described in Mullinax et al, J. Biomol Screen. 4, 151-55, 1999, or Litosch, Biochemistry 39, 7736-43, 2000).

Enzyme assays can be carried out after contacting either a purified human phospholipase C delta-like enzyme polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases an enzymatic activity of a human phospholipase C delta-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing human phospholipase C delta-like enzyme activity. A test compound which increases an enzymatic activity of a human phospholipase C delta-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) is identified as a potential therapeutic agent for increasing human phospholipase C delta-like enzyme activity.

Gene Expression

In another embodiment, test compounds that increase or decrease human phospholipase C delta-like enzyme gene expression are identified. A human phospholipase C delta-like enzyme polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the human phospholipase C delta-like enzyme polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression. The level of human phospholipase C delta-like enzyme mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a human phospholipase C delta-like enzyme polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmuno assay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a human phospholipase C delta- like enzyme polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a human phospholipase C delta-like enzyme polynucleotide can be used in a cell-based assay system. The human phospholipase C delta-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a human phospholipase C delta-like enzyme polypeptide, human phospholipase C delta-like enzyme polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a human phospholipase C delta-like enzyme polypeptide, or mimetics, activators, or inhibitors of a human phospholipase C delta-like enzyme polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%_>-2% sucrose, and 2-7%> mamiitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Therapeutic Indications and Methods

Human phospholipase C delta-like enzyme can be regulated to treat cancer, COPD, diabetes, and asthma.

Cancer

Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue. Most standard cancer therapies target cellular proliferation and rely on the differential proliferative capacities between transformed and normal cells for their efficacy. This approach is hindered by the facts that several important normal cell types are also highly proliferative and that cancer cells frequently become resistant to these agents. Thus, the therapeutic indices for traditional anti-cancer therapies rarely exceed 2.0.

The advent of genomics-driven molecular target identification has opened up the possibility of identifying new cancer-specific targets for therapeutic intervention that will provide safer, more effective treatments for cancer patients. Thus, newly discovered tumor-associated genes and their products can be tested for their role(s) in disease and used as tools to discover and develop innovative therapies. Genes playing important roles in any of the physiological processes outlined above can be characterized as cancer targets.

Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Activators and/or inhibitors of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.

COPD

Chronic obstructive pulmonary (or airways) disease (COPD) is a condition defined physiologically as airflow obstruction that generally results from a mixture of emphysema and peripheral airway obstruction due to chronic bronchitis (Senior &

Shapiro, Pulmonary Diseases and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp. 659-681, 1998; Barnes, Chest 117, 10S-14S, 2000). Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung. Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years, hi COPD, airflow obstruction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does occur in non-smokers.

Chronic inflammation of the airways is a key pathological feature of COPD (Senior & Shapiro, 1998). The inflammatory cell population comprises increased numbers of macrophages, neutrophils, and CD8⁺ lymphocytes. Inhaled irritants, such as cigarette smoke, activate macrophages which are resident in the respiratory tract, as well as epithelial cells leading to release of chemokines (e.g., interleukin-8) and other chemotactic factors. These chemotactic factors act to increase the neutrophil/- monocyte trafficking from the blood into the lung tissue and airways. Neutrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species.

Matrix degradation and emphysema, along with airway wall thickening, surfactant dysfunction, and mucus hypersecretion, all are potential sequelae of this inflammatory response that lead to impaired airflow and gas exchange.

PI-PLC isozymes are critical components of the transduction machinery responsible for cell activation in response to occupancy of G-protein coupled receptors. Jiang et al, Proc. Natl. Acad. Sci. 94, 7971-75, 1997; Sternweis & Srnrcka, Ciba Found. Symp. 176, 96-11, 1993. Hydrolysis of membrane inositol containing phospholipids by specific PLC isozymes produces inositol phosphates that can act as calcium releasing second messengers (e.g., inositol 1,4,5-trisphosphate), and the protein kinase C activator, diacylglycerol.

PLCs currently are classified into b, g and d families. Subtypes within these families have been identified. For example, the b family consists of 4 subtypes. There is clear evidence of differential tissue distribution of these isoforms and, importantly, differential activation by G-protein subunits. Studies on the inflammatory response in knock-out mice lacking the gene for PLC b-2 have shown selective abrogation of certain features of the inflammatory response. PI-PLC subtypes are therefore attractive therapeutic targets for the inhibition of the inflammatory response in

COPD.

Diabetes

Diabetes mellitus is a common metabolic disorder characterized by an abnormal elevation in blood glucose, alterations in lipids and abnormalities (complications) in the cardiovascular system, eye, kidney and nervous system. Diabetes is divided into two separate diseases: type 1 diabetes (juvenile onset), which results from a loss of cells which make and secrete insulin, and type 2 diabetes (adult onset), which is caused by a defect in insulin secretion and a defect in insulin action.

Type 1 diabetes is initiated by an autoimuune reaction that attacks the insulin secreting cells (beta cells) in the pancreatic islets. Agents that prevent this reaction from occurring or that stop the reaction before destruction of the beta cells has been accomplished are potential therapies for this disease. Other agents that induce beta cell proliferation and regeneration also are potential therapies.

Type U diabetes is the most common of the two diabetic conditions (6% of the population). The defect in insulin secretion is an important cause of the diabetic condition and results from an inability of the beta cell to properly detect and respond to rises in blood glucose levels with insulin release. Therapies that increase the response by the beta cell to glucose would offer an important new treatment for this disease.

The defect in insulin action in Type II diabetic subjects is another target for therapeutic intervention. Agents that increase the activity of the insulin receptor in muscle, liver, and fat will cause a decrease in blood glucose and a normalization of plasma lipids. The receptor activity can be increased by agents that directly stimulate the receptor or that increase the intracellular signals from the receptor. Other therapies can directly activate the cellular end process, i.e. glucose transport or various enzyme systems, to generate an insulin-like effect and therefore a produce beneficial outcome. Because overweight subjects have a greater susceptibility to Type II diabetes, any agent that reduces body weight is a possible therapy.

Both Type I and Type diabetes can be treated with agents that mimic insulin action or that treat diabetic complications by reducing blood glucose levels. Likewise, agents that reduces new blood vessel growth can be used to treat the eye complications that develop in both diseases.

Asthma

Allergy is a complex process in which environmental antigens induce clinically adverse reactions. The inducing antigens, called allergens, typically elicit a specific IgE response and, although in most cases the allergens themselves have little or no intrinsic toxicity, they induce pathology when the IgE response in turn elicits an IgE-dependent or T cell-dependent hypersensitivity reaction. Hypersensitivity reactions can be local or systemic and typically occur within minutes of allergen exposure in individuals who have previously been sensitized to an allergen. The hypersensitivity reaction of allergy develops when the allergen is recognized by IgE antibodies bound to specific receptors on the surface of effector cells, such as mast cells, basophils, or eosinophils, which causes the activation of the effector cells and the release of mediators that produce the acute signs and symptoms of the reactions. Allergic diseases include asthma, allergic rhinitis (hay fever), atopic dermatitis, and anaphylaxis.

Asthma is though to arise as a result of interactions between multiple genetic and environmental factors and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyperresponsiveness caused by a decreased control of airway caliber, and 3) airway inflammation. Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE. These effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to the tissue destruction related to the disorder. Other resident cells, such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to the pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually make asthma a chronic disabling disorder requiring long-term management.

Despite recent important advances in our understanding of the pathophysiology of asthma, the disease appears to be increasing in prevalence and severity (Gergen and Weiss, Am. Rev. Respir. Dis. 146, 823-24, 1992). It is estimated that 30-40% of the population suffer with atopic allergy, and 15%> of children and 5% of adults in the population suffer from asthma (Gergen and Weiss, 1992). Thus, an enormous burden is placed on our health care resources. However, both diagnosis and treatment of asthma are difficult. The severity of lung tissue inflammation is not easy to measure and the symptoms of the disease are often indistinguishable from those of respiratory infections, chronic respiratory inflammatory disorders, allergic rhinitis, or other respiratory disorders. Often, the inciting allergen cannot be determined, making removal of the causative environmental agent difficult. Current pharmacological treatments suffer their own set of disadvantages. Commonly used therapeutic agents, such as beta agonists, can act as symptom relievers to transiently improve pulmonary function, but do not affect the underlying inflammation. Agents that can reduce the underlying inflammation, such as anti-inflammatory steroids, can have major drawbacks that range from immuno suppression to bone loss (Goodman and Gilman's THE PHARMACOLOGIC BASIS OF THERAPEUTICS, Seventh Edition, MacMillan Publishing Company, NY, USA, 1985). In addition, many of the present therapies, such as inhaled corticosteroids, are short-lasting, inconvenient to use, and must be used often on a regular basis, in some cases for life, making failure of patients to comply with the treatment a major problem and thereby reducing their effectiveness as a treatment.

Because of the problems associated with conventional therapies, alternative treatment strategies have been evaluated. Glycophorin A (Chu and Sharom, Cell. Immunol. 145, 223-39, 1992), cyclosporin (Alexander et al, Lancet 339, 324-28, 1992), and a nonapeptide fragment of JL-2 (Zav'yalov et al, Immunol. Lett. 31, 285-88, 1992) all inhibit interleukin-2 dependent T lymphocyte proliferation; however, they are known to have many other effects. For example, cyclosporin is used as a immuno- suppressant after organ transplantation. While these agents may represent alternatives to steroids in the treatment of asthmatics, they inhibit interleukin-2 dependent T lymphocyte proliferation and potentially critical immune functions associated with homeostasis. Other treatments that block the release or activity of mediators of bronchochonstriction, such as cromones or anti-leukotrienes, have recently been introduced for the treatment of mild asthma, but they are expensive and not effective in all patients and it is unclear whether they have any effect on the chronic changes associated with asthmatic inflammation. What is needed in the art is the identification of a treatment that can act in pathways critical to the development of asthma hat both blocks the episodic attacks of the disorder and preferentially dampens the hyperactive allergic immune response without immunocompromising the patient.

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a human phospholipase C delta-like enzyme polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects human phospholipase C delta-like enzyme activity can be administered to a human cell, either in vitro or in vivo, to reduce human phospholipase C delta-like enzyme activity. The reagent preferably binds to an expression product of a human phospholipase C delta-like enzyme gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a Hposome. Preferably, the Hposome is stable in the animal into which it has been administered for at least about

30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A Hposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the Hposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A Hposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a Hposome is about

0.5 μg of DNA per 16 nmole of Hposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmole of Hposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of Hposome delivered to about 10⁶ cells. Preferably, a Hposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a Hposome comprises a compound capable of targeting the Hposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the Hposome.

Complexing a Hposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol 11, 202-05 (1993);

Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991). Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases human phospholipase C delta-like enzyme activity relative to the human phospholipase C delta-like enzyme activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in

50% of the population) and LD₅₀ (the dose lethal to 50%> of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅o.

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.

Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA. If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of a human phospholipase C delta-like enzyme gene or the activity of a human phospholipase C delta-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a human phospholipase C delta-like enzyme gene or the activity of a human phospholipase C delta-like enzyme polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to human phospholipase C delta-like enzyme-specific mRNA, quantitative RT-PCR, immunologic detection of a human phospholipase C delta-like enzyme polypeptide, or measurement of human phospholipase C delta-like enzyme activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Diagnostic Methods

Human phospholipase C delta-like enzyme also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding human phospholipase C delta-like enzyme in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85,

4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of human phospholipase C delta-like enzyme also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1

Detection of phospholipase C delta-like enzyme activity

The polynucleotide of SEQ LO NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-phospholipase C delta-like enzyme polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and phosphatidylinositol hydrolyzing activity is measured by incubation for 10 min at 37°C of a mixture containing lOmM Hepes, pH 7.0, 10 mM NaCI, 4 mM MgSO4, 100 mM KCl, 0.1% deoxycholate, 50 μM cell extract, 250 μM PE containing 25,000 dpm of [3H] PIP2, 10 μM free Ca++ in the presence of 2 mM EGTA in a total volume of 100 μl. The reaction is terminated by the addition of 500 μl of chloroform, methanol, 0.1 M HCl (200:100:0.6) followed by the addition of 150 μl of 1 N HCl, 5 mM EGTA. A 0.2-ml aliquot of the upper aqueous phase is removed for measurement of radioactivity. Free Ca++ concentrations are calculated as described previously. It is shown that the polypeptide of SEQ LD NO: 2 has a phospholipase C delta-like enzyme activity.

EXAMPLE 2

Expression of recombinant human phospholipase C delta-like enzyme

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human phospholipase C delta-like enzyme polypeptides in yeast. The human phospholipase C delta-like enzyme-encoding DNA sequence is derived from SEQ ID NO: 1. Before insertion into vector pPICZB, the

DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified

DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human phospholipase C delta-like enzyme polypeptide is obtained. EXAMPLE 3

Identification of test compounds that bind to human phospholipase C delta-like enzyme polypeptides

Purified human phospholipase C delta-like enzyme polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human phospholipase C delta- like enzyme polypeptides comprise the amino acid sequence shown in SEQ ID NO:

2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells.

Binding of a test compound to a human phospholipase C delta-like enzyme polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a human phospholipase C delta-like enzyme polypeptide.

EXAMPLE 4

Identification of a test compound which decreases human phospholipase C delta-like enzyme gene expression

A test compound is administered to a culture of human cells transfected with a human phospholipase C delta-like enzyme expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control. RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled human phospholipase C delta-like enzyme-specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1. A test compound that decreases the human phospholipase C delta-like enzyme-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of human phospholipase C delta-like enzyme gene expression.

EXAMPLE 5

Identification of a test compound which decreases human phospholipase C delta-like enzyme activity

A test compound is administered to a culture of human cells transfected with a human phospholipase C delta-like enzyme expression construct and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control, human phospholipase C delta-like enzyme activity is measured using the method of Mullinax et al, J. Biomol Screen. 4, 151-55, 1999, or Litosch, Biochemistry 39, 7736-43, 2000).

A test compound which decreases the human phospholipase C delta-like enzyme activity of the human phospholipase C delta-like enzyme relative to the human phospholipase C delta-like enzyme activity in the absence of the test compound is identified as an inhibitor of human phospholipase C delta-like enzyme activity. EXAMPLE 6

Tissue-specific expression of human phospholipase C delta-like enzyme

The qualitative expression pattern of human phospholipase C delta-like enzyme in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

To demonstrate that human phospholipase C delta-like enzyme is involved in cancer, expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes. Expression in the following cancer cell lines also is determined: DU- 145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205 (colon), A-549 (lung),

NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested.

To demonstrate that human phospholipase C delta-like enzyme is involved in the disease process of COPD, the initial expression panel consists of RNA samples from respiratory tissues and inflammatory cells relevant to COPD: lung (adult and fetal), trachea, freshly isolated alveolar type II cells, cultured human bronchial epithelial cells, cultured small airway epithelial cells, cultured bronchial sooth muscle cells, cultured H441 cells (Clara-like), freshly isolated neutrophils and monocytes, and cultured monocytes (macrophage-like). Body map profiling also is carried out, using total RNA panels purchased from Clontech. The tissues are adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, mammary gland, pancreas, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, trachea, thyroid, and uterus. To demonstrate that human phospholipase C delta-like enzyme is involved in the disease process of diabetes, the following whole body panel is screened to show predominant or relatively high expression: subcutaneous and mesenteric adipose tissue, adrenal gland, bone marrow, brain, colon, fetal brain, heart, hypothalamus, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, thyroid, trachea, and uterus. Human islet cells and an islet cell library also are tested. As a final step, the expression of human phospholipase C delta-like enzyme in cells derived from normal individuals with the expression of cells derived from diabetic individuals is compared.

To demonstrate that human phospholipase C delta-like enzyme is involved in the disease process of asthma, the following whole body panel is screened to show predominant or relatively high expression in lung or immune tissues: brain, heart, kidney, liver, lung, trachea, bone marrow, colon, small intestine, spleen, stomach, thymus, mammary gland, skeletal muscle, prostate, testis, uterus, cerebellum, fetal brain, fetal liver, spinal cord, placenta, adrenal gland, pancreas, salivary gland, thyroid, peripheral blood leukocytes, lymph node, and tonsil. Once this is established, the following lung and immune system cells are screened to localize expression to particular cell subsets: lung microvascular endothelial cells, bronchial/tracheal epithelial cells, bronchial/tracheal smooth muscle cells, lung fibroblasts, T cells (T helper 1 subset, T helper 2 subset, NKT cell subset, and cytotoxic T lymphocytes), B cells, mononuclear cells (monocytes and macrophages), mast cells, eosinophils, neutrophils, and dendritic cells. As a final step, the expression of human phospholipase C delta-like enzyme in cells derived from normal individuals with the expression of cells derived from asthmatic individuals is compared.

Quantitative expression profiling. Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis" firstly described in

Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.

If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).

The amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction. Hi this kind of experiment, the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.

All "real time PCR" measurements of fluorescence are made in the ABI Prism 7700.

RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy" were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.

Fifty μg of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM

MgCl₂; 50 mM NaCI; and 1 mM DTT. After incubation, RNA is extracted once with 1 volume of phenohchloroform:- isoamyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M aAcetate, pH5.2, and 2 volumes of ethanol.

Fifty μg of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectro- photometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is

200 ng/μL. Reverse transcription is carried out with 2.5μM of random hexamer primers.

TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy- tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification (multiplex

PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from

20X stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 μl.

Each of the following steps are carried out once: pre PCR, 2 minutes at 50°C, and 10 minutes at 95 °C. The following steps are carried out 40 times: denaturation, 15 seconds at 95°C, annealing/extension, 1 minute at 60°C. The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.

EXAMPLE 7

Proliferation inhibition assay: Antisense oligonucleotides suppress the growth of cancer cell lines

The cell line used for testing is the human colon cancer cell line HCT116. Cells are cultured in RPMI- 1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37°C in a 95% air/5%>CO₂ atmosphere.

Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamidite chemistry. A sequence of 24 bases complementary to the nucleotides at position 1 to 24 of SEQ ID NO: 1 is used as the test oligonucleotide. As a control, another (random) sequence is used: 5'-TCA ACT GAC TAG ATG TAC ATG GAC-3'. Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 μM once per day for seven days.

The addition of the test oligonucleotide for seven days results in significantly reduced expression of human phospholipase C delta-like enzyme as determined by Western blotting. This effect is not observed with the control oligonucleotide. After 3 to 7 days, the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30%> of control, indicating that the inhibition of human phospholipase C delta-like enzyme has an anti-proliferative effect on cancer cells.

EXAMPLE 8

In vivo testing of compounds/target validation

1. Acute Mechanistic Assays

1.1. Reduction in Mitogenic Plasma Hormone Levels

This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus. Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c). At a predetermined time after administration of test compound, blood plasma is collected. Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m. into mice at a dosage of 30 ng/mouse to induce a burst of testosterone synthesis). The timing of plasma collection would be adjusted to coincide with the peak of the induced hormone response. Compound effects are compared to a vehicle-treated control group. An F- test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value < 0.05 compared to the vehicle control group. 1.2. Hollow Fiber Mechanism of Action Assay

Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Ranlc Sum test after the variance between groups is compared by an F-test, with significance at p < 0.05 as compared to the vehicle control group.

2. Subacute Functional In Vivo Assays

2.1. Reduction in Mass of Hormone Dependent Tissues

This is another non-tumor assay that measures the ability of a compound to reduce the mass of a hormone dependent tissue (i.e., seminal vesicles in males and uteri in females). Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week). At termination of the study, animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded. Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent. Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value < 0.05 compared to the vehicle control group.

2.2. Hollow Fiber Proliferation Assay

Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol. Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p < 0.05 as compared to the vehicle control group.

2.3. Anti-angiogenesis Models

2.3.1. Comeal Angiogenesis

Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea. Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet). Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Ranlc Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p < 0.05 as compared to the growth factor or cells only group.

2.3.2. Matrigel Angiogenesis

Matrigel, containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p < 0.05 as compared to the vehicle control group. 3. Primary Antitumor Efficacy

3.1. Early Therapy Models

3.1.1. Subcutaneous Tumor

Tumor cells or fragments are implanted subcutaneously on Day 0. Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden. Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day. Anti- tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p < 0.05. The experiment may also be continued past the end of dosing in which case tumor measurements would continue to be recorded to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-

Meier curves from the times for individual tumors to attain the evaluation size. Significance is p < 0.05.

3.1.2. Intraperitoneal/Intracranial Tumor Models

Tumor cells are injected intraperitoneally or intracranially on Day 0. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long- term survivors is indicated separately. Survival times are used to generate Kaplan- Meier curves. Significance is p < 0.05 by a log-rank test compared to the control group in the experiment.

3.2. Established Disease Model

Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group. Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value< 0.05 compared to the vehicle control group.

3.3. Orthotopic Disease Models

3.3.1. Mammaiγ Fat Pad Assay

Tumor cells or fragments, of mammary adenocarcinoma origin, are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group.

Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value< 0.05 compared to the vehicle control group. In addition, this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p < 0.05 compared to the control group in the experiment.

3.3.2. Intraprostatic Assay

Tumor cells or fragments, of prostatic adenocarcinoma origin, are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents. The prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles. The successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p < 0.05 compared to the control group in the experiment.

3.3.3. Intrabronchial Assay

Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea. The trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p < 0.05 compared to the control group in the experiment. 3.3.4. Intracecal Assay

Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t- test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor.

Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p < 0.05 compared to the control group in the experiment.

4. Secondary (Metastatic) Antitumor Efficacy

4.1. Spontaneous Metastasis

Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p < 0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance determined at p < 0.05 compared to the control group in the experiment for both of these endpoints.

4.2. Forced Metastasis

Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p < 0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p < 0.05 compared to the vehicle control group in the experiment for both endpoints.

EXAMPLE 9

Identification of test compound efficacy in a COPD animal model

Guinea pigs are exposed on a single occasion to tobacco smoke for 50 minutes.

Animals are sacrificed between 10 minutes and 24 hour following the end of the exposure and their lungs placed in RNAlater™. The lung tissue is homogenized, and total RNA was extracted using a Qiagens RNeasy™ Maxi kit. Molecular Probes RiboGreen™ RNA quantitation method is used to quantify the amount of RNA in each sample.

Total RNA is reverse transcribed, and the resultant cDNA is used in a real-time polymerase chain reaction (PCR). The cDNA is added to a solution containing the sense and anti-sense primers and the 6-carboxy-tetramethyl-rhodamine labeled probe of the phospholipase C delta-like enzyme gene. Cyclophilin is used as the housekeeping gene. The expression of the phospholipase C delta-like enzyme gene is measured using the TaqMan real-time PCR system that generates an amplification curve for each sample. From this curve a threshold cycle value is calculated: the fractional cycle number at which the amount of amplified target reaches a fixed threshold. A sample containing many copies of the phospholipase C delta-like enzyme gene will reach this threshold earlier than a sample containing fewer copies.

The threshold is set at 0.2, and the threshold cycle C is calculated from the amplification curve. The Cx value for the phospholipase C delta-like enzyme gene is normalized using the Cj value for the housekeeping gene.

Expression of the phospholipase C delta-like enzyme gene is increased by at least 3- fold between 10 minutes and 3 hours post tobacco smoke exposure compared to air exposed control animals.

Test compounds are evaluated as follows. Animals are pre-treated with a test compound between 5 minutes and 1 hour prior to the tobacco smoke exposure and they are then sacrificed up to 3 hours after the tobacco smoke exposure has been completed. Control animals are pre-treated with the vehicle of the test compound via the route of administration chosen for the test compound. A test compound that reduces the tobacco smoke induced upregulation of phospholipase C delta-like enzyme gene relative to the expression seen in vehicle treated tobacco smoke exposed animals is identified as an inhibitor of phospholipase C delta-like enzyme gene expression.

EXAMPLE 10

Diabetes: In vivo testing of compounds/target validation

1. Glucose Production

Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injection of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle-treated control group.

Insulin Sensitivity

Both ob/ob and db/db mice as well as diabetic Zuclcer rats are hyperglycemic, hyperinsulinemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.

Insulin Secretion

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load. When measuring insulin levels, compounds are administered by different routes (p.o., i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60 and 90 minutes and plasma glucose levels determined. Compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load. When measuring insulin levels, test compounds which regulate phospholipase C delta-like enzyme are administered by different routes (p.o., i.p., s.c, or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Test compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60, and 90 minutes and plasma glucose levels determined. Test compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

4. Glucose Production

Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes.

Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injection of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle-treated control group.

5. Insulin Sensitivity

Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.

6. Insulin Secretion

EXAMPLE 11

Expression of human phospholipase C delta-like enzyme in various tissues

Total RNA used for Taqman quantitative analysis were either purchased (Clontech,

CA) or extracted from tissues using TRIzol reagent (Life Technologies, MD) according to a modified vendor protocol which utilizes the Rneasy protocol (Qiagen, CA). One hundred μg of each RNA were treated with DNase I using RNase free- DNase (Qiagen, CA) for use with RNeasy or QiaAmp columns. After elution and quantitation with Ribogreen (Molecular Probes Inc., OR), each sample was reverse transcribed using the GibcoBRL Superscript LI First Strand Synthesis System for RT-PCR according to vendor protocol (Life Technologies, MD). The final concentration of RNA in the reaction mix was 50ng/μL. Reverse transcription was performed with 50 ng of random hexamers.

Specific primers and probe were designed according to PE Applied Biosystems' Primer Express program recommendations.

Quantitation experiments were performed on 25 ng of reverse transcribed RNA from each sample. 18S ribosomal RNA was measured as a control using the Pre- Developed TaqMan Assay Reagents (PDAR)(PE Applied Biosystems, CA). The assay reaction mix was as follows:

TaqMan S YBR Green PCR Master Mix (2x) lx

(PE Applied Biosystems, CA)

Forward primer 300nM

Reverse primer 300nM cDNA 25ng

Water to 25uL

PCR conditions:

Once: 2' minutes at 50° C

10 minutes at 95 °C

40cycles: 15 secat 95°C ^*

1 minute at 60°C

The experiment was performed on an ABI Prism 7700 Sequence Detector (PE

Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR were processed as described in the ABI Prism 7700 user's manual. Fold change was calculated using the delta-delta CT method with normalization to the 18S values. Relative expression was calculated by normalizing to 18s (D Ct), then making the highest expressing tissue 100 and everything else relative to it. Copy number conversion was performed without normalization using the formula Cn=10(Ct- 40.007)/-3.623.

The results are shown in FIG. 13.

EXAMPLE 12

Therapeutic Indications and Methods

It was found by the present applicant that the novel human PLC-delta is expressed in various human tissues.

Gastro-intestinal disorders

The novel human PLC-delta is highly expressed in the following tissues of the gastro-intestinal system: rectum, stomach, esophagus. The expression in the above mentioned tissues demonstrates that the novel human PLC-delta or mRNA can be utilized to diagnose of gastro-intestinal disorders. Additionally the activity of the novel human PLC-delta can be modulated to treat gastro-intestinal disorders.

Gastrointestinal diseases comprise primary or secondary, acute or chronic diseases of the organs of the gastrointestinal tract which may be acquired or inherited, benign or malignant or metaplastic, and which may affect the organs of the gastrointestinal tract or the body as a whole. They comprise but are not limited to 1) disorders of the esophagus like achalasia, vigoruos achalasia, dysphagia, cricopharyngeal incoor- dination, pre-esophageal dysphagia, diffuse esophageal spasm, globus sensation, Barrett's metaplasia, gastroesophageal reflux, 2) disorders of the stomach and duodenum like functional dyspepsia, inflammation of the gastric mucosa, gastritis, stress gastritis, chronic erosive gastritis, atrophy of gastric glands, metaplasia of gastric tissues, gastric ulcers, duodenal ulcers, neoplasms of the stomach, 3) disorders of the pancreas like acute or chronic pancreatitis, insufficiency of the exocrinic or endocrinic tissues of the pancreas like steatorrhea, diabetes, neoplasms of the exocrine or endocrine pancreas like 3.1) multiple endocrine neoplasia syndrome , ductal adenocarcinoma, cystadenocarcinoma, islet cell tumors, insulinoma, gastrinoma, carcinoid tumors, glucagonoma, Zollinger-Ellison syndrome, Vipoma syndrome, malabsorption syndrome, 4) disorders of the bowel like chronic inflammatory diseases of the bowel, Crohn's disease, ileus, diarrhea and constipation, colonic inertia, megacolon, malabsorption syndrome, ulcerative colitis, 4.1) functional bowel disorders like irritable bowel syndrome, 4.2) neoplasms of the bowel like familial polyposis, adenocarcinoma, primary malignant lymphoma , carcinoid tumors, Kaposi's sarcoma, polyps, cancer of the colon and rectum.

Central Nervous System (CNS) Disorders

The novel human PLC-delta is highly expressed in the following brain tissues: retina, spinal cord, cerebellum (left), cerebral meninges, cerebellum, postcentral gyrus, dorsal root ganglia, alzheimer brain frontal lobe, frontal lobe, cerebellum (right). The expression in brain tissues and in particular the differential expression between diseased tissue alzheimer brain frontal lobe and healthy tissue frontal lobe demonstrates that the novel human PLC-delta or mRNA can be utilized to diagnose nervous system diseases. Additionally the activity of the novel human PLC-delta can be modulated to treat nervous system diseases.

CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system. CNS disorders include, but are not limited to brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease. Dementias, such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff s psychosis, within the meaning of the invention are also considered to be CNS disorders.

Similarly, cognitive-related disorders, such as mild cognitive impairment, age- associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities are also considered to be

CNS disorders.

Pain, within the meaning of the invention, is also considered to be a CNS disorder. Pain can be associated with CNS disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy,

Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, phantom feeling, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with peripheral nerve damage, central pain (i.e. due to cerebral ischemia) and various chronic pain i.e., lumbago, back pain (low back pain), inflammatory and/or rheumatic pain. Headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania are also CNS disorders.Nisceral pain such as pancreatits, intestinal cystitis, dysmenorrhea, irritable

Bowel syndrome, Crohn's disease, biliary colic, ureteral colic, myocardial infarction and pain syndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethral syndrome and protatodynia are also CNS disorders. Also considered to be a disorder of the nervous system are acute pain, for example postoperative pain, and pain after trauma.

Cardiovascular Disorders

The novel human PLC-delta is highly expressed in the following cardiovascular related tissues: heart ventricle (left), vein, coronary artery sclerotic, pericardium, heart atrium (left), aorta sclerotic. Expression in the above mentioned tissues and in particular the differential expression between diseased tissue coronary artery sclerotic and healthy tissue , between diseased tissue aorta sclerotic and healthy tissue aorta demonstrates that the novel human PLC-delta or mRNA can be utilized to diagnose of cardiovascular diseases. Additionally the activity of the novel human PLC-delta can be modulated to treat cardiovascular diseases.

Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included as well as the acute treatment of MI and the prevention of complications.

Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen. This group of diseases includes stable angina, unstable angina and asymptomatic ischemia. Arrhythmias include all forms of atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others. The genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.

Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.

Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel. The atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion. Although the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.

The formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition. Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear.

Thus, a need exists for therapeutic methods and agents to treat cardiovascular pathologies, such as atherosclerosis and other conditions related to coronary artery disease.

Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.

Genito-urinary disorders

The novel human PLC-delta is highly expressed in the following tissues of the genito-urinary system: bladder, prostata, penis, cervix. The expression in the above mentioned tissues demonstrates that the novel human PLC-delta or mRNA can be utilized to diagnose of genito-urinary disorders. Additionally the activity of the novel human PLC-delta can be modulated to treat genito-urinary disorders.

Genitourological disorders comprise benign and malign disorders of the organs constituting the genitourological system of female and male, renal diseases like acute or chronic renal failure, immunologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulo- pathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hyperplasia (BPH), neurogenic bladder syndrome, urinary incontinence like urge-, stress-, or overflow incontinence, pelvic pain, and erectile dysfunction. Metabolic Diseases

The novel human PLC-delta is highly expressed in the following metabolic disease related tissues: adipose. The expression in the above mentioned tissues demonstrates that the novel human PLC-delta or mRNA can be utilized to diagnose of metabolic diseases. Additionally the activity of the novel human PLC-delta can be modulated to treat metabolic diseases.

Metabolic diseases are defined as conditions which result from an abnormality in any of the chemical or biochemical transformations and their regulating systems essential to producing energy, to regenerating cellular constituents, to eliminating unneeded products arising from these processes, and to regulate and maintain homeostasis in a mammal regardless of whether acquired or the result of a genetic transformation.

Depending on which metabolic pathway is involved, a single defective trans- formation or disturbance of its regulation may produce consequences that are narrow, involving a single body function, or broad, affecting many organs, organ-systems or the body as a whole. Diseases resulting from abnormalities related to the fine and coarse mechanisms that affect each individual transformation, its rate and direction or the availability of substrates like amino acids, fatty acids, carbohydrates, minerals, cofactors, hormones, regardless whether they are inborn or acquired, are well within the scope of the definition of a metabolic disease according to this application.

Metabolic diseases often are caused by single defects in particular biochemical pathways, defects that are due to the deficient activity of individual enzymes or molecular receptors leading to the regulation of such enzymes. Hence in a broader sense disturbances of the underlying genes, their products and their regulation lie well within the scope of this definition of a metabolic disease. For example, but not limited to, metabolic diseases may affect 1) biochemical processes and tissues ubiquitous all over the body, 2) the bone, 3) the nervous system, 4) the endocrine system, 5) the muscle including the heart, 6) the skin and nervous tissue, 7) the urogenital system, 8) the homeostasis of body systems like water and electrolytes. For example, but not limited to, metabolic diseases according to 1) comprise obesity, amyloidosis, disturbances of the amino acid metabolism like branched chain disease, hyperaminoacidemia, hyperaminoaciduria, disturbances of the metabolism of urea, hyperammonemia, mucopolysaccharidoses e.g. Maroteaux-Lamy syndrom, storage diseases like glycogen storage diseases and lipid storage diseases, glycogenosis diseases like Cori's disease, malabsorption diseases like intestinal carbohydrate malabsorption, oligosaccharidase deficiency like maltase-, lactase-, sucrase- insufficiency, disorders of the metabolism of fructose, disorders of the metabolism of galactose, galactosaemia, disturbances of carbohydrate utilization like diabetes, hypoglycemia, disturbances of pyruvate metabolism, hypolipidemia, hypolipo- proteinemia, hyperlipidemia, hyperlipoproteinemia, carnitine or carnitine acyl- transferase deficiency, disturbances of the porphyrin metabolism, porphyrias, disturbances of the purine metabolism, lysosomal diseases, metabolic diseases of nerves and nervous systems like gangliosidoses, sphingolipidoses, sulfatidoses, leucodystrophies, Lesch-Nyhan syndrome. For example, but not limited to, metabolic diseases according to 2) comprise osteoporosis, osteomalacia like osteoporosis, osteopenia, osteogenesis imperfecta, osteopetrosis, osteonecrosis, Paget's disease of bone, hypophosphatemia. For example, but not limited to, metabolic diseases according to 3) comprise cerebellar dysfunction, disturbances of brain metabolism like dementia, Alzheimer's disease, Huntington's chorea, Parkinson's disease, Pick's disease, toxic encephalopathy, demyelinating neuropathies like inflammatory neuropathy, Guillain-Barre syndrome. For example, but not limited to, metabolic diseases according to 4) comprise primary and secondary metabolic disorders associated with hormonal defects like any disorder stemming from either an hyperfunction or hypofunction of some hormone-secreting endocrine gland and any combination thereof. They comprise Sipple's syndrome, pituitary gland dysfunction and its effects on other endocrine glands, such as the thyroid, adrenals, ovaries, and testes, acromegaly, hyper- and hypothyroidism, euthyroid goiter, euthyroid sick syndrome, thyroiditis, and thyroid cancer, over- or underproduction of the adrenal steroid hormones, adreno genital syndrome, Cushing's syndrome, Addison's disease of the adrenal cortex, Addison's pernicious anemia, primary and secondary aldosteronism, diabetes insipidus , carcinoid syndrome, disturbances caused by the dysfunction of the parathyroid glands, pancreatic islet cell dysfunction, diabetes, disturbances of the endocrine system of the female like estrogen deficiency, resistant ovary syndrome. For example, but not limited to, metabolic diseases according to 5) comprise muscle weakness, myotonia, Duchenne's and other muscular dystrophies, dystrophia myotonica of Steinert, mitochondrial myopathies like disturbances of the catabolic metabolism in the muscle, carbohydrate and lipid storage myopathies, glycogenoses, myoglobinuria, malignant hyperthermia, polymyalgia rheumatica, dermatomyositis, primary myocardial disease, cardiomyopathy. For example, but not limited to, metabolic diseases according to 6) comprise disorders of the ectoderm, neurofibromatosis, scleroderma and polyarteritis, Louis-Bar syndrome, von Hippel-Lindau disease, Sturge-Weber syndrome, tuberous sclerosis, amyloidosis, porphyria. For example, but not limited to, metabolic diseases according to 7) comprise sexual dysfunction of the male and female. For example, but not limited to, metabolic diseases according to 8) comprise confused states and seizures due to inappropriate secretion of antidiuretic hormone from the pituitary gland, Liddle's syndrome, Bartter's syndrome, Fanconi's syndrome, renal electrolyte wasting, diabetes insipidus.

Expression Profiling

Total cellular RNA was isolated from cells by one of two standard methods: 1) guanidine isothiocyanate/Cesium chloride density gradient centrifugation [ Kellogg et al. (1990)] ; or with the Tri-Reagent protocol according to the manufacturer's specificati ons (Molecular Research Center, Inc., Cincinatti, Ohio). Total RNA prepared by the Tri-reagent protocol was treated with DNAse I to remove genomic DNA contamination.

For relative quantitation of the mRNA distribution of the novel human PLC-delta, total RNA from each cell or tissue source was first reverse transcribed. 85 μ g of total

RNA was reverse transcribed using 1 μmole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680 μ 1. The first strand synthesis buffer and Omniscript reverse transcriptase (2 u/μl) were from (Qiagen, Hilden, Germany). The reaction was incubated at 37° C for 90 minutes and cooled on ice. The volume was adjusted to 6800 μl with water, yielding a final concentration of

12.5 ng/μl of starting RNA.

For relative quantitation of the distribution of the novel human PLC-delta mRNA in cells and tissues the Perkin Elmer ABI Prism RTM. 7700 Sequence Detection system or Biorad iCycler was used according to the manufacturer's specifications and protocols. PCR reactions were set up to quantitate the novel human PLC-delta and the housekeeping genes HPRT (hypoxanthine phosphoribosyltransferase), GAPDH (glyceraldehyde-3 -phosphate dehydrogenase), β -actin, and others. Forward and reverse primers and probes for the novel human PLC-delta were designed using the Perkin Elmer ABI Primer Express™ software and were synthesized by TibMolBiol

(Berlin, Germany). The novel human PLC-delta forward primer sequence was: Primerl (SE Q ED NO: 3). The novel human PLC-delta reverse primer sequence was Primer2 (SEQ ID NO: 5). Probel (SEQ ID NO: 4), labelled with FAM (carboxy- fluorescein succinimidyl ester) as the reporter dye and TAMRA (carboxytetra- methylrhodamine) as the quencher, is used as a probe for the novel human PLC- delta. The following reagents were prepared in a total of 25 μl : lx TaqMan buffer A, 5.5 mM MgCl ₂, 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/μl AmpliTaq Gold ™, 0.01 U/ μl AmpErase and Probel (SEQ ID NO: 4), novel human PLC-delta forward and reverse primers each at 200 nM, 200 nM , novel human PLC-delta FAM/TAMRA-labelled probe, and 5 μ 1 of template cDNA. Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. Calculation of corrected CT values

The CT (threshold cycle) value is calculated as described in the "Quantitative determination of nucleic acids" section. The CF-value (factor for threshold cycle correction) is calculated as follows:

1. PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA sample.

2. CT_HKG- values (threshold cycle for housekeeping gene) were calculated as described in the "Quantitative determination of nucleic acids" section.

3. CT _G-mean values (CT mean value of all HKG tested on one cDNAs) of all HKG for each cDNA are calculated (n = number of HKG):

CTHKG-_n-∞.ean value = (CTH_KGI -value + CTκκ_G2-value + ... + CT_HKG-_Π- value) / n

4. CTpann_ei mean value (CT mean value of all HKG in all tested cDNAs) = (CT_HKGI -mean value + CT_HKG2-mean value + ...+ CTu_KG-_y-mean value) / y (y = number of cDNAs)

5. CF_CDNA-n (correction factor for cDNA n) = CT_pannei-mean value - CT_Hκ_G-_n- mean value

6. CT_CDNA-n (CT value of the tested gene for the cDNA n) + CF_CDNA-_Π (correction factor for cDNA n) = CT CO_Γ-_CDNA-_Π (corrected CT value for a gene on cDNA n) Calculation of relative expression

Definition : highest CT_cor-CDNA-n ≠ 40 is defined as CT_COΓ-CDNA [high]

Relative Expression = ₂ ^(CTcor-^{cDNA[hlgh] " CTcor}-° ^DNA"n)

Human Tissues

skeletal muscle, rectum, retina, heart ventricle (left), vein, coronary artery sclerotic, pericardium, bladder, spinal cord, cerebellum (left), stomach, cerebral meninges, prostata, cerebellum, postcentral gyrus, adipose, dorsal root ganglia, liver liver cirrhosis, alzheimer brain frontal lobe, penis, cervix, skin, frontal lobe, cerebellum (right), kidney, pons, esophagus, heart atrium (left), aorta sclerotic, lung tumor, trachea, brain, testis, alzheimer cerebral cortex, occipital lobe, ileum, vermis cerebelli, lung COPD, cerebral cortex, hippocampus, temporal lobe, parietal lobe, thyroid, small intestine, aorta, artery, uterus, corpus callosum, fetal lung, thrombocytes, precentral gyrus, erythrocytes, tonsilla cerebelli , HEK 293 cells, breast tumor, alzheimer brain, heart, salivary gland, mammary gland, HUNEC cells, colon tumor, lymplmode, cerebral peduncles, coronary artery smooth muscle primary cells, heart atrium (right), colon, interventricular septum, thalamus, adrenal gland, fetal brain, fetal kidney, ileum chronic inflammation, ileum tumor, prostate BPH,

HEP G2 cells, spleen, thymus, fetal heart, MDA MB 231 cells (breast tumor), Jurkat (T-cells), liver, placenta, leukocytes (peripheral blood), fetal aorta, lung, pancreas, pancreas liver cirrhosis, bone marrow, breast, fetal liver, spleen liver cirrhosis, thyroid tumor, HeLa cells (cervix tumor)

Expression Profile

The results of the mRΝA-quantification (expression profiling) is shown in Table 1 Tissue Relative Expression skeletal muscle 1808 rectum 879 retina 755 heart ventricle (left) 680 vein 537 coronary artery sclerotic 519 pericardium 516 bladder 512 spinal cord 465 cerebellum (left) 465 stomach 443 cerebral meninges 410 prostata 385 cerebellum 338 postcentral gyrus 304 adipose 246 dorsal root ganglia 234 liver liver cirrhosis 176 alzheimer brain frontal lobe 170 penis 167 cervix 159 skin 154 frontal lobe 151 cerebellum (right) 138 kidney 133 pons 131 esophagus 127 heart atrium (left) 126 aorta sclerotic 121 lung tumor 115 trachea 111 brain 111 testis 87 alzheimer cerebral cortex 87 occipital lobe 83 ileum 74 vermis cerebelli 73 lung COPD 72 cerebral cortex 70 hippocampus 62 temporal lobe 59 parietal lobe 59 thyroid 58 small intestine 58 aorta 56 artery 53 uterus 52 corpus callosum 51 fetal lung 47 thrombocytes 44 precentral gyrus 41 erythrocytes 40 tonsiUa cerebelli 38

HEK 293 cells 38 breast tumor 37 alzheimer brain 36 heart 33 salivary gland 31 mammary gland 31

HUVEC cells 24 colon tumor 23 lymphnode 23 cerebral peduncles 22 coronary artery smooth muscle primary cells 20 heart atrium (right) 18 colon 17 interventricular septum 16 thalamus 16 adrenal gland 15 fetal brain 14 fetal kidney 13 ileum chronic inflammation 13 ileum tumor 13 prostate BPH 12

HEP G2 cells 12 spleen 9 thymus 6 fetal heart 5

MDA MB 231 cells (breast tumor) 5

Jurkat (T-cells) 5 liver 4 placenta 3 leukocytes (peripheral blood) 3 fetal aorta 3 lung 1 pancreas 1 pancreas liver cirrhosis 1 bone marrow 2 breast 1 fetal liver 1 spleen liver cirrhosis 1 thyroid tumor 1

HeLa cells (cervix tumor) 0 Sequences

Forward Primer

5'-gtgttgaggaggcccagata-3'

Backward Primer

5 '-tggtgacaagatccaccaac-3 '

Probe 5'-tggatgcgagggctccagct-3'

SEQUENCE LISTING

<110> Bayer AG

<120> REGUIiATION OF HUMAN PHOSPHOLIPASE C DELTA-LIKE ENZYME

<130> Lio356

<150> USSN 60/293,517 <151> 2001-05-29

<150> USSN 60/334,789 <151> 2002-01-07

<160> 7

<170> Patentln version 3.1

<210> 1

<211> 2289

<212> DNA

<213> Homo sapiens

<400> 1 atggcgtccc tgctgcaaga ccagctgacc aαtgatcagg acttgctgct gatgcaggaa 60 ggcatgccga tgcgcaaggt gaggtccaaa agctggaaga agctaagata cttcagactt 120 cagaatgacg gcatgacagt ctggcatgca cggcaggcca ggggαagtgc caagαccagc 180 ttctcaatct ctgatgtgga gacaatacgt aatggccatg attccgagtt gctgcgtagc 240 ctggcagagg agctccccαt ggagcagggc ttcaccattg tcttccatgg ccgccgc cc 300 aacctggacc tgatggccaa cagtgttgag gaggcccaga tatggatgcg agggctccag 360 ctgttggtgg atcttgtcac cagcatggac catcaggagc gcctggacca atggctgagσ 420 gattggtttc aacgtggaga caaaaatcag gatggtaaga tgagtttcca agaagttcag 480 cggttattgc acαtaatgaa tgtggaaatg gaccaagaat atgccttcag tctttttcag 540 gcagcagaca cgtcccagtc tggaaccctg gaaggagaag aattcgtaca gttctataag 600 gcattgacta aacgtgctga ggtgcaggaa ctgtttgaaa gtttttcagc tgatgggcag 660 aagctgactc tgctggaatt tttggatttc ctccaagagg agcagaagga gagagactgc 720 acctctgagc ttgctctgga actcattgac cgctatgaac cttcagacag tggcaaactg 780 cggcatgtgc tgagtatgga tggcttcctc agctacctct gctctaagga tggagacatc 840 ttcaacccag cctgcctccc catctateag gatatgactc aacccctgaa ccactacttc 900 atctgctctt ctcataacac ctacctagtg ggggaccagα tttgcggcca gagcagcgtc 960 gagggatata tacgggccct gaagcggggg tgccgctgcg tggaggtgga tgtatgggat 1020 ggacctagcg gggaacctgt cgtttaccac ggacacaccc tgacctcccg eatcctgttc 1080 aaagatgtcg tggccacagt agcacagtat gccttccaga catcagacta cccagtcatc 1140 ttgtccctgg agacccactg cagctgggag cagcagcaga ccatggcccg tcatctgact 1200 gagatcctgg gggagαagct gctgagcacc accttggatg gggtgctgcc cactcagctg 1260 ccctcgcctg aggagcttcg gaggaagatc ctggtgaagg ggaagaagtt aacacttgag 1320 gaagacctgg aatatgagga agaggaagca gaacctgagt tggaagagtc agaattggcg 1380 ctggagtccc agtttgagac tgagcctgag ccccaggagc agaaσcttca gaataaggac 1440 aaaaagaaga aatccaagcc catcttgtgt ccagccctct cttccctggt tatctacttg 1500 aagtctgtct cattccgcag cttcacacat tcaaaggagc actaccactt ctacgagata 1560 tcatctttct ctgaaaccaa ggccaagcgc ctcatcaagg aggctggcaa tgagtttgtg 1620 cagcacaata cttggcagtt aagcσgtgtg tatcαcagcg gσctgaggac agactcttcc 1680 aactacaacc cccaggaact ctggaatgca ggαtgccaga tggtggccat gaatatgcag 1740 actgcagggc ttgaaatgga catctgtgat gggcatttcc gccagaatgg cggctgtggc 1800 tatgtgctga agccagactt cαtgcgtgat atccagagtt ctttccaccc tgagaagccc 1860 atcagccctt tcaaagccca gactctctta atαcaggtga tcagσggtca gcaactcccc 1920 aaagtggaca agaccaaaga ggggtccatt gtggatccac tggtgaaagt gcagatcttt 1980 ggcgttcgtc tagaσacagc acggcaggag accaactatg tggagaacaa tggttttaat 2040 ccataαtggg ggcagacact atgtttccgg gtgctggtgc ctgaacttgc catgctgσgt 2100 tttgtggtaa tggattatga ctggaaatcc cgaaatgact ttattggtca gtacaccctg 2160 αcttggacαt gcatgcaaca aggttaccgc cacattcacc tgctgtccaa agatggcatc 2220 agcctccgcc cagcttccat ctttgtgtat atctgcatcc aggaaggcct ggagggggat 2280 gagtcctga 2289

<210> 2

<211> 762

<212> PRT

<213> Homo sapiens

<400> 2

Met Ala Ser Leu Leu Gin Asp Gin Leu Thr Thr Asp Gin Asp Leu Leu 1 5 10 15

Leu Met Gin Glu Gly Met Pro Met Arg Lys Val Arg Ser Lys Ser Trp 20 25 30 Lys Lys Leu Arg Tyr Phe Arg Leu Gin Asn Asp Gly Met Thr Val Trp 35 40 45

His Ala Arg Gin Ala Arg Gly Ser Ala Lys Pro Ser Phe Ser He Ser 50 55 60

Asp Val Glu Thr He Arg Asn Gly His Asp Ser Glu Leu Leu Arg Ser 65 70 75 80

Leu Ala Glu Glu Leu Pro Leu Glu Gin Gly Phe Thr He Val Phe His 85 90 95

Gly Arg Arg Ser Asn Leu Asp Leu Met Ala Asn Ser Val Glu Glu Ala 100 105 110

Gin He Trp Met Arg Gly Leu Gin Leu Leu Val Asp Leu Val Thr Ser 115 120 125

Met Asp His Gin Glu Arg Leu Asp Gin Trp Leu Ser Asp Trp Phe Gin 130 135 140

Arg Gly Asp Lys Asn Gin Asp Gly Lys Met Ser Phe Gin Glu Val Gin 145 150 155 160

Arg Leu Leu His Leu Met Asn Val Glu Met Asp Gin Glu Tyr Ala Phe 165 170 175

Ser Leu Phe Gin Ala Ala Asp Thr Ser Gin Ser Gly Thr Leu Glu Gly 180 185 190

Glu Glu Phe Val Gin Phe Tyr Lys Ala Leu Thr Lys Arg Ala Glu Val 195 200 205

Gin Glu Leu Phe Glu Ser Phe Ser Ala Asp Gly Gin Lys Leu Thr Leu 210 215 220

Leu Glu Phe Leu Asp Phe Leu Gin Glu Glu Gin Lys Glu Arg Asp Cys 225 230 235 240

Thr Ser Glu Leu Ala Leu Glu Leu He Asp Arg Tyr Glu Pro Ser Asp 245 250 255

Ser Gly Lys Leu Arg His Val Leu Ser Met Asp Gly Phe Leu Ser Tyr 260 265 270 Leu Cys Ser Lys Asp Gly Asp He Phe Asn Pro Ala Cys Leu Pro He 275 280 285

Tyr Gin Asp Met Thr Gin Pro Leu Asn His Tyr Phe He Cys Ser Ser 290 295 300

His Asn Thr Tyr Leu Val Gly Asp Gin Leu Cys Gly Gin Ser Ser Val 305 310 315 320

Glu Gly Tyr He Arg Ala Leu Lys Arg Gly Cys Arg Cys Val Glu Val 325 330 335

Asp Val Trp Asp Gly Pro Ser Gly Glu Pro Val Val Tyr His Gly His 340 345 350

Thr Leu Thr Ser Arg He Leu Phe Lys Asp Val Val Ala Thr Val Ala 355 360 365

Gin Tyr Ala Phe Gin Thr Ser Asp Tyr Pro Val He Leu Ser Leu Glu 370 375 380

Thr His Cys Ser Trp Glu Gin Gin Gin Thr Met Ala Arg His Leu Thr 385 390 395 400

Glu He Leu Gly Glu Gin Leu Leu Ser Thr Thr Leu Asp Gly Val Leu 405 410 415

Pro Thr Gin Leu Pro Ser Pro Glu Glu Leu Arg Arg Lys He Leu Val 420 425 430

Lys Gly Lys Lys Leu Thr Leu Glu Glu Asp Leu Glu Tyr Glu Glu Glu 435 440 445

Glu Ala Glu Pro Glu Leu Glu Glu Ser Glu Leu Ala Leu Glu Ser Gin 450 455 460

Phe Glu Thr Glu Pro Glu Pro Gin Glu Gin Asn Leu Gin Asn Lys Asp 465 470 475 480

Lys Lys Lys Lys Ser Lys Pro He Leu Cys Pro Ala Leu Ser Ser Leu 485 490 495

Val He Tyr Leu Lys Ser Val Ser Phe Arg Ser Phe Thr His Ser Lys 500 505 510 Glu His Tyr His Phe Tyr Glu He Ser Ser Phe Ser Glu Thr Lys Ala 515 520 525

Lys Arg Leu He Lys Glu Ala Gly Asn Glu Phe Val Gin His Asn Thr 530 535 540

Trp Gin Leu Ser Arg Val Tyr Pro Ser Gly Leu Arg Thr Asp Ser Ser 545 550 555 560

Asn Tyr Asn Pro Gin Glu Leu Trp Asn Ala Gly Cys Gin Met Val Ala 565 570 575

Met Asn Met Gin Thr Ala Gly Leu Glu Met Asp He Cys Asp Gly His 580 585 590

Phe Arg Gin Asn Gly Gly Cys Gly Tyr Val Leu Lys Pro Asp Phe Leu 595 600 605

Arg Asp He Gin Ser Ser Phe His Pro Glu Lys Pro He Ser Pro Phe 610 615 620

Lys Ala Gin Thr Leu Leu He Gin Val He Ser Gly Gin Gin Leu Pro 625 630 635 640

Lys Val Asp Lys Thr Lys Glu Gly Ser He Val Asp Pro Leu Val Lys 645 650 655

Val Gin He Phe Gly Val Arg Leu Asp Thr Ala Arg Gin Glu Thr Asn 660 665 670

Tyr Val Glu Asn Asn Gly Phe Asn Pro Tyr Trp Gly Gin Thr Leu Cys 675 680 685

Phe Arg Val Leu Val Pro Glu Leu Ala Met Leu Arg Phe Val Val Met 690 695 700

Asp Tyr Asp Trp Lys Ser Arg Asn Asp Phe He Gly Gin Tyr Thr Leu 705 710 715 720

Pro Trp Thr Cys Met Gin Gin Gly Tyr Arg His He His Leu Leu Ser 725 730 735 Lys Asp Gly He Ser Leu Arg Pro Ala Ser He Phe Val Tyr He Cys 740 745 750

He Gin Glu Gly Leu Glu Gly Asp Glu Ser 755 760

<210> 3

<211> 762

<212> PRT

<213> Homo sapiens

<400> 3

Met Ala Ser Leu Leu Gin Asp Gin Leu Thr Thr Asp Gin Asp Leu Leu 1 5 10 15

Leu Met Gin Glu Gly Met Pro Met Arg Lys Val Arg Ser Lys Ser Trp 20 25 30

Lys Lys Leu Arg Tyr Phe Arg Leu Gin Asn Asp Gly Met Thr Val Trp 35 40 45

His Ala Arg Gin Ala Arg Gly Ser Ala Lys Pro Ser Phe Ser He Ser 50 55 60

Asp Val Glu Thr He Arg Asn Gly His Asp Ser Glu Leu Leu Arg Ser 65 70 75 80

Leu Ala Glu Glu Leu Pro Leu Glu Gin Gly Phe Thr He Val Phe His 85 90 95

Gly Arg Arg Ser Asn Leu Asp Leu Met Ala Asn Ser Val Glu Glu Ala 100 105 110

Gin He Trp Met Arg Gly Leu Gin Leu Leu Val Asp Leu Val Thr Ser 115 120 125

Met Asp His Gin Glu Arg Leu Asp Gin Trp Leu Ser Asp Trp Phe Gin 130 135 140

Arg Gly Asp Lys Asn Gin Asp Gly Lys Met Ser Phe Gin Glu Val Gin 145 150 155 160

Arg Leu Leu His Leu Met Asn Val Glu Met Asp Gin Glu Tyr Ala Phe 165 170 175 Ser Leu Phe Gin Ala Ala Asp Thr Ser Gin Ser Gly Thr Leu Glu Gly 180 185 190

Glu Glu Phe Val Gin Phe Tyr Lys Ala Leu Thr Lys Arg Ala Glu Val 195 200 205

Gin Glu Leu Phe Glu Ser Phe Ser Ala Asp Gly Gin Lys Leu Thr Leu 210 215 220

Leu Glu Phe Leu Asp Phe Leu Gin Glu Glu Gin Lys Glu Arg Asp Cys 225 230 235 240

Thr Ser Glu Leu Ala Leu Glu Leu He Asp Arg Tyr Glu Pro Ser Asp 245 250 255

Ser Gly Lys Leu Arg His Val Leu Ser Met Asp Gly Phe Leu Ser Tyr 260 265 270

Leu Cys Ser Lys Asp Gly Asp He Phe Asn Pro Ala Cys Leu Pro He 275 280 285

Tyr Gin Asp Met Thr Gin Pro Leu Asn His Tyr Phe He Cys Ser Ser 290 295 300

His Asn Thr Tyr Leu Val Gly Asp Gin Leu Cys Gly Gin Ser Ser Val 305 310 315 320

Glu Gly Tyr He Arg Ala Leu Lys Arg Gly Cys Arg Cys Val Glu Val 325 330 335

Asp Val Trp Asp Gly Pro Ser Gly Glu Pro Val Val Tyr His Gly His 340 345 350

Thr Leu Thr Ser Arg He Leu Phe Lys Asp Val Val Ala Thr Val Ala 355 360 365

Gin Tyr Ala Phe Gin Thr Ser Asp Tyr Pro Val He Leu Ser Leu Glu 370 375 380

Thr His Cys Ser Trp Glu Gin Gin Gin Thr Met Ala Arg His Leu Thr 385 390 395 400

Glu He Leu Gly Glu Gin Leu Leu Ser Thr Thr Leu Asp Gly Val Leu 405 410 415 Pro Thr Gin Leu Pro Ser Pro Glu Glu Leu Arg Arg Lys He Leu Val 420 425 430

Lys Gly Lys Lys Leu Thr Leu Glu Glu Asp Leu Glu Tyr Glu Glu Glu 435 440 445

Glu Ala Glu Pro Glu Leu Glu Glu Ser Glu Leu Ala Leu Glu Ser Gin 450 455 460

Phe Glu Thr Glu Pro Glu Pro Gin Glu Gin Asn Leu Gin Asn Lys Asp 465 470 475 480

Lys Lys Lys Lys Ser Lys Pro He Leu Cys Pro Ala Leu Ser Ser Leu 485 490 495

Val He Tyr Leu Lys Ser Val Ser Phe Arg Ser Phe Thr His Ser Lys 500 505 510

Glu His Tyr His Phe Tyr Glu He Ser Ser Phe Ser Glu Thr Lys Ala 515 520 525

Lys Arg Leu He Lys Glu Ala Gly Asn Glu Phe Val Gin His Asn Thr 530 535 540

Trp Gin Leu Ser Arg Val Tyr Pro Ser Gly Leu Arg Thr Asp Ser Ser 545 550 555 560

Asn Tyr Asn Pro Gin Glu Leu Trp Asn Ala Gly Cys Gin Met Val Ala 565 570 575

Met Asn Met Gin Thr Ala Gly Leu Glu Met Asp He Cys Asp Gly His 580 585 590

Phe Arg Gin Asn Gly Gly Cys Gly Tyr Val Leu Lys Pro Asp Phe Leu 595 600 605

Arg Asp He Gin Ser Ser Phe His Pro Glu Lys Pro He Ser Pro Phe 610 615 620

Lys Ala Gin Thr Leu Leu He Gin Val He Ser Gly Gin Gin Leu Pro 625 630 635 640 Lys Val Asp Lys Thr Lys Glu Gly Ser He Val Asp Pro Leu Val Lys 645 650 655

Val Gin He Phe Gly Val Arg Leu Asp Thr Ala Arg Gin Glu Thr Asn 660 665 670

Tyr Val Glu Asn Asn Gly Phe Asn Pro Tyr Trp Gly Gin Thr Leu Cys 675 680 685

Phe Arg Val Leu Val Pro Glu Leu Ala Met Leu Arg Phe Val Val Met 690 695 700

Asp Tyr Asp Trp Lys Ser Arg Asn Asp Phe He Gly Gin Tyr Thr Leu 705 710 715 720

Pro Trp Thr Cys Met Gin Gin Gly Tyr Arg His He His Leu Leu Ser 725 730 735

Lys Asp Gly He Ser Leu Arg Pro Ala Ser He Phe Val Tyr He Cys 740 745 750

He Gin Glu Gly Leu Glu Gly Asp Glu Ser 755 760

<210> 4

<211> 3116

<212> DNA

<213> Homo sapiens

<400> 4 ggcacgaggg cαagctgctg tagaagaggg gaggaaacaa gccagtgcaa ggggagcaaa 60 agagaaaagg agccaggαtg ggcttcctga tcccacagca tcgcagagct αgggaggcac 120 agctcacaga cacaggaaac acaggaσtgc tattctgctα tcctgcccac ggtgatctgg 180 tgccagctgg tggaaαagtg ggtgatggcg tαcαtgctgc aagacαagct gaccaαtgat 240 caggacttgc tgctgatgca ggaaggcatg ccgatgcgca aggtgaggtα αaaaagαtgg 300 aagaagαtaa gatacttcag acttcagaat gacggcatga cagtctggca tgcacggαag 360 gccaggggca gtgccaagcc cagcttσtca atctctgatg tggagacaat acgtaatggc 420 catgattccg agttgctgαg tagαctggca gaggagctcc ccctggagca gggcttcacc 480 attgtαttcc atggccgccg ctccaacctg gacctgatgg ccaacagtgt tgaggaggcc 540 cagatatgga tgcgagggct cαagctgttg gtggatcttg tcaccagcat ggaccatcag 600 gagcgcctgg accaatggct gagcgattgg tttcaacgtg gagacaaaaa tcaggatggt 660 - I l l -

aagatgagtt tαcaagaagt tcagcggtta ttgcacctaa tgaatgtgga aatggacαaa 720 gaatatgcct tcagtctttt tσaggcagca gacacgtccc agtctggaac ασtggaagga 780 gaagaattcg tacagttcta taaggcattg aαtaaacgtg ctgaggtgca ggaaαtgttt 840 gaaagttttt cagαtgatgg gcagaagαtg aαtctgctgg aatttttgga tttcctccaa 900 gaggagcaga aggagagaga ctgcacctct gagcttgctα tggaactcat tgaccgctat 960 gaaccttcag aαagtggαaa actgcggcat gtgctgagta tggatggctt cctcagσtac 1020 ctctgσtcta aggatggaga catcttcaac cαagcctgcc tαccαatcta tcaggatatg 1080 actσaacccc tgaaccaαta cttcatctgα tcttctcata acacctacαt agtgggggac 1140 cagctttgαg gccagagcag cgtcgaggga tatatacggg ccctgaagcg ggggtgcαgα 1200 tgcgtggagg tggatgtatg ggatggacct agcggggaac ctgtcgttta ccacggacac 1260 aσαctgacct cccgcatααt gttαaaagat gtcgtggcαa cagtagcaca gtatgαcttc 1320 cagacatcag actacccagt αatcttgtcc ctggagaccc actgcagctg ggagcagαag 1380 cagacσatgg ccαgtcatct gactgagatc ctgggggagc agctgctgag αaccaccttg 1440 gatggggtgα tgcccaαtca gαtgccctcg cctgaggagα ttcggaggaa gatcαtggtg 1500 aaggggaaga agttaacact tgaggaagac ctggaatatg aggaagagga agcagaacct 1560 gagttggaag agtcagaatt ggcgctggag tcccagtttg agaαtgagcα tgagccccag 1620 gagαagaacc ttcagaataa ggacaaaaag aagaaatcca agαcαatctt gtgtccagcc 1680 ctctcttccc tggttatαta cttgaagtct gtσtcattcc gcagcttcaα acattcaaag 1740 gagcactacα aαttctacga gatatcatct ttσtctgaaa ccaaggccaa gcgcctcatc 1800 aaggaggctg gcaatgagtt tgtgcagcac aataαttggc agttaagccg tgtgtatccα 1860 agcggαctga ggacagactc ttccaactac aacccαcagg aaαtctggaa tgcaggctgα 1920 cagatggtgg ccatgaatat gcagactgca gggcttgaaa tggacatctg tgatgggαat 1980 ttccgαcaga atggcggctg tggctatgtg ctgaagcαag acttcctgcg tgatatccag 2040 agttctttαc accctgagaa gcααatcagc cctttcaaag cccagactct cttaatccag 2100 gtgatcagcg gtcagcaact ccccaaagtg gacaagacca aagaggggtc αattgtggat 2160 ccaσtggtga aagtgcagat ctttggαgtt cgtctagaca αagcacggca ggagaccaac 2220 tatgtggaga acaatggttt taatαcatac tgggggcaga cactatgttt ccgggtgctg 2280 gtgcctgaac ttgccatgct gcgttttgtg gtaatggatt atgaαtggaa atcccgaaat 2340 gactttattg gtcagtacac cctgccttgg acσtgαatgc aacaaggtta ccgccacatt 2400 cacctgctgt cαaaagatgg αatcagcctα cgccαagctt ccatctttgt gtatatctgα 2460 atccaggaag gcαtggaggg ggatgagtcα tgaggtgggc atttcacggg aagggttggt 2520 atgctggctt tagacgggga gaaacatctg gaaggatgct cgagagaaca aatggaggtg 2580 gtgaaaatca agαtttggat tgtgcattαc taggcacaaa attacctcat tcttcctaac 2640 aagcaatctg ggacctgatt ttccaccttt tttctctttt cttcααttcc tttgttt ca 2700 taagcctttg gtatctttαc tgσccttttα ctttgtgtaα tctatactgg agttcccttc 2760 ttcctαttgc tgtaggαtca atcccatacc gacatαtaca aαtaatcttt cccatcaaαt 2820 ctgtgtgaag gαaggttgca aσtagaaatt σagaggggct tggaatagag aaacσtaaag 2880 aagcatcatc cσctccatcc ccaacttcct caaagccoaa agccaaggga aggataaatc 2940 aaggctcaag gcttccccag caaagattag ggaaagagac ttgaαααcag gactgtacta 3000 cgaαtcttaa gagaacaαtg cacagcaαtc aaagtccccc actggactgα ttcctcαtta 3060 gccccactgg tataaataca tctctαtcca atttggcaaa aaaaaaaaaa aaaaaa 3116

<210> 5

<211> 20

<212> DNA

<213> Homo sapiens

<400> 5 gtgttgagga ggαccagata 20

<210> 6

<211> 20

<212> DNA

<213> Homo sapiens

<400> 6 tggtgacaag atccacαaac 20

<210> 7

<211> 20

<212> DNA

<213> Homo sapiens

<400> 7 tggatgcgag ggctccagct 20

Claims

1. An isolated polynucleotide being selected from the group consisting of:

a. a polynucleotide encoding a phospholipase C delta-like enzyme polypeptide comprising an amino acid sequence selected form the group consisting of:

i. amino acid sequences which are at least about 72% identical to the amino acid sequence shown in SEQ ID NO: 2; and ii. the amino acid sequence shown in SEQ ID NO: 2.

b. a polynucleotide comprising the sequence of SEQ ID NO: 1 ;

c. a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a phospholipase C delta-like enzyme polypeptide;

d. a polynucleotide the sequence of which deviates from the poly- nucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a phospholipase C delta-like enzyme polypeptide; and

e. a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a phospholipase C delta-like enzyme polypeptide.

2. An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

. A substantially purified phospholipase C delta-like enzyme polypeptide encoded by a polynucleotide of claim 1.

5. A method for producing a phospholipase C delta-like enzyme polypeptide, wherein the method comprises the following steps:

a. culturing the host cell of claim 3 under conditions suitable for the expression of the phospholipase C delta-like enzyme polypeptide; and

b. recovering the phospholipase C delta-like enzyme polypeptide from the host cell culture.

6. A method for detection of a polynucleotide encoding a phospholipase C deltalike enzyme polypeptide in a biological sample comprising the following steps:

a. hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and

b. detecting said hybridization complex.

7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or a phospholipase

C delta-like enzyme polypeptide of claim 4 comprising the steps of:

a. contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the phospholipase C delta-like enzyme polypeptide; and b. detecting the interaction.

9. A diagnostic kit for conducting the method of any one of claims 6 to 8.

10. A method of screening for agents which decrease the activity of a phospholipase C delta-like enzyme, comprising the steps of:

a. contacting a test compound with any phospholipase C delta-like enzyme polypeptide encoded by any polynucleotide of claim 1;

b. detecting binding of the test compound to the phospholipase C deltalike enzyme polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a phospholipase C delta-like enzyme.

11. A method of screening for agents which regulate the activity of a phospholipase C delta-like enzyme, comprising the steps of:

a. contacting a test compound with a phospholipase C delta-like enzyme polypeptide encoded by any polynucleotide of claim 1; and

b. detecting a phospholipase C delta-like enzyme activity of the polypeptide, wherein a test compound which increases the phospholipase C delta-like enzyme activity is identified as a potential therapeutic agent for increasing the activity of the phospholipase C delta-like enzyme, and wherein a test compound which decreases the phospholipase C delta-like enzyme activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the phospholipase C delta-like enzyme.

2. A method of screening for agents which decrease the activity of a phospholipase C delta-like enzyme, comprising the steps of:

a. contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of phospholipase C delta-like enzyme.

13. A method of reducing the activity of phospholipase C delta-like enzyme, comprising the steps of:

a. contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any phospholipase C delta-like enzyme polypeptide of claim 4, whereby the activity of phospholipase C deltalike enzyme is reduced.

14. A reagent that modulates the activity of a phospholipase C delta-like enzyme polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.

15. A pharmaceutical composition, comprising the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the expression vector of claim 2 or the reagent of claim 14 in the preparation of a medicament for modulating the activity of a phospholipase C delta-like enzyme in a disease.

17. Use of claim 16 wherein the disease is a CNS disorder, cancer, COPD, diabetes or asthma.