WO2002066623A2 - Regulation of human phosphatidic acid-preferring phospholipase a1 - Google Patents

Abstract

Reagents that regulate human phosphatidic acid-preferring phospholipase A1 and reagents which bind to human phosphatidic acid-preferring phospholipase A1 gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, CNS disorders, genitourinary disorders, cardiovascular disorders, hematological disorders, cancer, asthma or COPD.

Description

REGULATION OF HUMAN PHOSPHATIDIC ACID-PREFERRING PHOSPHOLIPASE Al

This application incorporates by reference co-pending provisional application Serial

No. 60/269,856 filed February 21, 2001.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the regulation of human phosphatidic acid-preferring phospholipase Al.

BACKGROUND OF THE INVENTION

Phospholipase Al is a hydro lytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. There is a need in the art to identify related 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 phosphatidic acid-preferring phospholipase Al. 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 phosphatidic acid-preferring phospholipase Al polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2 and; the amino acid sequence shown in SEQ D 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 phosphatidic acid-preferring phospholipase Al polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2 and; the amino acid sequence shown in SEQ ID NO: 2;

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

Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a poly- nucleotide encoding a phosphatidic acid-preferring phospholipase Al 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 ED 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 phosphatidic acid-preferring phospholipase Al through interacting with the phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2 and; the amino acid sequence shown in SEQ ID NO: 2.

A phosphatidic acid-preferring phospholipase Al activity of the polypeptide is detected. A test compound which increases phosphatidic acid-preferring phospholipase Al activity of the polypeptide relative to phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al activity of the polypeptide relative to phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 TD NO: 1 and; the nucleotide sequence shown in SEQ ID NO: 1 ;

Binding of the test compound to the phosphatidic acid-preferring phospholipase Al product is detected. A test compound which binds to the phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 ID NO: 1 and; the nucleotide sequence shown in SEQ ID NO: 1 ;

Phosphatidic acid-preferring phospholipase Al activity in the cell is thereby decreased.

The invention thus provides a human phosphatidic acid-preferring phospholipase Al that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site. Human phosphatidic acid-preferring phospho- lipase Al 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 phosphatidic acid-preferring phospholipase Al 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 trembl AF045022*AF045022_1 (SEQ ID NO: 3).

Fig. 4 shows the DNA-sequence encoding a phosphatidic acid-preferring phospholipase Al Polypeptide (SEQ ID NO: 4).

Fig. 5 shows the DNA-sequence encoding a phosphatidic acid-preferring phospholipase Al Polypeptide (SEQ ID NO: 5).

Fig. 6 shows the DNA-sequence encoding a phosphatidic acid-preferring phospholipase Al Polypeptide (SEQ ID NO: 6).

Fig. 7 shows the DNA-sequence encoding a phosphatidic acid-preferring phospholipase Al Polypeptide (SEQ ID NO: 7).

Fig. 8 shows the DNA-sequence encoding a phosphatidic acid-preferring phospholipase Al Polypeptide (SEQ ID NO: 8).

Fig. 9 shows the BLASTP - alignment of 425 (SEQ ID NO: 2) against trembl* AF045022* AF045022 (SEQ ID NO: 3).

Fig. 10 shows the BLASTN SNP alignments.

Fig. 11 shows the Genewise analysis.

Fig. 12 shows the Expression profile of phosphatidic acid-preferring phospholipase Al.

Fig. 13 shows the Expression profile of phosphatidic acid-preferring phospho- lipase Al. Fig. 14 shows the Expression profile of phosphatidic acid-preferring phospholipase Al.

DETAILED DESCRIPTION OF THE INVENTION

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

a) a polynucleotide encoding a phosphatidic acid-preferring phospholipase Al polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2 and; the amino acid sequence shown in SEQ TD 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide. Furthermore, it has been discovered by the present applicant that a novel phosphatidic acid-preferring phospholipase Al, paticularly a human phosphatidic adid- preferring phospholipase Al, can be used in therapeutic methods to treat a CNS disorder, a genitourinary disorder, a cardiovascular disorder, asthma, cancer, COPD or a hematological disorder. Human phosphatidic acid-preferring phospholipase Al comprises the amino acid sequence shown in SEQ ID NO: 2. A coding sequence for human phosphatidic acid-preferring phospholipase Al is shown in SEQ ID NO: 1. This sequence is located on chromosome 14. Related ESTs (SEQ ID NOS: 4-8) are expressed in testis, lymph, pooled tissues, infant brain, primitive neuroectoderm, nervous tumor, and fetal liver, spleen, and bone. Expression in CNS tissues indicates that this protein may play a role in brain-specific disorders, such as Alzheimer's disease and Parkinson's disease.

Human phosphatidic acid-preferring phospholipase Al is 88% identical over 876 amino acids to phosphatidic acid-preferring phospholipase Al (FIG. 1). Human phosphatidic acid-preferring phospholipase Al contains an amino acid sequence, Ser535-His-Ser-Leu-Gly539, which resembles the consensus sequence containing the active serine nucleophile present in most lipases, GXSXG.

Human phosphatidic acid-preferring phospholipase Al of the invention is expected to be useful for the same purposes as previously identified phosphatidic acid- preferring phospholipase Al enzymes. Human phosphatidic acid-preferring phospholipase Al is believed to be useful in therapeutic methods to treat disorders such as CNS disorders, genitourinary disorders, cardiovascular disorders, and hematological disorders. Human phosphatidic acid-preferring phospholipase Al also can be used to screen for human phosphatidic acid-preferring phospholipase Al activators and inhibitors. Polypeptides

Human phosphatidic acid-preferring phospholipase Al 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, 525, 550, 575, 600, 625, 650,

675, 700, 725, 750, 775, 800, 825, 850, or 852 contiguous amino acids selected from the amino acid sequence shown in SEQ TD NO: 2 or a biologically active variant thereof, as defined below. A phosphatidic acid-preferring phospholipase Al polypeptide of the invention therefore can be a portion of a phosphatidic acid-preferring phospholipase Al protein, a full-length phosphatidic acid-preferring phospholipase

Al protein, or a fusion protein comprising all or a portion of a phosphatidic acid- preferring phospholipase Al protein.

Biologically Active Variants

Human phosphatidic acid-preferring phospholipase Al polypeptide variants that are biologically active, e.g., retain a phospholipase Al activity, also are phosphatidic acid-preferring phospholipase Al polypeptides. Preferably, naturally or non- naturally occurring phosphatidic acid-preferring phospholipase Al polypeptide variants have amino acid sequences which are at least about 88, 90, 96, 96, 98, or

99% identical to the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof. Percent identity between a putative phosphatidic acid-preferring phospholipase Al polypeptide variant and an amino acid sequence of SEQ ID NO: 2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).

Variations 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 phosphatidic acid-preferring phospholipase Al polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active phosphatidic acid-preferring phospholipase Al polypeptide can readily be determined by assaying for phospholipase Al activity, as described for example, in U.S. Patent 5,538,874.

Fusion Proteins

Fusion proteins are useful for generating antibodies against phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al poly- peptide. 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 phosphatidic acid-preferring phospholipase Al 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, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, or 852 contiguous amino acids of SEQ DO NO: 2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length phosphatidic acid-preferring phospholipase Al 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, horseradish 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 (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the phosphatidic acid-preferring phospholipase Al polypeptide-encoding sequence and the heterologous protein sequence, so that the phosphatidic acid-preferring phospholipase Al 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 DNA methods can be used to prepare fusion proteins, for example, by making a DNA 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 homo logs of human phosphatidic acid-preferring phospholipase Al polypeptide can be obtained using phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides

A phosphatidic acid-preferring phospholipase Al polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a phosphatidic acid-preferring phospholipase Al polypeptide. A coding sequence for human phosphatidic acid-preferring phospholipase Al is shown in SEQ

LD NO: 1.

Degenerate nucleotide sequences encoding human phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polynucleotides that encode biologically active phosphatidic acid- preferring phospholipase Al polypeptides also are phosphatidic acid-preferring phospholipase Al polynucleotides. Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO: 1 or its complement also are phosphatidic acid-preferring phospholipase Al polynucleotides. 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 phosphatidic acid-preferring phospholipase Al polynucleotides described above also are phosphatidic acid-preferring phospholipase Al polynucleotides. Typically, homologous phosphatidic acid-preferring phospholipase Al polynucleotide sequences can be identified by hybridization of candidate poly- nucleotides to known phosphatidic acid-preferring phospholipase Al polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions-2X SSC (0.3 M NaCl, 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 phosphatidic acid-preferring phospholipase Al polynucleo- tides 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polynucleotides or phosphatidic acid-preferring phospholipase Al polynucleotides of other species can therefore be identified by hybridizing a putative homologous phosphatidic acid- preferring phospholipase Al polynucleotide with a polynucleotide having a nucleo- tide 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 polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to phosphatidic acid-preferring phospholipase

Al polynucleotides or their complements following stringent hybridization and/or wash conditions also are phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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) - 60011), 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid- preferring phospholipase Al polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise phosphatidic acid-preferring phospholipase Al nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.

Human phosphatidic acid-preferring phospholipase Al cDNA molecules can be made with standard molecular biology techniques, using phosphatidic acid-preferring phospholipase Al mRNA as a template. Human phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a phosphatidic acid-preferring phospholipase Al 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 PROMOTERFINDER 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 phosphatidic acid-preferring phospholipase Al polypeptides can be obtained, for example, by purification from human cells, by expression of phosphatidic acid- preferring phospholipase Al polynucleotides, or by direct chemical synthesis.

Protein Purification

Human phosphatidic acid-preferring phospholipase Al polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with phosphatidic acid-preferring phospholipase Al expression constructs. A purified phosphatidic acid-preferring phospholipase Al polypeptide is separated from other compounds that normally associate with the phosphatidic acid- preferring phospholipase Al polypeptide in the cell, such as certain proteins, carbo- hydrates, 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polynucleotide, the polynucleotide 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide. For example, when a large quantity of a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospho- lipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 (RSN) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DΝA 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 phosphatidic acid-preferring phospholipase Al polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid- preferring phospholipase Al sequences. Resistant clones of stably transformed 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 aprf 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 at 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 phosphatidic acid- preferring phospholipase Al polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a phosphatidic acid-preferring phospholipase Al polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the phosphatidic acid-preferring phospholipase Al polynucleotide.

Alternatively, host cells which contain a phosphatidic acid-preferring phospholipase

Al polynucleotide and which express a phosphatidic acid-preferring phospholipase Al 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 mem- brane, 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 phosphatidic acid-preferring phospholipase Al polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a phosphatidic acid-preferring phospholipase Al polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a phosphatidic acid- preferring phospholipase Al polypeptide to detect transformants that contain a phosphatidic acid-preferring phospholipase Al polynucleotide.

A variety of protocols for detecting and measuring the expression of a phosphatidic acid-preferring phospholipase Al 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 immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a phos- phatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a phosphatidic acid- preferring phospholipase Al polypeptide can be cloned into a vector for the production of an rnRNA 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 phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid- preferring phospholipase Al polypeptides can be designed to contain signal sequences which direct secretion of soluble phosphatidic acid-preferring phospho- lipase Al polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound phosphatidic acid-preferring phospholipase Al polypeptide.

As discussed above, other constructions can be used to join a sequence encoding a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a phosphatidic acid-preferring phospholipase Al polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by EVIAC (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 phosphatidic acid-preferring phospholipase Al polypeptide from the fusion protein. Vectors that contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 72, 441-453, 1993. Chemical Synthesis

Sequences encoding a phosphatidic acid-preferring phospholipase Al 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, 4980; Horn et al. Nucl.

Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide can be used therapeutically, as well as in immuno- chemical 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid- preferring phospholipase Al polypeptides do not detect other proteins in immuno- chemical assays and can immunoprecipitate a phosphatidic acid-preferring phospholipase Al polypeptide from solution.

Human phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, 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-Gueriή) and Corynebacterium parvum are especially useful.

Monoclonal antibodies that specifically bind to a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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, Eur. 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; Νicholls et al, 1993, J. Immunol. Meth. 165, 81-91).

Antibodies which specifically bind to phosphatidic acid-preferring phospholipase Al 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 multivalent 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the phosphatidic acid-preferring phospholipase

Al gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are prefeπed. 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 phosphatidic acid- preferring phospholipase Al polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a phosphatidic acid-preferring phospholipase Al polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent phosphatidic acid-preferring phospholipase Al nucleotides, can provide sufficient targeting specificity for phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid- preferring phospholipase Al polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a phosphatidic acid-preferring phospholipase Al 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.

Ribozymes

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 phosphatidic acid-preferring phospholipase Al polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase

Al 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 phosphatidic acid-preferring phospholipase Al RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleo- tides 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 phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, CNS disorders, genitourinary disorders, cardiovascular disorders, and hematological disorders. 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 phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human phosphatidic acid- preferring phospholipase Al. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide or a phosphatidic acid-preferring phospholipase Al polynucleotide. A test compound preferably binds to a phosphatidic acid-preferring phospholipase Al polypeptide or polynucleotide. More preferably, a test compound decreases or increases phospholipase Al activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound. Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The com- pounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, 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 phosphatidic acid- preferring phospholipase Al polypeptides or polynucleotides or to affect phospha- tidic acid-preferring phospholipase Al activity or phosphatidic acid-preferring phospholipase Al 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 microtiter 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 combina- torial 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. In 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide. A microphysio- meter (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 phosphatidic acid-preferring phospholipase Al polypeptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a phosphatidic acid-preferring phospholipase Al 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.

In yet another aspect of the invention, a phosphatidic acid-preferring phospholipase

Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 proteinrdependent 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 phosphatidic acid- preferring phospholipase Al polypeptide.

It may be desirable to immobilize either the phosphatidic acid-preferring phospho- lipase Al 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 phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide (or polynucleotide) 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 phosphatidic acid-preferring phospholipase Al polypeptide is a fusion protein comprising a domain that allows the phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Bio- tinylated phosphatidic acid-preferring phospholipase Al polypeptides (or polynucleotides) 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 phosphatidic acid-preferring phospholipase Al polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the phosphatidic acid-preferring phospholipase Al polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a phosphatidic acid-preferring phospholipase Al polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a phosphatidic acid-preferring phospholipase Al polypeptide or polynucleotide can be used in a cell-based assay system. A phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide or polynucleotide is determined as described above.

Enzyme Assays

Test compounds can be tested for the ability to increase or decrease the activity of a human phosphatidic acid-preferring phospholipase Al polypeptide. Phospholipase

Al activity can be measured, for example, as described in U.S. Patent 5,538,874.

Enzyme assays can be carried out after contacting either a purified phosphatidic acid- preferring phospholipase Al polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases a phospholipase Al activity of a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al activity. A test compound which increases a phospholipase Al activity of a human phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al activity.

Gene Expression

In another embodiment, test compounds that increase or decrease phosphatidic acid- preferring phospholipase Al gene expression are identified. A phosphatidic acid- preferring phospholipase Al polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radio- immunoassay, 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 phosphatidic acid-preferring phospholipase Al polypeptide. Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a phosphatidic acid-preferring phospholipase Al polynucleotide can be used in a cell-based assay system. The phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide, phosphatidic acid-preferring phospholipase Al polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a phosphatidic acid-preferring phospholipase Al polypeptide, or mimetics, activators, or inhibitors of a phosphatidic acid-preferring phospholipase Al 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% mannitol, 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 phosphatidic acid-preferring phospholipase Al can be regulated to treat CNS disorders, genitourinary disorders, cardiovascular disorders, and hematological disorders.

CNS disorders

Central and peripheral nervous system disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain.

Pain that is associated with CNS disorders also can be treated by regulating the activity of human phosphatidic acid-preferring phospholipase Al. Pain which can be treated includes that associated with central nervous system 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 mal- formation). Non-central neuropathic pain includes that associated with post mastectomy pain, 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 cancer and cancer treatment also can be treated, as can 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.

Cardiovascular Disorders.

Cardiovascular diseases include the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular aπhythmias, hypertensive vascular diseases, and peripheral vascular diseases.

Heart failure is defined as a pathophysiologic 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 failure, 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.

Aπhythmias include all forms of atrial and ventricular tachyaπhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexcitation syndrome, ventricular tachycardia, ventricular flutter, and ventricular fibrillation), as well as bradycardic forms of arrhythmias.

Vascular diseases include primary as well as all kinds of secondary arterial hyper- tension (renal, endocrine, neurogenic, others). The disclosed gene and its product may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications. 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.

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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al activity can be administered to a human cell, either in vitro or in vivo, to reduce phosphatidic acid-preferring phospholipase Al activity. The reagent preferably binds to an expression product of a human phosphatidic acid-preferring phospholipase Al 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 liposome. Preferably, the liposome 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 liposome 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 liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome 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 liposome is about

0.5 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10° cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome 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 prefeπed liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome. Complexing a liposome 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 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 phosphatidic acid-preferring phospholipase Al activity relative to the phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al gene or the activity of a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al gene or the activity of a phosphatidic acid-prefeπing phospholipase Al polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to phosphatidic acid-prefemng phospholipase Al- specific mRNA, quantitative RT-PCR, immunologic detection of a phosphatidic acid-preferring phospholipase Al polypeptide, or measurement of phosphatidic acid- preferring phospholipase Al 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 syner- gistically 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase A 1 activity

The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-phosphatidic acid-preferring phospholipase Al polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and phosphatidic acid-preferring phospholipase Al- activity is measured in the following assay: 0.05 ml of a 0.1M aqueous solution of calcium chloride and 0.25 ml of a 0.2M aqueous acetate buffer soultion (pH 4.0) are added to 0.5 ml of a solution formed by mixing a 2.0% (w/v) suspension of SLP- White (manufactured by True Lecithin Kogyo Co., Ltd.) and 4% (v/v) Triton X-100. TM. In water. Next, 0.1 ml of the cell extracts is added to the resulting mixture, and the mixture is stirred until a homogeneous mixture results. The mixture obtained in this manner is left to stand at 37°C for 10 minutes, in order to allow the enzyme reaction to take place. At the end of this time, 0.1 ml of a IN aqueous solution of hydrochloric acid is added to the reaction mixture to stop the enzyme reaction. A sample of 0.02 ml of the reaction mixture is then taken, and this is used to determine the amount of free fatty acid. Free fatty acid is quantified using a free fatty acid quantitative reagent, Determiner NEFA (Kyowa Medex Co, Ltd.). It is shown that the polypeptide of SEQ ID NO: 2 has a phosphatidic acid-preferring phospholipase Al activity.

EXAMPLE 2

Expression of recombinant human phosphatidic acid-preferring phospholipase A 1

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human phosphatidic acid-preferring phos- pholipase Al polypeptides in yeast. The phosphatidic acid-preferring phospholipase

A 1 -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 phosphatidic acid-preferring phospholipase Al polypeptide is obtained.

EXAMPLE 3

Identification of test compounds that bind to phosphatidic acid-preferring phospholipase Al polypeptides

Purified phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide.

EXAMPLE 4

Identification of a test compound which decreases phosphatidic acid-preferring phospholipase Al gene expression

A test compound is administered to a culture of human cells transfected with a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-prefeπing phospholipase Al -specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ED NO: 1. A test compound that decreases the phosphatidic acid-preferring phospholipase Al -specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of phosphatidic acid-preferring phospholipase Al gene expression. EXAMPLE 5

Identification of a test compound which decreases phosphatidic acid-preferring phospholipase A 1 activity

A test compound is administered to a culture of human cells transfected with a phosphatidic acid-preferring phospholipase Al 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, phosphatidic acid-preferring phospholipase Al activity is measured using the method of U.S. Patent 5,538,874.

A test compound which decreases the phospholipase Al activity of the phosphatidic acid-preferring phospholipase Al relative to the phospholipase Al activity in the absence of the test compound is identified as an inhibitor of phosphatidic acid- prefeπing phospholipase Al activity.

EXAMPLE 6

Tissue-specific expression of phosphatidic acid-preferring phospholipase Al

Total cellular RNA was isolated from cells by one of two standard methods:

(1) guanidine isothiocyanate/Cesium chloride density gradient centrifugation

(Maniatis et al, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, 1982) or (2) with the Tri-Reagent protocol according to the manufacturer's specifications (Molecular Research Center, Inc., Cincinnati, Ohio). Total RNA prepared by the Tri-reagent protocol was treated with DNase I to remove genomic DNA contamination. For library construction, poly A+ mRNA was selected using Oligotex kit from Qiagen (Santa Clara, Calif.) according to the manufacturer's specifications. Libraries were constructed using standard methods (Maniatis et al, 1982). For relative quantitation of the mRNA distribution of phosphatidic acid-preferring phospholipase Al, total RNA from each cell or tissue source was first reverse transcribed. Two μg of total RNA was reverse transcribed using 25 pmole random hexamer primers and 100 pmole poly dTι₅ (Boehringer Mannheim, Indianapolis,

Ind.), 0.5 mM each of dATP, dCTP, dGTP and dTTP (Pharmacia, Uppsala, Sweden), 5 mM DTT, 1 μl RNAsin (Promega, Madison Wis.) in a final volume of 20 μl. The first strand synthesis buffer and Superscript TJ (1 μl/2μl) reverse transcriptase were from Gibco/BRL (Gaithersburg, Md.). Replicate samples were also prepared similarly with the exception that no Superscript TT reverse transcriptase was added; these samples served as controls for genomic contamination. The reaction was incubated at 42-45°C for 90 minutes, heated to 95°C for 5 minutes and cooled on ice. The volume was adjusted to 200 μl with Tris HCI pH 7.4, yielding a final concentration of 10 ng/μl of starting RNA.

For relative quantitation of the distribution of phosphatidic acid-preferring phospholipase Al mRNA in cells and tissues the Perkin Elmer ABI Prism.RTM. A 7700 Sequence Detection system was used according to the manufacturer's specifications and protocols. Multiplex PCR reactions were set up to quantitate X and the housekeeping genes HPRT, GAPDH, beta-actin, according to ABI 7700 Sequence

Detection System User Bulletin #2. For primers the PE kit 4310890E was used. Forward and reverse primers and probe for phosphatidic acid-preferring phospholipase Al were designed using the Perkin Elmer ABI Primer Express™ software and were synthesized by PE Biosystems (Foster City, Calif). The phosphatidic acid-preferring phospholipase Al forward primer sequence was: Primerl

GGAGATGGTGGAGCTTGTGAA. The phosphatidic acid-preferring phospholipase Al reverse primer sequence was Primer2 ATCGAGCCTGTGTGCGTGCGG. The fluorogenic probe, labeled with FAM as the reporter dye, is Probe 1 TTGGGTCACATCCACCTCGTA. The following reactions in a final volume of 50 μl were set up in duplicate for each cDNA (RNA) sample (where the final concentrations of each component are indicated): lx TaqMan buffer A, 5.5 mM MgCl₂, 200 nM each of dATP, dCTP, dGTP and dUTP, 0.025 U/μl AmpliTaq Gold™, 0.01 U/μl AmpErase UNG.RTM., HPRT forward, reverse primers and probe IX, phosphatidic acid-preferring phospholipase Al forward and reverse primers each at 200 nM, 100 nM X FAM-labeled probe, and 20 ng of template cDNA (from cells and tissue or 40 ng from libraries). Thermal cycling parameters were 2 min HOLD at 50°C, 10 min HOLD at 95°C, followed by melting at 95 °C for 15 sec and annealing/extending at 60°C for 1 min for each of 40 cycles.

Relative quantitation of the phosphatidic acid-preferring phospholipase Al mRNA levels was done using the comparative CT method described in the ABI 7700 Sequence Detection System User Bulletin #2 for multiplex reactions. Following derivation of the .DELTA.Rn value, representing the normalized reporter signal for each gene (phosphatidic acid-preferring phospholipase Al and housekeeping genes) minus the baseline signal for each gene established in the first few cycles of PCR, C_T (threshold cycle) values, representing the first PCR cycle at which an increase in reporter fluorescence signal above baseline is detected, were determined for each gene. For each sample the phosphatidic acid-preferring phospholipase Al values were normalized to that housekeeping genes. Duplicate values were then averaged.

The following human tissues were used: coronary smooth muscle cells, brain, testis, pancreas, stomach, cerebellum, trachea, adrenal gland, skeletal muscle, salivary gland, small intestine, prostate, fetal liver, placenta, fetal brain, uterus, mammary gland, heart, spleen, lung, HeLa cells, liver, kidney, thymus, bone marrow, thyroid, colon, bladder, spinal cord, peripheral blood, liver, ciπhotic liver, pancreas liver cirrhosis, spleen liver ciπhosis, total Alzheimer brain, fetal lung, breast tumor, colon tumor, lung tumor, HEK 293 cells, adipose, pericardium, fetal heart, thyroid tumor, MDA MB 231 cells, HEP G2 cells, HUVEC cells, fetal kidney, breast, Jurkat T-cells,

Alzheimer brain cortex, cervix, esophagus, thalamus, precentral gyrus, hippocampus, occipital lobe, cerebral peduncles, postcentral gyrus, temporal lobe, parietal lobe, cerebellum (right), cerebellum (left), tonsilla cerebelli, cerebral meninges, pons, frontal lobe, cerebral cortex, corpus callosum, vermis cerebelli, Alzheimer brain frontal lobe, interventricular septum, heart atrium (right), heart atrium (left), and heart ventricle (left)

The results of the mRNA-quantification (expression profiling) are shown in Figs.12- 14. The phosphatidic acid-preferring phospholipase Al is highly expression in fetal brain, cerebral cortex, Alzheimer brain cortex, frontal lobe, Alzheimer brain frontal lobe, cerebellum, cerebellum (right), cerebellum (left), tonsilla cerebelli, precentral gyrus, hippocampus, occipital lobe, cerebral peduncles, postcentral gyrus, temporal lobe, parietal lobe, cerebral meninges, pons, corpus callosum, vermis cerebelli, spinal cord, thalamus, interventricular septum, HEK293 cells and cervix. The results of the expression profiling suggest an association between phosphatidic acid-preferring phospholipase Al and the indications cardiovascular diseases, CNS diseases, and genitourinary diseases.

REFERENCES

1. Cloning of a phosphatidic acid-preferring phospholipase Al from bovine testis. J Biol Chem 1998 Mar 6;273(10):5468-77

2. Identification of a phosphatidic acid-preferring phospholipase Al from bovine brain and testis. Proc Natl Acad Sci U S A 1994 Sep 27;91(20):9574-8

3. Membrane lipids have multiple effects on interfacial catalysis by a phosphatidic acid-preferring phospholipase Al from bovine testis. Biochemistry 2000 Aug 8;39(31):9335-44 SEQUENCE LISTING

<110> Bayer AG

<120> REGULATION OF HUMAN PHOSPHATIDIC ACID-PREFERRING PHOSPHOLIPASE Al

<130> Lio303

<150> USSN 60/269,856 <151> 2001-02-21

<150> USSN 60/301,483 <151> 2001-06-29

<160> 8

<170> Patentln version 3.1

<210> 1

<211> 2618

<212> DNA

<213> Homo sapiens

<400> 1 atgaattacc cgggccgcgg gtccccacgg agccccgagc ataacggccg aggcggcggc 60 ggcggcgcct gggagctggg ctcagacgcg aggccagcgt tcggcggcgg cgtctgctgc 120 ttcgagcacc tgcccggcgg ggacccggac gacggcgacg tgcccctggc cctgctgcgc 180 ggggaacccg ggctgcattt ggcgccgggc accgacgacc acaaccacca cctcgcgctg 240 gacccctgcc tcagtgacga gaactatgac ttcagctccg ccgagtcggg ctcctcgctg 300 cgctactaca gcgagggtga gagcggcggc ggcggcagct ccttgtcgct gcacccgccg 360 cagcagcctc cgctggtccc gacgaactcg gggggcggcg gcgcgacagg agggtccccc 420 ggggaaagga aacgtacccg gcttggcggc ccggcggccc ggcaccgcta tgaggtagtg 480 acggagctgg gcccggagga ggtacgctgg ttctacaagg aggacaagaa gacctggaag 540 cccttcatcg gctacgactg cgtccgcatc gagctcgcct tccggaccct gctgcagacc 600 acgggtgccc ggccccaggg cggggaccgg gacggcgacc atgtgtgctc ccccacgggc 660 ccagcctcca gttccggaga agatgacgat gaggaccgcg cctgcggctt ctgccagagt 720 acgacggggc acgagccgga gatggtggag cttgtgaaca tcgagcctgt gtgcgtgcgg 780 ggcggcctct acgaggtgga tgtgacccaa ggagagtgct acccggtgta ctggaaccag 840 gctgataaaa taccagtaat gcgtggacag tggtttattg acggcacttg gcagcctcta 900 gaagaggaag aaagtaattt aattgagcaa gaacatctca attgttttag gggccagcag 960 atgcaggaaa atttcgatat tgaagtgtca aaatccatag atggaaaaga tgctgttcat 1020 agtttcaagt tgagtcgaaa ccatgtggac tggcacagtg tggatgaagt atatctttat 1080 agtgatgcaa caacatctaa aattgcaaga acagttaccc aaaaactggg attttctaaa 1140 gcatcaagta gtggtaccag acttcataga ggttatgtag aagaagccac attagaagac 1200 aagccatcac agactaccca tattgtattt gttgtgcatg gcattgggca gaaaatggac 1260 caaggaagaa ttatcaaaaa tacagctatg atgagagaag ctgcaagaaa aatagaagaa 1320 aggcattttt ccaaccatgc aacacatgtt gaatttctgc ctgttgagtg gcggtcaaaa 1380 cttactcttg atggagacac tgttgattcc attactcctg acaaagtacg aggtttaagg 1440 gatatgctga acagcagtgc aatggacata atgtattata ctagtccact ttatagagat 1500 gaactagtta aaggccttca gcaagagctg aatcgattgt attccctttt ctgttctcgg 1560 aatccagact ttgaagaaaa agggggtaaa gtctcaatag tatcacattc cttgggatgt 1620 gtaattactt atgacataat gactggctgg aatccagttc ggctgtatga acagttgctg 1680 caaaaggaag aagagttgcc tgatgaacga tggatgagct atgaagaacg acatcttctt 1740 gatgaactct atataactaa acgacggctg aaggaaatag aagaacggct tcacggattg 1800 aaagcatcat ctatgacaca aacacctgcc ttaaaattta aggttgagaa tttcttctgt 1860 atgggatccc cattagcagt tttcttggcg ttgcgtggca tccgcccagg aaatactgga 1920 agtcaagacc atattttgcc tagagagatt tgtaaccggt tactaaatat ttttcatcct 1980 acagatccag tggcttatag attagaacca ttaatactga aacactacag caacatttca 2040 cctgtccaga tccactggta caatacttca aatcctttac cttatgaaca tatgaagcca 2100 agctttctca acccagctaa agaacctacc tcagtttcag agaatgaagg catttcaacc 2160 ataccaagcc ctgtgacctc accagttttg tcccgccgac actatggaga atctataaca 2220 aatataggca aagcaagcat attaggggct gctagcattg gaaagggact tggaggaatg 2280 ttgttctcaa gatttggacg ttcatctaca acacagtcat ctgaaacatc aaaagactca 2340 atggaagatg agaagaagcc agttgcctca ccttctgcta ccaccgtagg gacacagacc 2400 cttccacata gcagttctgg cttcctcgat tctgcattgg agttggatca caggattgat 2460 tttgaactca gagaaggcct tgtggagagc cgctattggt cagctgtcac gtcgcatact 2520 gcctattggt catccttgga tgttgccctt tttcctttaa ccttcatgta taaacatgag 2580 cacgatgatg atgcaaaacc caatttagat ccaatctg 2618

<210> 2

<211> 872

<212> PRT

<213> Homo sapiens <400> 2

Met Asn Tyr Pro Gly Arg Gly Ser Pro Arg Ser Pro Glu His Asn Gly 1 5 10 15

Arg Gly Gly Gly Gly Gly Ala Trp Glu Leu Gly Ser Asp Ala Arg Pro 20 25 30

Ala Phe Gly Gly Gly Val Cys Cys Phe Glu His Leu Pro Gly Gly Asp 35 40 45

Pro Asp Asp Gly Asp Val Pro Leu Ala Leu Leu Arg Gly Glu Pro Gly 50 55 60

Leu His Leu Ala Pro Gly Thr Asp Asp His Asn His His Leu Ala Leu 65 70 75 80

Asp Pro Cys Leu Ser Asp Glu Asn Tyr Asp Phe Ser Ser Ala Glu Ser 85 90 95

Gly Ser Ser Leu Arg Tyr Tyr Ser Glu Gly Glu Ser Gly Gly Gly Gly 100 105 110

Ser Ser Leu Ser Leu His Pro Pro Gin Gin Pro Pro Leu Val Pro Thr 115 120 125

Asn Ser Gly Gly Gly Gly Ala Thr Gly Gly Ser Pro Gly Glu Arg Lys 130 135 140

Arg Thr Arg Leu Gly Gly Pro Ala Ala Arg His Arg Tyr Glu Val Val 145 150 155 160

Thr Glu Leu Gly Pro Glu Glu Val Arg Trp Phe Tyr Lys Glu Asp Lys 165 170 175

Lys Thr Trp Lys Pro Phe lie Gly Tyr Asp Cys Val Arg lie Glu Leu 180 185 190

Ala Phe Arg Thr Leu Leu Gin Thr Thr Gly Ala Arg Pro Gin Gly Gly 195 200 205

Asp Arg Asp Gly Asp His Val Cys Ser Pro Thr Gly Pro Ala Ser Ser 210 215 220 Ser Gly Glu Asp Asp Asp Glu Asp Arg Ala Cys Gly Phe Cys Gin Ser 225 230 235 240

Thr Thr Gly His Glu Pro Glu Met Val Glu Leu Val Asn lie Glu Pro 245 250 255

Val Cys Val Arg Gly Gly Leu Tyr Glu Val Asp Val Thr Gin Gly Glu 260 265 270

Cys Tyr Pro Val Tyr Trp Asn Gin Ala Asp Lys lie Pro Val Met Arg 275 280 285

Gly Gin Trp Phe lie Asp Gly Thr Trp Gin Pro Leu Glu Glu Glu Glu 290 295 300

Ser Asn Leu lie Glu Gin Glu His Leu Asn Cys Phe Arg Gly Gin Gin 305 310 315 320

Met Gin Glu Asn Phe Asp lie Glu Val Ser Lys Ser lie Asp Gly Lys 325 330 335

Asp Ala Val His Ser Phe Lys Leu Ser Arg Asn His Val Asp Trp His 340 345 350

Ser Val Asp Glu Val Tyr Leu Tyr Ser Asp Ala Thr Thr Ser Lys lie 355 360 365

Ala Arg Thr Val Thr Gin Lys Leu Gly Phe Ser Lys Ala Ser Ser Ser 370 375 380

Gly Thr Arg Leu His Arg Gly Tyr Val Glu Glu Ala Thr Leu Glu Asp 385 390 395 400

Lys Pro Ser Gin Thr Thr His lie Val Phe Val Val His Gly lie Gly 405 410 415

Gin Lys Met Asp Gin Gly Arg lie lie Lys Asn Thr Ala Met Met Arg 420 425 430

Glu Ala Ala Arg Lys lie Glu Glu Arg His Phe Ser Asn His Ala Thr 435 440 445

His Val Glu Phe Leu Pro Val Glu Trp Arg Ser Lys Leu Thr Leu Asp 450 455 460 Gly Asp Thr Val Asp Ser lie Thr Pro Asp Lys Val Arg Gly Leu Arg 465 470 475 480

Asp Met Leu Asn Ser Ser Ala Met Asp lie Met Tyr Tyr Thr Ser Pro 485 490 495

Leu Tyr Arg Asp Glu Leu Val Lys Gly Leu Gin Gin Glu Leu Asn Arg 500 505 510

Leu Tyr Ser Leu Phe Cys Ser Arg Asn Pro Asp Phe Glu Glu Lys Gly 515 520 525

Gly Lys Val Ser lie Val Ser His Ser Leu Gly Cys Val lie Thr Tyr 530 535 540

Asp lie Met Thr Gly Trp Asn Pro Val Arg Leu Tyr Glu Gin Leu Leu 545 550 555 560

Gin Lys Glu Glu Glu Leu Pro Asp Glu Arg Trp Met Ser Tyr Glu Glu 565 570 575

Arg His Leu Leu Asp Glu Leu Tyr lie Thr Lys Arg Arg Leu Lys Glu 580 585 590

lie Glu Glu Arg Leu His Gly Leu Lys Ala Ser Ser Met Thr Gin Thr 595 600 605

Pro Ala Leu Lys Phe Lys Val Glu Asn Phe Phe Cys Met Gly Ser Pro 610 615 620

Leu Ala Val Phe Leu Ala Leu Arg Gly lie Arg Pro Gly Asn Thr Gly 625 630 635 640

Ser Gin Asp His He Leu Pro Arg Glu He Cys Asn Arg Leu Leu Asn 645 650 655

He Phe His Pro Thr Asp Pro Val Ala Tyr Arg Leu Glu Pro Leu He 660 665 670

Leu Lys His Tyr Ser Asn He Ser Pro Val Gin He His Trp Tyr Asn 675 680 685

Thr Ser Asn Pro Leu Pro Tyr Glu His Met Lys Pro Ser Phe Leu Asn 690 695 700 Pro Ala Lys Glu Pro Thr Ser Val Ser Glu Asn Glu Gly He Ser Thr 705 710 715 720

He Pro Ser Pro Val Thr Ser Pro Val Leu Ser Arg Arg His Tyr Gly 725 730 735

Glu Ser He Thr Asn He Gly Lys Ala Ser He Leu Gly Ala Ala Ser 740 745 750

He Gly Lys Gly Leu Gly Gly Met Leu Phe Ser Arg Phe Gly Arg Ser 755 760 765

Ser Thr Thr Gin Ser Ser Glu Thr Ser Lys Asp Ser Met Glu Asp Glu 770 775 780

Lys Lys Pro Val Ala Ser Pro Ser Ala Thr Thr Val Gly Thr Gin Thr 785 790 795 800

Leu Pro His Ser Ser Ser Gly Phe Leu Asp Ser Ala Leu Glu Leu Asp 805 810 815

His Arg He Asp Phe Glu Leu Arg Glu Gly Leu Val Glu Ser Arg Tyr 820 825 830

Trp Ser Ala Val Thr Ser His Thr Ala Tyr Trp Ser Ser Leu Asp Val 835 840 845

Ala Leu Phe Pro Leu Thr Phe Met Tyr Lys His Glu His Asp Asp Asp 850 855 860

Ala Lys Pro Asn Leu Asp Pro He 865 870

<210> 3

<211> 875

<212> PRT

<213> Homo sapiens

<400> 3

Met Asn Tyr Pro Gly His Gly Ser Pro Arg Ser Ser Glu Arg Asn Gly 1 5 10 15

Gly Arg Gly Gly Asp Gly Ala Ala Trp Glu Leu Gly Ser Asp Thr Glu 20 25 30 Pro Ala Phe Gly Gly Ser Val Cys Arg Phe Asp His Leu Pro Val Gly 35 40 45

Glu Pro Gly Asp Asp Glu Val Pro Leu Ala Leu Leu Arg Gly Glu Pro 50 55 60

Gly Leu His Leu Ala Pro Gly Ala Glu Asp His Asn His His Leu Ala 65 70 75 80

Leu Asp Pro Cys Leu Ser Asp Asp Asn Tyr Asp Phe Ser Ser Ala Glu 85 90 95

Ser Gly Ser Ser Leu Arg Tyr Tyr Ser Glu Gly Glu Ser Gly Gly Gly 100 105 110

Gly Ser Ser Ser Ser Leu His Pro Pro Gin Gin Pro Leu Val Pro Ser 115 120 125

Asn Ser Gly Gly Gly Gly Ala Ala Gly Gly Gly Pro Gly Glu Arg Lys 130 135 140

Arg Thr Arg Pro Gly Gly Ala Ala Ala Arg His Arg Tyr Glu Val Val 145 150 155 160

Thr Glu Leu Gly Pro Glu Glu Val Arg Trp Phe Tyr Lys Glu Asp Lys 165 170 175

Lys Thr Trp Lys Pro Phe He Gly Tyr Asp Ser Leu Arg He Glu Leu 180 185 190

Ala Phe Arg Thr Leu Leu Gin Ala Thr Gly Ala Arg Ala Arg Ala Gin 195 200 205

Asp Pro Asp Gly Asp His Val Cys Gly Pro Ala Ser Pro Ala Gly Pro 210 215 220

Ala Ser Ser Ser Val Glu Asp Glu Asp Glu Asp Arg Val Cys Gly Phe 225 230 235 240

Cys Pro Arg He Ala Gly His Gly Arg Glu Met Glu Glu Leu Val Asn 245 250 255 Ile Glu Arg Val Cys Val Arg Gly Gly Leu Tyr Glu Val Asp Val Thr 260 265 270

Gin Gly Glu Cys Tyr Pro Val Tyr Trp Asn Gin Ser Asp Lys He Pro 275 280 285

Val Met Arg Gly Gin Trp Phe He Asp Gly Thr Trp Gin Pro Leu Glu 290 295 300

Glu Glu Glu Ser Asn Leu He Glu Gin Glu His Leu Ser Arg Phe Arg 305 310 315 320

Gly Gin Gin Met Gin Glu Ser Phe Asp He Glu Val Ser Lys Pro He 325 330 335

Asp Gly Lys Asp Ala He His Ser Phe Lys Leu Ser Arg Asn His Val 340 345 350

Asp Trp His Ser Val Asp Glu Val Tyr Leu Tyr Ser Asp Ala Thr Thr 355 360 365

Ser Lys He Ala Arg Thr Val Thr Gin Lys Leu Gly Phe Ser Lys Ala 370 375 380

Ser Ser Ser Gly Thr Arg Leu His Arg Gly Tyr Val Glu Glu Ala Thr 385 390 395 400

Leu Glu Asp Lys Pro Ser Gin Thr Thr His He Val Phe Val Val His 405 410 415

Gly He Gly Gin Lys Met Asp Gin Gly Arg He He Lys Asn Thr Ala 420 425 430

Met Met Arg Glu Ala Ala Arg Lys He Glu Glu Arg His Phe Ser Asn 435 440 445

His Ala Thr His Val Glu Phe Leu Pro Val Glu Trp Arg Ser Lys Leu 450 455 460

Thr Leu Asp Gly Asp Thr Val Asp Ser He Thr Pro Asp Lys Val Arg 465 470 475 480

Gly Leu Arg Asp Met Leu Asn Ser Ser Ala Met Asp He Met Tyr Tyr 485 490 495 Thr Ser Pro Leu Tyr Arg Asp Glu Leu Val Lys Gly Leu Gin Gin Glu 500 505 510

Leu Asn Arg Leu Tyr Ser Leu Phe Cys Ser Arg Asn Pro Asn Phe Glu 515 520 525

Glu Lys Gly Gly Lys Val Ser He Val Ser His Ser Leu Gly Cys Val 530 535 540

He Thr Tyr Asp He Met Thr Gly Trp Asn Pro Val Arg Leu Tyr Glu 545 550 555 560

Gin Leu Leu Gin Lys Glu Glu Glu Leu Pro Asp Glu Arg Trp Met Ser 565 570 575

Tyr Glu Glu Arg His Leu Leu Asp Glu Leu Tyr He Thr Lys Arg Arg 580 585 590

Leu Arg Glu He Glu Glu Arg Leu His Gly Leu Lys Ala Ser Ser Met 595 600 605

Thr Gin Thr Pro Ala Leu Lys Phe Lys Val Glu Asn Phe Phe Cys Met 610 615 620

Gly Ser Pro Leu Ala Val Phe Leu Ala Leu Arg Gly He Arg Pro Gly 625 630 635 640

Asn Thr Gly Ser Gin Asp His He Leu Pro Arg Glu He Cys Asn Arg 645 650 655

Leu Leu Asn He Phe His Pro Thr Asp Pro Val Ala Tyr Arg Leu Glu 660 665 670

Pro Leu He Leu Lys His Tyr Ser Asn He Ser Pro Val Gin He His 675 680 685

Trp Tyr Asn Thr Ser Asn Pro Leu Pro Tyr Glu Tyr Met Lys Pro Ser 690 695 700

Phe Leu His Pro Ala Lys Asp Pro Thr Ser He Ser Glu Asn Glu Gly 705 710 715 720

He Ser Thr He Pro Ser Pro Val Thr Ser Pro Val Leu Ser Arg Arg 725 730 735 His Tyr Gly Glu Ser He Thr Asn He Gly Lys Ala Ser He Leu Gly 740 745 750

Ala Ala Ser He Gly Lys Gly Leu Gly Gly Met Leu Phe Ser Arg Phe 755 760 765

Gly Arg Ser Ser Ala Ser Gin Pro Ser Glu Thr Ser Arg Asp Ser He 770 775 780

Glu Asp Glu Lys Lys Pro Val Ala Ser Pro Pro Met Thr Thr Val Ala 785 790 795 800

Thr Gin Thr Leu Pro His Ser Ser Ser Gly Phe Leu Asp Ser Ala Leu 805 810 815

Glu Leu Asp His Arg He Asp Phe Glu Leu Arg Glu Gly Leu Val Glu 820 825 830

Ser Arg Tyr Trp Ser Ala Val Thr Ser His Thr Ala Tyr Trp Ser Ser 835 840 845

Leu Asp Val Ala Leu Phe Leu Leu Thr Phe Met Tyr Lys His Glu His 850 855 860

Asp Asn Asn Val Lys Pro Ser Leu Asp Pro Val 865 870 875

<210> 4

<211> 470

<212> DNA

<213> Homo sapiens

<400> 4 ttgagattct tctgtatggg atccccatta gcagttttct tggcgttgcg tggcatccgc 60 ccaggaaata ctggaagtca agaccatatt ttgcctagag agatttgtaa ccggttacta 120 aatatttttc atcctacaga tccagtggct tatagattag aaccattaat actgaaacac 180 tacagcaaca tttcacctgt ccagatccac tggtacaata cttcaaatcc tttaccttat 240 gaacatatga agccaagctt tctcaaccca gctaaagaac ctacctcagt ttcagagaat 300 gaaggcattt caaccatacc aagccctgtg acctcaccag ttttgtcccg ccgacactat 360 ggagaatcta taacaaatat aggcaaagca agcatattag gggctgctag cattggaaag 420 ggacttggag gaatgttgtt ctcaagattt ggacgttcat ctacaacaca 470 <210> 5

<211> 421

<212> DNA

<213> Homo sapiens

<400> 5 aataaagtgc ttttaaattc aaatatatgt tgccctaaag aaaactgaaa tatactttaa 60 ccctgaaatc tccgtatctt gacacacaca ttttaacgga aaaaaaaaaa aatcagtttt 120 aggccattca tgtccttcaa gagttcagat tggatctaaa ttgggttttg catcatcatc 180 gtgctcatgt ttatacatga aggttaaagg aaaaagggca acatccaagg atgaccaata 240 ggcagtatgc gacgtgacag ctgaccaata gcggctctcc acaaggcctt ctctgagttc 300 aaaatcaatc ctgtgatcca actccaatgc agaatcgagg aagccagaac tgctatgtgg 360 aagggtctgt gtccctacgg tggtagcaga aggtgaggca actggcttct tctcatcttc 420 c 421

<210> 6

<211> 357

<212> DNA

<213> Homo sapiens

<400> 6 cacgagggcg gctgcgggtc tcgtgggggc ggagcggtcg ccgctgccgc cgcagctcgg 60 gtcgggattt gaaagattag aaacttcggg tggagagggc ggcggcgttg aatgtgtggc 120 ggaagcgctg ggggtcacgg ctccgcgcgc cgccggacag ccggcggcgt ctccacagca 180 tgaattaccc gggccgcggg tccccacgga gccccgagca taacggccga ggcggcggcg 240 gcggcgcctg ggagctgggc tcagacgcga ggccagcgtt cggcggcggc gtctgctgct 300 tcgagcacct gcccggcggg gacccggacg acggcgacgt gcccctggcc ctgctgc 357

<210> 7

<211> 407

<212> DNA

<213> Homo sapiens

<400> 7 tttttttttt ttttttatgt tccatatgcc tttatatttg ggaggaatca gattgtgatt 60 tgtagccttt acatgcagtt tacccaaacc ttcttcacat ccaggtcttg cttgattctc 120 tggagaaggt tttgtcagac tcataattaa aatgatgcaa actgtaaatg acttcttcag 180 aactgaaaac ttcttttttt tttaaataaa gtgcttttaa attcaaatat atgttgccct 240 aaagaaaact gaaatatact ttaaccctga aatctccgta tcttgacaca cacattttaa 300 cggaaaaaaa aaaaatcagt tttaggccat tcatgtcctt caagagagtt cagattggat 360 ctaaattggg ttttgcatca tcatcgtgct catgtttata catgaag 407

<210> 8

<211> 420

<212> DNA

<213> Homo sapiens

<400> 8 tttttttttg aacttcagga aaagaacatt gaaatattaa tgaaacttgt cttgatataa 60 caacaaggaa tttttgggtt aagttcaatt tcctttgata actaaaaaat tactaaaaaa 120 gattaaatag ggagtgcatt gtaacaatct gtacatttat agtaaactgt gttaatatca 180 tgacacttac catacatatt aaaataaaaa ttgagatagt tcagatattt atagtagtta 240 atttttaaat tctagaataa tcaaatgttt aaaaaaatca aattttctaa ttctaaacct 300 cctgaattat tatgtcaaat ttaccaacca ctctaaactt tatcaacaac ttacttggtt 360 tgtagaatat attaaatgta tcaattgatc ttaccatctt ttccatctat ggattttgac 420

Claims

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

a) a polynucleotide encoding a phosphatidic acid-preferring phospholipase Al polypeptide comprising an amino acid sequence selected form the group consisting of:

amino acid sequences which are at least about 88% 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 ED NO: 1 ;

c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a phosphatidic acid-preferring phospholipase Al polypeptide;

d) a polynucleotide the sequence of which deviates from the polynucleo- tide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a phosphatidic acid-preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide.

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

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

4. A substantially purified phosphatidic acid-preferring phospholipase Al polypeptide encoded by a polynucleotide of claim 1.

5. A method for producing a phosphatidic acid-preferring phospholipase Al polypeptide, wherein the method comprises the following steps:

a) culturing the host cell of claim 3 under conditions suitable for the expression of the phosphatidic acid-preferring phospholipase Al polypeptide; and

b) recovering the phosphatidic acid-preferring phospholipase Al polypeptide from the host cell culture.

6. A method for detection of a polynucleotide encoding a phosphatidic acid- preferring phospholipase Al 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 phosphatidic acid-preferring phospholipase Al polypeptide of claim 4 comprising the steps of: contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the phosphatidic acid-preferring phospholipase Al polypeptide.

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 phosphatidic acid-preferring phospholipase Al, comprising the steps of:

contacting a test compound with any phosphatidic acid-preferring phospholipase Al polypeptide encoded by any polynucleotide of claiml;

detecting binding of the test compound to the phosphatidic acid-preferring phospholipase Al polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a phosphatidic acid-preferring phospholipase Al.

11. A method of screening for agents which regulate the activity of a phosphatidic acid-preferring phospholipase Al, comprising the steps of:

contacting a test compound with a phosphatidic acid-preferring phospholipase Al polypeptide encoded by any polynucleotide of claim 1; and

detecting a phosphatidic acid-preferring phospholipase Al activity of the polypeptide, wherein a test compound which increases the phosphatidic acid- preferring phospholipase Al activity is identified as a potential therapeutic agent for increasing the activity of the phosphatidic acid-preferring phospholipase Al, and wherein a test compound which decreases the phosphatidic acid-prefeπing phospholipase Al activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the phosphatidic acid-preferring phospholipase Al.

12. A method of screening for agents which decrease the activity of a phosphatidic acid-preferring phospholipase Al, comprising the steps of:

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 phosphatidic acid-preferring phospholipase Al.

13. A method of reducing the activity of phosphatidic acid-preferring phospholipase Al, comprising the steps of:

contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any phosphatidic acid-preferring phospholipase Al polypeptide of claim 4, whereby the activity of phosphatidic acid-preferring phospholipase

Al is reduced.

14. A reagent that modulates the activity of a phosphatidic acid-preferring phospholipase Al polypeptide or a polynucleotide wherein said reagent is iden- tified 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 pharma- ceutically acceptable earner.

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 phosphatidic acid-prefeπing phospholipase Al in a disease.

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

18. A cDNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

19. The cDNA of claim 18 which comprises SEQ ID NO : 1.

20. The cDNA of claim 18 which consists of SEQ ED NO: 1.

21. An expression vector comprising a polynucleotide which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

22. The expression vector of claim 21 wherein the polynucleotide consists of

SEQ TD NO: 1.

23. A host cell comprising an expression vector which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

24. The host cell of claim 23 wherein the polynucleotide consists of SEQ ID NO: 1.

25. A purified polypeptide comprising the amino acid sequence shown in SEQ ED NO: 2.

26. The purified polypeptide of claim 25 which consists of the amino acid sequence shown in SEQ ED NO: 2.

27. A fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.

28. A method of producing a polypeptide comprising the amino acid sequence shown in SEQ ED NO: 2, comprising the steps of:

culturing a host cell comprising an expression vector which encodes the polypeptide under conditions whereby the polypeptide is expressed; and

isolating the polypeptide.

29. The method of claim 28 wherein the expression vector comprises SEQ ED

NO: 1.

30. A method of detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, comprising the steps of:

hybridizing a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NO: 1 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and

detecting the hybridization complex.

31. The method of claim 30 further comprising the step of amplifying the nucleic acid material before the step of hybridizing.

32. A kit for detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ED NO: 2, comprising:

a polynucleotide comprising 11 contiguous nucleotides of SEQ ED NO: 1 ; and

instructions for the method of claim 30.

33. A method of detecting a polypeptide comprising the amino acid sequence shown in SEQ ED NO: 2, comprising the steps of:

contacting a biological sample with a reagent that specifically binds to the polypeptide to form a reagent-polypeptide complex; and

detecting the reagent-polypeptide complex.

34. The method of claim 33 wherein the reagent is an antibody.

35. A kit for detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, comprising:

an antibody which specifically binds to the polypeptide; and

instructions for the method of claim 33.

36. A method of screening for agents which can modulate the activity of a human phosphatidic acid-preferring phospholipase Al, comprising the steps of:

contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2 and (2) the amino acid sequence shown in SEQ ED NO: 2; and

detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the human phosphatidic acid-preferring phospholipase Al.

37. The method of claim 36 wherein the step of contacting is in a cell.

38. The method of claim 36 wherein the cell is in vitro.

39. The method of claim 36 wherein the step of contacting is in a cell-free system.

40. The method of claim 36 wherein the polypeptide comprises a detectable label.

41. The method of claim 36 wherein the test compound comprises a detectable label.

42. The method of claim 36 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.

43. The method of claim 36 wherein the polypeptide is bound to a solid support.

44. The method of claim 36 wherein the test compound is bound to a solid support.

45. A method of screening for agents which modulate an activity of a human phosphatidic acid-preferring phospholipase Al, comprising the steps of:

contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 88% identical to the amino acid sequence shown in

SEQ ID NO: 2 and (2) the amino acid sequence shown in SEQ ED NO: 2; and

detecting an activity of the polypeptide, wherein a test compound which increases the activity of the polypeptide is identified as a potential agent for increasing the activity of the human phosphatidic acid-prefeπing phospholipase Al, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human phosphatidic acid-preferring phospholipase Al.

46. The method of claim 45 wherein the step of contacting is in a cell.

47. The method of claim 45 wherein the cell is in vitro.

48. The method of claim 45 wherein the step of contacting is in a cell- free system.

49. A method of screening for agents which modulate an activity of a human phosphatidic acid-preferring phospholipase Al, comprising the steps of:

contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ID NO: 1; and

detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of the human phosphatidic acid-preferring phos- pholipase Al.

50. The method of claim 49 wherein the product is a polypeptide.

51. The method of claim 49 wherein the product is RNA.

52. A method of reducing activity of a human phosphatidic acid-preferring phospholipase Al, comprising the step of:

contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1, whereby the activity of a human phosphatidic acid-preferring phospholipase Al is reduced.

53. The method of claim 52 wherein the product is a polypeptide.

54. The method of claim 53 wherein the reagent is an antibody.

55. The method of claim 52 wherein the product is RNA.

56. The method of claim 55 wherein the reagent is an antisense oligonucleotide.

57. The method of claim 56 wherein the reagent is a ribozyme.

58. The method of claim 52 wherein the cell is in vitro.

59. The method of claim 52 wherein the cell is in vivo.

60. A pharmaceutical composition, comprising:

a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ED NO: 2; and

a pharmaceutically acceptable carrier.

61. The pharmaceutical composition of claim 60 wherein the reagent is an antibody.

62. A pharmaceutical composition, comprising:

a reagent which specifically binds to a product of a polynucleotide comprising the nucleotide sequence shown in SEQ ED NO: 1; and a pharmaceutically acceptable carrier.

63. The pharmaceutical composition of claim 62 wherein the reagent is a ribozyme.

64. The pharmaceutical composition of claim 62 wherein the reagent is an anti- sense oligonucleotide.

65. The pharmaceutical composition of claim 62 wherein the reagent is an antibody.

66. A pharmaceutical composition, comprising: an expression vector encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; and a pharmaceutically acceptable carrier.

67. The pharmaceutical composition of claim 66 wherein the expression vector comprises SEQ ID NO: 1.

68. A method of treating a phosphatidic acid-preferring phospholipase Al dysfunction related disease, wherein the disease is selected from a CNS disorder, a genitourinary disorder, a cardiovascular disorder, asthma, cancer, COPD or hematological disorder comprising the step of:

administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a human phosphatidic acid-preferring phospholipase Al, whereby symptoms of the phosphatidic acid-preferring phospholipase Al disfunction related disease are ameliorated.

69. The method of claim 68 wherein the reagent is identified by the method of claim 36.

70. The method of claim 68 wherein the reagent is identified by the method of claim 45.

71. The method of claim 68 wherein the reagent is identified by the method of claim 49.