WO2000000635A1 - Phosphatidylcholine phospholipase d - Google Patents

Abstract

Three new isoforms of phosphatidylcholine phospholipase D, hPLD2.1, hPLD2.2 and hPLD1.5, can be produced recombinantly and are useful for screening compounds, as drug candidates, for an ability to modify PCPLD activity.

Description

PHOSPHATroYLCHOLINE PHOSPHOLIPASE D

Cross Reference to Related Applications

This is a continuing application, under 35 USC §120, based on U.S. application serial No. 08/768,147, filed December 15, 1995, the contents of which are hereby incorporated by reference.

Technical Field of the Invention

In general, the invention pertains to human polynucleotide sequences encoding for polypeptides having enzymatic activity relevant in cell signaling. The present application pertains in particular to mammalian phosphatidylcholine phospholipase D (PCPLD), specifically, human PCPLD (hPCPLD), to fragments and polypeptide analogs thereof and to polynucleotides encoding the same.

Background of the Invention

Cell activation is associated with rapid upregulation of synthesis of phospholipids (PL) that includes phosphatidic acid (PA), diacylglycerol (DAG) and phosphatidylinositol (PI).

PA's are a molecularly diverse group of phospholipid second messengers coupled to cellular activation and mitogenesis. Singer et al , Exp. Opin. Invest. Drugs 3:631-643, 1994.

Compounds capable of modulating PA generation and hence altering a signal involved in cell activation may therefore be of therapeutic interest in the area of inflammation and oncology. Lysophosphatidic acid acyltransferase (LPAAT) is an important enzyme in the synthesis of a specific species of PA in activated monocytic cells. Rice et al., Proc. Nat 'I

Acad. Sci. USA 91:3857-3861, 1994. PCPLD is another major enzyme class involved in the generation of PA through hydrolysis of phosphatidyl choline (PC) into PA and choline.

Exton, Biochim Biophys Ada 1212:26-42, 1994). Okamura et al , J. Biol. Chem. 269:31207-31213, 1994, report PCPLD protein purification from pig lung. Brown et al,

J. Biol. Chem. 270:14935-14943, 1995, report PCPLD protein purification from porcine brain, and Vinggaard et al. discuss PCPLD isolation from human placenta. Biochim Biophys Ada 1258: 169-176, 1995.

In plant species, Wang et al. published results of cloning efforts with castor bean

PCPLDs. J. Biol. Chem. 269:20312-20317, 1994. Ueki et al. disclose PCPLD purified from rice and maize, Plant Cell Physiol. 36:903-914, 1995, and there also are reports on

PCPLD isolation and purification from yeast. Ella et al , Biochem. J. 314, 15-19, 1996;

Rose et al , Proc. Natl. Acad. Sci. 92: 12151-12155, 1995.

Most recently, Hammond et al. report cloning of a human isoform of PCPLD, hPLDl. J. Biol. Chem. 270: 29640-29643, 1995. SEQ ID NO. 3 is a sequence listing of the amino acids of hPLDl. Based on a variety of biochemical studies including differential subcellular fractionation, distinct mechanism of activation, substrate specificity and different chromatographic properties, evidence for the existence of multiple phospholipase D (PLD) isoforms in mammalian cells is growing rapidly. Liscovitch et al , Chem. Phys. Lipids 80: 37-44, 1996; Kiss, Chem. Phys. Lipids 80: 81-102. hPLDl has approximately a 40% sequence homology with hPCPLD.

Although other mammalian PLD sequences have been cloned, heretofore the sequence of the disclosed PCPLD has not been obtained. Therefore, cloning cDNA isoforms of PLD that are closely related to other mammalian and plant isoforms of PLD would be useful in conducting discovery research to identify specific agents capable of modulating this enzyme.

Summary of the Invention

This present invention relates to three, previously unknown isoforms, hPCPLD2.1, hPCPLD2.2, and hPCPLD1.5, which are hereafter called "hPLD2.1," "hPLD2.2" and "hPLD1.5," respectively. Thus, the invention provides cDNA sequences, polypeptide sequences, and transformed cells for producing isolated, recombinant hPLD2.1, hPLD2.2 or hPLDl .5. The invention contemplates, inter alia, the incorporation of codons "preferred" for expression by selected nonmammalian hosts, the provision of sites for cleavage by restriction endonuclease enzymes, and the provision of initial, terminal or intermediate DNA sequences which facilitate construction of readily expressed vectors. The invention also provides DNA sequences coding for microbial expression of polypeptide analogs or derivatives of hPLD2.1, hPLD2.2 or hPLD1.5, which differ from naturally-occurring forms, in terms of the identity or location of one or more amino acid residues, and which share some or all properties of naturally-occurring forms. Accordingly, the invention encompasses deletion analogs that contain fewer than all of the residues specified for hPLD2.1, hPCD 2.2, or hPLD1.5; substitution analogs, such as [Ser¹⁷]PLD, where one or more amino acid residues are added to a terminal or medial portion of the polypeptide.

As described in greater detail below, hPLD2.1 and hPLD2.2 polypeptides display PCPLD activity in a particular fluorescent assay. Accordingly, the present invention includes polynucleotide sequences that are useful for expressing, in procaryotic or eucaryotic host cells, polypeptide products that have at least a primary structure and a biological property in common with naturally-occurring hPLD2.1 or hPLD2.2.

With the aforementioned assay, the present inventors have not observed activity associated with hPLD1.5, under circumstances where hPLD2.1 and hPLD2.2 were active in the assay (see below). Because hPLDl and hPLD1.5 share substantial aspects of primary structure, however, hPLD1.5 may display PCPLD activity under other circumstances. In any event, the present invention encompasses assays for screening test compounds for their ability to inhibit hPLD2.1 or hPLD2.2. Accordingly, hPLD1.5 protein can be used as a negative control in the context of screening compounds for inhibition of PCPLD 1 activity, pursuant to the present invention. Also, a polynucleotide encoding hPLD1.5 can be used as a probe to identify genes encoding other PCPLD isoforms. More generally, the present invention contemplates a category of polynucleotides that includes, without limitation, (a) an isolated DNA that encodes hPLD2.1, hPLD2.2 or hPLD1.5; (b) a DNA that hybridizes, under conditions such as are illustrated herein or are more stringent conditions, to a DNA set forth in this specification or to a fragment thereof; (c) a DNA that, but for the degeneracy of the genetic code, would hybridize to DNA sequences disclosed herein; and (d) an antisense oligonucleotide for modulating expression of hPLD2.1, hPLD2.2 or hPLD1.5. Subcategory (b) includes, without limitation, genomic DNA sequences encoding allelic variants of hPLD2.1, hPLD2.2 or hPLD1.5. Subcategory (c) includes, without limitation, manufactured DNAs encoding hPLD2.1, hPLD2.2 or hPLD1.5, fragments of these proteins, and analogs of the proteins, which DNAs optionally incorporate codons facilitating translation messenger RNA in a prescribed microbial or other host. To these ends, the present invention provides, in accordance with one of its aspects, a polynucleotide (i) that codes for a PCPLD isoform selected from group consisting of hPLD2.1, hPLD2.2, and hPLD1.5 or (ii) that hybridizes to a polynucleotide encoding said isoform. In a preferred embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID Nos. 1, 15 or 16, respectively.

In accordance with another aspect of the present invention, an isolated PCPLD isoform is provided, selected from a group consisting of hPLD2.1, hDLD2.2, and hPLD1.5. Pursuant to one preferred embodiment, the isolated PCPLD isoform comprises the amino acid sequence of SEQ ID NO. 1 or of SEQ ID NO. 15, or an enzymatically active fragment thereof. According to another embodiment, the isolated PCPLD isoform comprises the amino acid sequence of SEQ ID NO. 16.

In accordance with yet another aspect of the present invention, a method is provided for screening a drug candidate, comprising (a) providing at least one of the aforementioned isoforms that displays PCPLD activity, (b) contacting that isoform with the drug candidate, and then (c) determining whether the drug candidate affects PCPLD activity of the isoform.

In a preferred embodiment, step (c) comprises measuring the PCPLD activity of the isoform against a control sample, which can contain a PCPLD isoform comprising the amino acid sequence of SEQ ID NO. 16. In another embodiment, the drug candidate is a pool of compounds from combinatorial library expression.

Brief Description of the Drawings

Figure 1 shows TLC analysis of PCPLD activity in Sf9 cell extracts transfected with various Baculoviral constructs expressing hPLD2.1, also referred to as hPCPLD, and hPLDl by using a fluorescent-labeled PC substrate. Figure 2 demonstrates a screening assay for PCPLD activity in Sf9 cell extracts transfected with various Baculoviral constructs expressing hPLD2.2, hPLD2.1 and a human isoform of PLD1, hPLDljS (Hammond et al , J. Biol. Chem. 272: 3860-3868, 1997), designated as hPLD1.4 here, using a fluorescent-labeled PC substrate.

Figure 3 displays the effect of CT-2584 on PCPLD activity in insect cell extracts transfected with a Baculoviral construct expressing hPLDl. As shown in figure 3, an increase in the concentration of CT-2584 correlates to an increase in fluorescent intensity of the products corresponding to NBD-Pa-Bt, NBD-LPA-Bt, and NBD-PA bands on the TLC plate.

Detailed Description of Preferred Embodiments In the description that follows, a number of terms are utilized extensively.

Definitions are provided to facilitate understanding of the invention.

Definitions

The term "isolated" applied to a polypeptide throughout the specification refers to the purity of the polypeptide that is sufficiently free of other materials endogenous to the host, from which the polypeptide is isolated, such that any remaining materials do not materially affect the biological properties of the polypeptide.

The term "derived" as used throughout the specification in relation to a polypeptide of the invention, encompasses (a) a polypeptide obtained by isolation or purification from host cells, as well as a polypeptide obtained by manipulation and expression of a polynucleotide prepared from host cells; (b) a polynucleotide including genomic DNA, mRNA, cDNA synthesized from mRNA, and a synthetic oligonucleotide having a sequence corresponding to an inventive polynucleotide; and (c) a synthetic polypeptide antigen prepared based on any known polypeptide sequence of the invention. The term "expression product" as used throughout the specification refers to materials produced by recombinant DNA techniques.

PCPLD catalyzes the hydrolysis of phospholipids to PA. The preferred substrate for this reaction is PC, a major mammalian cell-membrane constituent. Recombinant hPCPLD is useful in screening drug candidates which inhibit or activate hPCPLD activity. The invention provides (a) a polynucleotide, which encodes a polypeptide, comprising a DNA sequence set forth in SEQ ID NO. 1 (hPLD2.1), NO. 15 (hPLD2.2) or NO. 16 (hPLD1.5); (b) a shortened polynucleotide thereof, or an additional polynucleotide, which due to the degeneracy of the genetic code encodes a polypeptide of SEQ ID NO. 1, NO. 15 or NO. 16, or a biologically active fragment thereof; (c) a polynucleotide capable of hybridizing thereto; and (d) a polypeptide which comprises a polypeptide sequence of SEQ ID NO. 1, NO. 15 or NO. 16, or a biologically active fragment thereof. The invention also provides a vector containing a DNA sequence encoding hPLD2.1, hPLD2.2, or hPLD1.5 in operative association with an expression control sequence, and a host cell transformed with such a vector to produce recombinant hPLD2.1, hPLD2.2, or hPLD1.5. An inventive vector and a transformed cell are employed in a process to produce recombinant hPLD2.1, hPLD2.2, or hPLD1.5. In this process, a cell line transformed with a DNA sequence encoding hPLD2.1, hPLD2.2, or hPLD1.5 in operative association with an expression control sequence, is cultured. The process may employ a number of known cells as host cells for the expression of hPLD2.1, hPLD2.2, or hPLD1.5, including, for example, mammalian cells, yeast cells, insect cells and bacterial cells. The invention further includes a method to select a pharmaceutically active compound by determining whether the compound is capable of inhibiting the enzymatic activity of hPLD2.1, hPLD2.2, hPLD1.5. A selected compound could be a pharmaceutical drug useful to inhibit a signal cascade in an inflammatory response.

The invention further provides a transformed cell that expresses active hPLD2.1 or hPLD2.2, and further comprises a means for determining whether a drug candidate is therapeutically active by inhibiting or activating the enzymatic activity of a recombinant PCPLD.

Accordingly, hPLD2.1 is characterized by the 933 amino acids of SEQ ID NO. 1; hPLD2.2 is characterized by the 933 amino acids of SEQ ID NO. 15; and hPLD1.5 is characterized by the 971 amino acids of SEQ ID NO. 16. The invention includes an allelic variant (naturally-occurring base changes in the species which may or may not result in an amino acid change) of a DNA sequence herein encoding hPLD2.1, hPLD2.2, or hPLD1.5 polypeptide, or an active fragment thereof. The inventive polynucleotide sequences further comprise a sequence which hybridizes under stringent conditions to the coding region (e.g., nucleotide #66 to nucleotide #2864) . Regarding hybridization conditions, see Maniatis et al , MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, pages 387-389, 1982. For example, one such stringent hybridization condition is 4 X SSC at 65°C, followed by a wash in 0.1 X SSC at 65°C for 30 minutes. Another stringent hybridization condition is 50% formamide and 4 X SSC at 42°C. The present invention further includes a polynucleotide encoding an enzymatic active polypeptide whose codon is the same as hPLD2.1 or hPLD2.2, or differs due to the degeneracy of genetic code. In addition, the invention encompasses a variant with a point mutation or an induced modification of a polynucleotide sequence set forth in SEQ ID NO. 1, NO. 15 or NO. 16, which enhances the enzymatic activity or the production of the encoded polypeptide.

Identification of Coding Sequences

A search of the Genbank with the blastp program, using the castor bean PCPLD protein sequence as a probe, disclosed a yeast DNA sequence (Genbank Accession # Z28256) that encodes a S. cerevisiae reading frame (ORF YKR031c) mapped to chromosome XI. This sequence contains several short stretches of amino acids homologous to the plant PCPLD protein sequence. Recently, this yeast sequence has been identified to encode the SPO14 gene (Genbank Accession# L46807), which is essential for yeast meiosis. Honigberg et al, Genetics 130:703-716, 1992. The gene product for SP014 has been recently found to contain PCPLD activity. Ella, et al, Biochem. J.314, 15-19, 1996; Rose, et al, Proc. Nat'l Acad. Sci.92: 12151-12155, 1995. This yeast protein sequence was then used to search for homologous sequences in the Genbank database of expressed sequence tag

(dbEST). Translated polypeptide sequence of several short stretches of human cDNA sequences were found homologous to the plant PCPLD and the yeast SPO14 protein sequence. These cDNA sequences of interest were derived from single-run partial sequencing of random human cDNA clones carried out by either the WashU-Merck EST or the Genexpress-Genethon program. 1. hPLD2 (A)hPLD2.1

An example of a short stretch of amino acid sequence homology alignment among the plant, yeast and two overlapping human cDNA clones (Genbank #R83570 and dbEST #261972) is shown, wherein castor bean PCPLD fragment is SEQ ID NO.3, yeast is SEQ

ID NO.4, and R83570 is SEQ ID NO 5.

Castor bean PCPLD Q R S M D G A R DS E I A M G A Y Q P

Yeast SPO14 E R S Q L G N R DS E V A I L I R D T

R83570 / 261972 D R S L L G K R DS E L A V L I E D T

The top row refers to the partial protein sequence of the castor bean PCPLD from amino acid 679 to 698; the middle row refers to the partial protein sequence of the yeast SPO14 from amino acid 813 to 831; and the bottom row refers to a homologous protein sequence derived from human cDNA clone genbank #R83570 and dbEST #261972. Identical amino acids sequences among these three sequences are shown in bold letters.

Accordingly , synthetic oligonucleotides 5 '- GTATTC AATCCTGC ATCGCCTTAA-3 ' (O.R83570.1) (SEQ ID NO. 6) and 5'-GTCATCTGCGATGAGCACCTTGCTGTG-3' (O.R83570. IR) (SEQ ID NO. 7), were synthesized (Life Technologies, Gaithersburg, MD) based on the putative coding sequence corresponding to nucleotides 44-67 and complement sequence corresponding to nucleotides 168-194 of the human cDNA clone genbank#R83570, respectively.

O.R83570. 1 was used in combination with the primer 5 '- CTAGCTTATAATACGACTCA C-3' (o.sport. lR) (SEQ ID NO. 8) corresponding to the vector sequence just downstream of the cDNA cloning region of the plasmid pCMV. SPORT (Life Technologies, Gaithersburg, MD) to isolate the 3' -region of the human PCPLD cDNA from a human lung cDNA library (Life Technologies, Gaithersburg, MD) using Expand™ long template PCR (Boehringer Mannheim, Indianapolis, IN). The PCR fragments generated were cleaved with Nco I and Xho I prior to subcloning into the Litmus28 vector (New England Biolab, Beverly, MA). DNA sequence analysis showed that cDNA clone pL28.NX has a 1,200 bp Nco I - Xho I insert, a polyA tail at one end and an open reading frame with several stretches homologous to amino acids 766-862, 1228-1275 and 1338-1360 of SPO14, suggesting that this clone contained the C-terminal coding region of human PCPLD and its 3 '-untranslated region.

O.R83570.1R was used in combination with the primer 5'-GACTCTAGCC TAGGCTTTTG C-3' (o.sport. l) (SEQ ID NO. 9) corresponding to the vector sequence just upstream of the cDNA clomng region of the plasmid pCMV. SPORT (Life Technologies, Gaithersburg, MD) to isolate the 5 '-region of the human PCPLD cDNA from a human lung cDNA library (Life Technologies, Gaithersburg, MD) using Expand™ long template PCR (Boehringer Mannheim, Indianapolis, IN). The PCR fragments generated were either cleaved with Sst I and AspllS I or with Sst I alone prior to subcloning into the pBluescriptKS(-) vector (Stratagene, LaJoUa, CA). DNA sequence analysis displayed that the cDNA clone pKS.Sst has a 1,190 bp Sst I - Sst I insert and an open reading frame homologous to amino acids 401-780 of SPO14, indicating that this clone contained the central coding region of human PCPLD. To isolate the 5'-region of the human PCPLD cDNA, a synthetic oligonucleotide 5'- CTCAGGACTCAACCACCAGT C-3' (o.pld3.2R, SEQ ID NO 10) was designed (Life Technologies, Gaithersburg, MD) based on the complement sequence corresponding to the region about 50 bp downstream of the Sst I site on the 5'-side of the 1190 bp Sst I fragment. o.pld3.2R was used in combination with the primer 5'-GACTCTAGCC TAGGCTTTTG C-3' (o. sport.1) corresponding to the vector sequence just upstream of the cDNA cloning region of the plasmid pCMV. SPORT (Life Technologies, Gaithersburg, MD) to isolate the 5'-region of the human PCPLD cDNA from a human lung cDNA library (Life Technologies, Gaithersburg, MD) using Expand™ long template PCR (Boehringer Mannheim, Indianapolis, IN). The PCR fragments generated were cleaved with EcoR I prior to subcloning into the pBluescript(II)SK(-) vector (Stratagene, LaJolla, CA) between the EcoR I site and Sma I site. DNA sequence analysis disclosed that cDNA clone pSK.R83.16 has a 1,240 bp insert containing an ATG near the 5 '-end and an open reading frame with several major stretches homologous to amino acids 1-10, 153-262, and 328-410 of SPO14, implying that this cDNA clone contained the N-terminal coding region of human PCPLD. It has recently been reported that SPO14 protein has PCPLD activity (Engebrecht et al. , ASBMB Fall Symposium, 1995), again suggesting that a human sequence with extensive homology to the yeast SPO14 protein probably has PCPLD activity.

To assemble the human PCPLD cDNA clone, the following fragments were isolated: 1) The 1197 bp Hind III - Sst I fragment from pSK.R83.16.

2) The 512 bp Sst I - Sfu I fragment from pKS.Sst.

3) The 660 bp Sfu I - Ban 1 fragment from pKS.Sst.

4) The 1129 bp Ban I - Xho I fragment from pL28.NX.

Fragments 1 and 2 were inserted via a three-part ligation into pLitmus28 (New England Biolab, Beverly, MA) cleaved with Sfu I and Hind III to generate pL28.HS. Fragments 3 and 4 were inserted via a three-part ligation into pLitmus28 cleaved with Sfu I and Xho I to generate pL28.SX. The 1700 bp Hind III - Sfiέ I fragment, derived from pL28.HS and the 1780 bp Sfu I - Notl fragment, derived from pL28.SX, were then inserted via a three-part ligation into the expression vector pCE2, cleaved with Hind III and Notl to generate pCE2.PLD. pCE2.PLD is transfected into various mammalian cells to assay for PCPLD activity using labeled-PC as a substrate. Ella et al , Anal. Biochem. 218: 136-142, 1994. The plasmid pCE2 was derived from pREP7b (Leung et al , Proc. Nat'l Acad. Sci. USA, 92:4813-4817, 1995) with the RSV promoter region replaced by the CMV enhancer and the elongation factor-la (EF-la) promoter and intron. The CMV enhancer came from a 380 bp Xba l-Sph I fragment produced by PCR from pCEP4 (Invitrogen, San Diego, CA) using primers 5'-GGCTCTAGATATTAATAGTAATCAATTAC-3' (SEQ ID NO. 11) and

5'-CCTCACGCATGCACCATGGTAATAGC-3' (SEQ ID NO 12). The EF-la promoter and intron (Uetsuki et al , J. Biol. Chem., 264: 5791-5798, 1989) came from a 1200 bp Sph I- Aspl 8 I fragment produced by PCR from human genomic DNA using the primers 5'- GGTGCATGCGTGAGGCTCCGGTGC-3 ' (SEQ ID NO . 13)and 5 '- GTAGTTTTCACGGTACCTGAAATGGAAG-3' (SEQ ID NO. 14). These two fragments were ligated into a Xba \IAspl 8 I digested vector derived from pREP7b to generate pCE2

Nucleotide sequencing analysis of various human PCPLD cDNA inserts was performed. SEQ ID NO. 1 shows the DNA sequence of the cDNA insert of the hPLD2.1 isolated herein and the predicted amino acids sequence using the first ATG (nucleotide positions 66-68) from the 5'-end of the sequence for the start of translation. This open reading frame encodes a 933 amino acid polypeptide (SEQ ID NO. 1) and followed by a 3'- untranslated region of > 550 bp. Although the putative initiation site for translation at nucleotide positions 66-68 fulfilled the requirement for an adequate initiation site (Kozak, Critical Rev. Biochem. Mol. Biol. 27:385-402, 1992), translation may still start further upstream of the sequence shown here, as there is no in frame stop codon preceding the 933 amino acid shown here.

The sequence of the 933 amino acid open reading frame in pCE2.PLD was used as the query sequence to search for homologous sequences in protein databases. Search of the database based on Genbank Release 91 from the National Center for Biotechnology Information (NCBI) using the blastp program showed that the protein encoded by pCE2.PLD was homologous to the yeast SPO14 and the various plant PCPLDs.

(B) hPLD2.2

Three overlapping hPLD2.2 cDNA fragments were isolated from a human liver cDNA library (Life Technologies, Gaithersburg, MD) using the hPLD2.1 cDNA fragment as a probe. For the assembly of a full-length hPLD2.2 cDNA clone, the 1,600 bp

EcoRI— BstBI fragment, the 660 bp BstBI— Banl fragment, and the 1,145 bp Banl— Notl fragment were ligated into the EcoRI— Notl vector of pBluescriptSK (Stratagene, La Jolla, CA) to generate plasmid pSK.PLD2.2. DNA sequence analysis showed that the sequence of hPLD2.2 (SEQ ID NO. 15) was identical to that of hPLD2.1 (SEQ ID NO. 1) with the exceptions of 8 nucleotide changes scattered throughout the entire molecule.

Nucleotide acids Amino acids position change position change

660 A - C 184 Glu - Asp

718 T - C 504 Ser - Pro

1296 A - G 404 Lys - Arg

1614 T - G 502 Asn - Lys

2154 G - C 682 Lys - Asn

2372 A - G 755 Asp - Gly

2524 A - G 806 Ser - Gly

2840 A - T 911 Lys - Met

2. hPLDl.5

Another example of alignment of short stretch of amino acid sequences from plant PLD, yeast PLD and a human cDNA clone (dbEST#204986) is shown below:

Castor bean PLD LKILSKIAAGERFTVYiVVPMWPE dbEST# 204986 ORILKAHRENOKYRVYVVIPLLPG Yeast PLD DPJVKANOEKKPWKAFILIPLMPG

The top row refers to the castor bean PCPLD sequence (Wang, et al. , J. Biol. Chem. 269: 20312-20317, 1994) of amino acids 551-574, the middle row refers to homologous translated sequence derived from a human cDNA clone dbEST#204986, and the bottom row refers to the yeast PCPLD sequence of amino acids 1002-1025. Identical amino acids among these three sequences are doubly underlined, whereas conservative amino acids are singly underlined.

Accordingly, an oligonucleotides 5'- GTCCATGCTA ATGTACAGTT GCTC -3' (o. 204986.1), was synthesized (Life Technologies, Gaithersberg, MD) based on the putative coding sequence of the human cDNA clone dbEST#204986, in which one nucleotide was changed from C to T to generate a BsrG I site. o.204986.1 was used in combination with o. sport. IR to isolate the 3'-region of the human PCPLD cDNA from a human liver cDNA library (Life Technologies, Gaithersburg, MD) using Expand™ long template PCR (Boehringer Mannheim, Indianapolis, IN). Two PCR fragments, 1,300 bp and 900 bp, were generated. These two fragments were cleaved with BsrG I and Xho I prior to subcloning into the Litmus28 vector between the Acc65 I and the Xho I site. DNA sequence analysis showed that the open reading frame of the cDNA clone pL28.Li.29 with the 1,300 bp insert matched perfectly with amino acids 742-1074 of hPLDl sequence (Hammond, et al. , J. Biol. Chem. 270: 29640-29643, 1995), whereas the cDNA clone pL28.Li.8 with the 900 bp insert contained a divergent coding sequence after amino acid 961 of hPLDl and only the first 650 bp of the insert matched with hPLDl DNA sequence, suggesting that pL28.Li.8 represented an alternatively spliced variant of hPLDl encoding a protein with a different C-terminal sequence. To isolate the 5 '-region of the human PCPLD cDNA from a human liver cDNA library (Life Technologies, Gaithersburg, MD) using Expand™ long template PCR (Boehringer Mannheim, Indianapolis, IN), a primer 5'-TTCCCTGTGA GCTTTCAGGA TCCT-3' (o.pldl.R) complementary to the region corresponding to amino acids 804-810 of hPLDl was used in combination with the primer 5'-CGCCAACGC GAGGTGCTAG C-3' (o.pldl. lF) corresponding to the region near the Nhe I site in the 5 '-untranslated region of hPLDl. The PCR fragments generated were cleaved with Nhe I and BamH I. The fragments of about 2,400 bp were isolated from agarose gel prior to subcloning into the pLitmus38 vector (New England Biolab, Beverly, MA). DNA sequence analysis showed that cDNA clone pL38.1.6 with a 2,450 bp Nhe I - BamH I insert contained an open reading frame with perfect match to amino acids 1-805 of hPLDl

To assemble the various hPLDl isoforms, the following two fragments were isolated:

1) The 2,500 bp BsrG I - BamH I fragment from pL38.1.6.

2) The 662 bp BamH I - Not I fragment from pL28.Li.8.

Fragments 1 and 2 were inserted via a three-part ligation into pBluescriptSK(-)II cleaved with Acc65 I and Not I to generate pskPLD1.5.

Nucleotide sequencing of hPLD1.5 insert was performed. (SEQ ID NO. 16) shows that the DNA sequence and amino acid sequence of hPLD1.5. The first 961 amino acids of the 971 amino acids of hPLD1.55 is identical to the first 961 amino acids of hPLDl (SEQ ID NO. 2).

Peptide Sequencing of Polypeptides Purified polypeptides prepared by the methods described above can be sequenced using methods well known in the art, for example using a gas phase peptide sequencer (Applied Biosy stems, Foster City, CA). Because the proteins of the present invention may be glycosylated, it is preferred that the carbohydrate groups are removed from the proteins prior to sequencing. This can be achieved by using glycosidase enzymes. Preferably, glycosidase F (Boehringer-Mannheim, Indianapolis, IN) is used. To determine as much of the polypeptide sequence as possible, it is preferred that the polypeptides of the present invention be cleaved into smaller fragments more suitable for gas-phase sequence analysis. This can be achieved by treatment of the polypeptides with selective peptidases, and in a particularly preferred embodiment, with endoproteinase lys-C (Boehringer). The fragments so produced can be separated by reversed-phase HPLC chromatography.

Production of Polypeptides

Once the entire coding sequence of a gene is determined, the DNA sequence of the gene can be inserted into an appropriate expression system. Gene expression can be achieved in any number of different recombinant DNA expression systems to generate large amount of such polypeptide. The present invention includes a polypeptide with a native glycosylation sequence, or a deglycosylated or unglycosylated polypeptide prepared by the methods described below. Expression systems known to the skilled practitioner in the art include bacteria such as E. coli, yeast such as Pichia pastoris, baculovirus, and mammalian expression systems such as Cos or CHO cells. In a preferred embodiment, a recombinant protein is expressed in E. coli or baculovirus expression system. A complete gene or, alternatively, fragments of the gene encoding an antigenic determinant can be expressed. In a first preferred embodiment, the DNA sequence encoding the polypeptide is analyzed to detect putative transmembrane sequences. Such sequences are typically very hydrophobic and readily detected by using standard sequence analysis software, such as MacDNASIS (Hitachi, San Bruno, CA). The presence of transmembrane sequences is often deleterious when a recombinant protein is synthesized in many expression systems, especially E. coli, as it leads to insoluble aggregates which are difficult to be renatured into a native conformation of the polypeptide. Deletion of transmembrane sequences normally does not significantly alter the conformation of the remaining polypeptide structure. Moreover, a transmembrane sequence, by definition embedded within a membrane, is inaccessible as an antigenic determinant to a host immune system. Antibodies to such a sequence will not, therefore, provide immunity to the host and, hence, little information is lost in terms of immunity by omitting such a sequence from a recombinant polypeptide of the invention. Deletion of a transmembrane-encoding sequence from a gene used for expression can be achieved by standard techniques. See Ausubel et al , supra, Chapter 8. For example, fortuitously-placed restriction enzyme sites can be used to excise the desired gene fragment, or the PCR can be used to amplify only the desired part of the gene.

Alternatively, computer sequence analysis is used to determine the location of the predicted major antigenic determinant epitopes of a recombinant polypeptide. Software capable of carrying out this analysis is readily available commercially, for example MacDNASIS (Hitachi, San Bruno, CA). The software typically uses standard algorithms such as the Kyte/Doolittle or Hopp/Woods methods to locate hydrophilic sequences which are characteristically found on the surface of polypeptides and are, therefore, likely to act as antigenic determinants. Once this analysis is completed, a polypeptide can be prepared to contain at least the essential features of an antigenic determinant. A polynucleotide encoding such a determinant can be constructed and inserted into an expression vector by a standard method, for example, using PCR cloning methodology. Polypeptide sequence variants can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native polypeptide which are not essential for PCPLD activity, and are exemplified by the variants lacking a transmembrane sequence described above. Another common type of deletion variant is one lacking a secretory signal sequence or a signal sequence directing a polypeptide to bind a particular part of a cell.

A substitutional variant typically contains an exchange of one amino acid for another at one or more sites within the polypeptide, and is designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. A substitution is preferably conservative, that is, one amino acid is replaced with one of similar shape and charge. A conservative substitution is well known in the art and includes, for example, changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. An insertional variant could be a fusion protein used for rapid purification of a polypeptide, a hybrid polypeptide containing a sequence of another protein, or a polypeptide which is homologous to an inventive polypeptide. For example, an insertional variant could contain a portion of the amino acid sequence of the polypeptide from one species, together with a portion of the homologous polypeptide from another species. An insertional variant could also be one with additional amino acids introduced within the coding sequence. Such an insertion is typically smaller than a fusion protein described above and is introduced, for example, to disrupt a protease cleavage site.

A gene or gene fragment encoding a desired polypeptide can be inserted into an expression vector by standard subcloning techniques. In a preferred embodiment, an E. coli expression vector is used to produce a recombinant protein as a fusion protein, allowing rapid affinity purification of the protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). Some of these systems produce recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the LPAAT ability of the recombinant polypeptide. For example, both the FLAG system and the 6xHis system add only short sequences. The two systems are known to be poorly antigenic and do not adversely affect folding of the polypeptide to its native conformation. Other fusion systems produce a protein where it is desirable to excise the fusion partner from the desired protein. In a preferred embodiment, the fusion partner is linked to the recombinant polypeptide by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA) or enterokinase. LaVallie et al , J. Biol. Chem. 268:23311-17, 1993.

In another preferred embodiment, the expression system used is one driven by the baculovirus polyhedron promoter. A gene encoding a polypeptide can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector. See Ausubel et al , supra. A preferred baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, CA). A vector carrying polynucleotide encoding a polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by standard protocols, and the cells are cultured and processed to produce a recombinant polypeptide. See Summers et al. , A MANUAL FOR METHODS

OF BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station.

Purification of Polypeptides In accordance with the invention, a protein is isolated from host cells, and tested for their ability to produce a desired biological response. Polypeptide extracts can be prepared from host cells by standard methods known to the art. See, for example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, 1987 and Current Protocols in Molecular Biology, John Wiley & Sons 1995. In a preferred embodiment, host cells are extracted into a buffer, and the extracts are separated into membrane and soluble fractions. Each fraction is tested for biological activity. Fractions which elicit a desired biological activity are then purified further to determine which components are responsible for the activity. At each step of the purification, fractions can be assayed for enzymatic activity by the means described above. Purification of the active fractions can be carried out by methods known to the art. See, for example, Protein Purification Methods - A Practical Approach, Harris et al , Eds. (IRL Press, Oxford, 1989).

In a preferred embodiment, extracts prepared as above are purified by sequential size exclusion chromatography isoelectric focusing, HPLC size exclusion chromatography, and chromatography on an affinity column. Fractions which display PCPLD activity can be analyzed further by SDS-PAGE analysis to determine the approximate molecular mass of the active component. It is known that many naturally occurring polypeptides are glycosylated to varying degrees and, as a consequence, a single protein often appears as a pattern of bands of differing electrophoretic mobility on SDS-PAGE analysis. In such situations, it can be difficult to determine whether such a pattern is due to heterogeneity in glycosylation of a single amino acid chain or due to the presence of contaminating polypeptides. To distinguish between these two situations, the polypeptide fraction under study can be treated with a glycosidase to remove some or all the carbohydrate moieties from the protein. The SDS- PAGE analysis is repeated under both reducing and non-reducing conditions, and the resulting banding patterns compared. If the electrophoretic bands observed on the gel show similar or identical shifts in mobility after enzyme treatment, this is an indication that the electrophoretic heterogeneity observed in the purified protein fraction is due to variations in glycosylation. Conversely, if the electrophoretic mobilities differ significantly, it is evident that contaminated polypeptides are present. In a preferred embodiment of the invention, the glycosidase is glycosidase F (Boehringer-Mannheim, Indianapolis, IN) and the peptidase is endoproteinase glu-C (Boehringer). A polypeptide may also be treated with a peptidase to be cleaved into fragments for reversed phase HPLC mapping. Some polypeptides have previously been purified from host cells and it is important therefore to exclude the possibility that biological activity in a particular fraction is due to the presence of these polypeptides. The presence of known polypeptides in a mixture can be detected by methods well known to the art, for instance, by Western blotting with an antiserum specific for the known polypeptide. In a preferred embodiment of the invention, previously identified polypeptides are removed from fractions containing antigenic activity by passage over affinity columns prepared using antibodies or antiserum specific for the known polypeptides.

A polypeptide expressed in any of a number of different recombinant DNA expression systems can be obtained in large amounts and tested for biological activity. Recombinant bacterial cells, for example E. coli, are grown in any of a number of suitable media, for example LB, and the expression of a recombinant polypeptide is induced by adding IPTG to the media or switching incubation to a higher temperature. After culturing the bacteria for a further period of between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual media. The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. The centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a selective speed. If a recombinant polypeptide is expressed in the inclusion, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions with high concentration of urea (e.g., 8 M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as β-mercaptoethanol or DTT (dithiothreitol).

At this stage it may be advantageous to incubate the polypeptide for several hours under conditions suitable for the polypeptide to undergo a refolding process into a conformation which more closely resembles that of the native polypeptide. Such conditions generally include a polypeptide at a concentration less than 500 mg/ml, a reducing agent at low concentration, urea of less than 2 M and often reagents, such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the protein molecule. The refolding process can be monitored, for example, by SDS-PAGE or with antibodies which are specific for the native molecule. Following refolding, the polypeptide can then be purified further and separated from the refolding mixture by chromatography with ion exchange resins, or gel permeation resins, or by a variety of affinity columns.

More specifically, an oligonucleotide and a polynucleotide encoding a polypeptide of the invention can be used as hybridization probes, capable of recognizing and specifically binding to a complementary polynucleotide nucleotide sequence, providing thereby a means of detecting, identifying, locating and measuring a complementary polynucleotide sequence in a biological sample.

Biological samples include, among a great many others, blood or blood serum, lymph, ascites fluid, urine, microorganism or tissue culture medium, cell extracts, or the like, derived from a biological source, or a solution containing chemically synthesized protein, or an extract or solution prepared from such biological-sourced fluid. It is further intended to include cells, tissue and other organic matter such as feces, food and plants.

An oligonucleotide containing a modified nucleotide of the invention can be used as a primer to initiate nucleic acid synthesis at locations in a DNA or RNA molecule comprising the sequence complementary to an inventive oligonucleotide sequence (SEQ ID NO. 1, NO. 15, or NO.16). The synthesized polynucleotide would have incorporated, at its 5' terminus, the oligonucleotide primer bearing an inventive sequence and would, therefore, be detectable by exploitation of the characteristics of a detectable label. Two such primers, specific for different nucleotide sequences on complementary strands of dsDNA, can be used in the polymerase chain reaction (PCR) to synthesize and amplify a polynucleotide. A detectable label present on a primer will facilitate the identification of desired PCR products. PCR, combined with techniques for preparing complementary DNA (cDNA) can be used to amplify various RNAs, with oligonucleotide primers to provide points for initiation of synthesis in the cDNA duplex flanking the desired sequence and to identify a desired product. Primers labeled with the invention may also be utilized for enzymatic nucleic acid sequencing by the dideoxy chain-termination technique. Alternatively, expression vectors are introduced into Brassica tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Gruber et al , supra; Miki et al , supra; Klein et al , Biotechnology 10:268, 1992.

PCR Background Information

Polymerase chain reaction (PCR) technology is employed in a growing variety of ways, including preparation of cDNAs and constructing cDNA libraries. An early use of PCR to generate a cDNA library was reported by Belyavsky et al , Nucleic Acids Res. 17:2919-32, 1989. The Belyavsky method utilized oligo (dT) as a primer for reverse transcriptase reaction, followed by poly (dG) tailing via the action of terminal deoxynucleotidyl transferase (TdT). The resulting dG-tailed cDNAs were subsequently amplified with poly (dT) and poly (dC) primers. The cDNA pool obtained was cloned into a vector for subsequent cDNA screening. Since an oligo (dT) primer can anneal at any position of the poly (A) tail of a (+) strand of cDNA, and an oligo (dC) primer can anneal at any position of the poly(G) tail of a (-) strand of cDNA, the amplified cDNAs generated by the Belyavsky method often have varying lengths. Accordingly, these products cannot be analyzed directly, and instead require subcloning and screening of a cDNA library, a time-consuming technique. Furthermore, the use of primers containing homopolymers on the 3' end typically yields a high background of non-specific product. A technique for rapid amplification of cDNA ends (RACE) was in Frohman et al ,

Proc. Nat'l Acad. Sci. USA 85:8998-9002, 1988, and Frohman, PCR Protocols, A Guide to Methods and Applications, 28-38 (Academic Press 1990). The RACE protocol produces specific cDNAs by using PCR to amplify the region between a single point on a transcript and the 3' or the 5' ends. One requires knowledge of the sequence of an internal portion of the transcript, however, in order to design a primer for use in conjunction with either the polyT or polyG primers to amplify the ends. This protocol yields specific cDNAs products only, not whole libraries. A modification to the RACE protocol introduced by Borson et al. , PCR Methods and Applications 2:144-48, 1992, entails the use of a "lock-docking oligo (dT)." The locking mechanism involves extending the poly dT primer, by either one nucleotide (A, C or G) or by two nucleotides (also A, C or G) and yet one more of the four possible nucleotides, at the 3'-end of the primer. This "locks" the primer to the beginning of the poly dT tail, either the natural dT or a poly dT tail attached to the first strand cDNA 3 '-end, by use of TdT, resulting in the synthesis of cDNA's of discrete lengths. Subcloning and screening of subclone library is not necessary before analysis, which can speed up the inquiry. Like the RACE protocol, however, Borson's protocol uses a gene-specific internal primer and, hence, produces only specific cDNAs, not whole libraries.

Approaches are described in the literature to identify mRNA expressed differentially, either in only some cell types, or at certain times of a biological process, or during infection by a parasite or a virus, etc. Those studies generally employ subtractive hybridization to reveal the differentially expressed mRNA(s). Liang and colleagues have used the anchored- end technique to look for specific differences in mRNA populations. Liang et al , Nucleic Acids Res. 21:3269-75, 1993. The Liang method, called "differential display," employs a decanucleotide of arbitrary sequence as a primer for PCR, internal to the mRNA, and a polyTMN primer on the 3 '-end of mRNAs; "M" in this context is randomly G, C or A, but N is chosen as one of the four possible nucleotides. When such sets of primers are employed, patterns of mRNAs can be visualized, upon polyacrylamide gel electrophoresis of the PCR product, and the comparison of such patterns produced by mRNAs from two sources reveal the differentially expressed mRNAs.

The differential display method can indicate the individual, differently expressed mRNA's, but cannot constitute a complete library of such mRNA's. As a further consequence of having one primer of an arbitrary sequence, and therefore probably not having an exact match, low copy number mRNAs may not be picked up by this method. Finally, the cDNA candidates identified would still require recovery from the gel and subcloning, if the individual cDNA is desired for further analysis.

Lisitsyn et al , Science 259:946-51, 1993, have described a representational differences analysis (RDA) which uses subtractive hybridization and PCR technology to define the differences between two genomes. Like other subtractive hybridization protocols, in RDA there are defined two sets of DNAs, the "tester" DNA and the "driver" DNA. According to the RDA protocol, the DNA of the two genomes to be compared are digested by restriction endonucleases, and a dephoshorylated double-stranded oligonucleotide adapter is ligated. After denaturation and hybridization of driver and tester DNA, oligonucleotides from the adapters covalently linked to tester DNA were used to amplify unique DNA sequences of tester library. The adapters are partially double-stranded DNAs made by partially complementary oligos, where the single-stranded sequence at one end of the double stranded adapter is complementary to the single-strand tail of the digested genomic DNA. The combined use of (i) restriction enzyme, digested DNA as PCR substrate and (ii) the preferential amplification of shorter substrates results in a population of fairly short, amplified DNA molecules. The adapters then are removed by cleavage with the restriction enzymes used originally to digest the DNA. To the tester DNA, new adapters with novel sequences are ligated, the tester and driver DNA are mixed, the DNA strands are separated by heating ("melting"), and the DNA's are cooled to allow for reannealing. PCR is performed with primers complementary to the adapters on tester DNA, thereby amplifying only target DNA, i.e. , only DNA unique to the tester DNA. By restriction enzyme digestion of the adapters from the amplified DNA and ligation of additional, novel adapters, followed by PCR, the target DNA is amplified to become the dominant fraction.

The RDA procedure does not use any physical method of separation between the tester and driver DNA which, if used, would allow enhanced purification of target DNA. The method is used only to identify differences between genomes and was not used to identify differential cDNA expression.

Expression vectors that are suitable for production of PCPLD polypeptide typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence. PCPLD polypeptide of the present invention preferably is expressed in eukaryotic cells, such as mammalian, insect and yeast cells. Mammalian cells are especially preferred eukaryotic hosts because mammalian cells provide suitable post-translational modifications such as glycosylation. Examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH,; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H- 4-II-E; ATCC CRL1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658). For a mammalian host, the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al , J. Molec. Appl. Genet. 1:273,1982); the TK promoter of Herpes virus (McKnight, Cell 31: 355, 1982); the SV40 early promoter (Benoist et al. , Nature 290:304, 1981); the Rous sarcoma virus promoter (Gorman et al. , Proc. Nat'l. Acad. Sci. USA 79:6777, 1982); and the cytomegalovirus promoter (Foecking et al , Gene 45:101, 1980). Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used to control fusion gene expression if the prokaryotic promoter is regulated by a eukaryotic promoter. Zhou et al. , Mol Cell Biol 10:4529, 1990; Kaufman et al , Nucl. Acids Res. 19:4485, 1991.

An expression vector can be introduced into host cells using a variety of techniques including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, transfected cells are selected and propagated wherein the expression vector is stably integrated in the host cell genome to produce stable transformants. Techniques for introducing vectors into eukaryotic cells and techniques for selecting stable transformants using a dominant selectable marker are described, for example, by Ausubel and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991). DNA molecules encoding the human PCPLD gene can be used to detect the level of

PCPLD gene expression in tissue samples. Such a detection method can be used, for example, to compare the amount of PCPLD RNA in a sample obtained from normal tissue and in a sample isolated from methotrexate-resistant tumor tissue. The presence of relatively low levels of PCPLD RNA in the tumor sample would indicate that methotrexate resistance is due, at least in part, to underexpression of the PCPLD gene. This result also would indicate that treatment of a mammal having such a tumor with methotrexate should be augmented by PCPLD gene therapy. In testing a tissue sample for PCPLD RNA using a nucleic acid hybridization assay, RNA can be isolated from tissue by sectioning on a cryostat and lysing the sections with a detergent such as SDS and a chelating agent such as EDTA, optionally with overnight digestion with proteinase K. Such tissue is obtained by biopsy. A preferred quantity of tissue is in the range of 1-10 milligrams. Protein is removed by phenol and chloroform extractions, and nucleic acids are precipitated with ethanol. RNA is isolated by chromatography on an oligo dT column and then eluted from the column. Further fractionation also can be carried out according to methods well known to those of ordinary skill in the art. A number of techniques for molecular hybridization are used for the detection of

DNA or RNA sequences in tissues. When large amounts of tissue are available, analysis of hybridization kinetics provides the opportunity to accurately quantitate the amount of DNA or RNA present, as well as to distinguish sequences that are closely related but not identical to the probe. Reactions are run under conditions of hybridization (Tm-25 °C) in which the rate of reassociation of the probe is optimal. Wetmur et al , J. Mol. Biol. 31:349, 1968. The kinetics of the reaction are second order when the sequences in the tissue are identical to those of the probe; however, the reaction exhibits complex kinetics when probe sequences have partial homology to those in the tissue. Sharp et al , J. Mol. Biol 86:709, 1974.

The concentration of probe to cellular RNA is determined by the sensitivity desired. To detect one transcript per cell would require about 100 pg of probe per mg of total cellular DNA or RNA. The nucleic acids are mixed, denatured, brought to the appropriate salt concentration and temperature, and allowed to hybridize for various periods of time. The rate of reassociation can be determined by quantitating the amount of probe hybridized either by hydroxyapatite chromatography (Britten et al , Science 161:529, 1968) or by SI nuclease digestion (Sutton, Biochim. Biophys. Acta 240:522, 1971).

A more flexible method of hybridization is the northern blot technique. The particular hybridization technique is not essential to the invention, and any technique commonly used in the art being within the scope of the present invention. Typical probe technology is described in United States Patent 4,358,535 to Falkow et al , incorporated by reference herein. For example, hybridization can be carried out in a solution containing 6 x SSC (10 x SSC: 1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0), 5 x Denhardt's (1 x Denhardt's: 0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone, 0.02% Ficoll 400), 10 mM EDTA, 0.5% SDS and about 10⁷ cpm of nick-translated DNA for 16 hours at 65 °C.

The hybridization assays of the present invention are particularly well suited for preparation and commercialization in kit form, the kit comprising a carrier means compartmentalized to receive one or more container means (vial, test tube, etc.) in close confinement, with each container means comprising one of the separate elements to be used in hybridization assay. For example, there may be a container means containing PCPLD DNA molecules suitable for labeling by "nick translation," or containing labeled PCPLD DNA or labeled PCPLD RNA molecules. Further container means may contain standard solutions for nick translation of DNA comprising DNA polymerase I/DNase I and unlabeled deoxyribonucleotides .

Antibodies to human PCPLD protein can be obtained using the product of an PCPLD expression vector as an antigen. The preparation of polyclonal antibodies is well-known to those of skill in the art. See, for example, Green et al , "Production of Polyclonal Antisera, " in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992).

Alternatively, PCPLD antibody of the present invention may be derived from a rodent monoclonal antibody (MAb). Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256:495, 1975, and Coligan et al (eds.), Current Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter "Coligan"]. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a variety of well- established techniques. Such isolation techniques include affinity chromatography with Protein- A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al, "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, 10:79- 104 Humana Press, Inc. 1992. A PCPLD antibody of the present invention may also be derived from a subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al , international patent publication No. WO 91/11465 (1991), and in Losman et al , Int. J. Cancer 46:310, 1990. Alternatively, a therapeutically useful PCPLD antibody may be derived from a

"humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al , Proc. Nat'l. Acad. Sci. USA 86:3833, 1989. Techniques for producing humanized MAbs are described, for example, by Jones et al , Nature 321:522, 1986, Riechmann et al , Nature 332:323, 1988, Verhoeyen et al. , Science 239: 1534, 1988,

Carter et al , Proc. Nat'l Acad. Sci. USA 89:4285, 1992, Sandhu, Crit. Rev. Biotech. 12: 437, 1992, and Singer et al , J. Immun. 150:2844, 1993, each of which is hereby incorporated by reference.

As an alternative, a PCPLD antibody of the present invention may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al , METHODS: A Companion to Methods in Enzymology 2: 119 1991, and Winter et al , Ann. Rev. Immunol. 12:433, 1994, which are incorporated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA). In addition, a PCPLD antibody of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody- secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al , Nature Genet. 7: 13, 1994; Lonberg et al. , Nature 368:856, 1994, and Taylor et al, Int. Immun. 6:579, 1994.

The invention, illustrated by the following examples, should not be deemed as limited in any way by the following representative examples.

Example 1

This example illustrates that the recombinant human PCPLD enzymes are useful in developing a screening assay for compounds that modulate PCPLD activity. Figures 1 and 3 show two examples of a screening assay for PCPLD activity in cell extracts based on a fluorecent asssay (Ella, et al. , Anal Biochem. 218: 136-142, 1994) with the major exception that, instead of using the substrate BPC (Molecular Probes, Eugene, OR), we used a synthetic phosphatidylcholine (PC) substrate with a fluorescent NBD moiety incorporated into the end of the acyl-chain at the SNl position of PC (NBD-PC). BPC contains an ether linkage at the sn-l position, while NBD-PC contains an acyl linkage at the sn-l position. Having an acyl linkage at the snl position provides the additional opportunity to examine PLA1 activity along with other PC-hydrolysing phospholipases such as PCPLD, PCPLC, and PLA2 at the same time.

The assay for PCPLD uses the transphosphatidylation (Saito, et al. , Arch. Biochem. Biophys. 169: 318-323, 1975) reaction as a means of defining PCPLD activity. This reaction occurs when PCPLD hydrolyses PC into PA and choline in presence of a primary alcohol, such as butanol, where PA will be converted to phosphatidylbutanol (PBt). PBt is more resistant to hydrolysis by enzymes such as PA phosphohydrolase (PAPh) and can be easily separated from PC and other products by thin layer chromatography.

In figures 1 and 2, cell lysate was prepared from Sf9 cells transfected with Baculoviral constructs expressing different PCPLD enzymes. The samples were then incubated with NBD-PC for 30 min at 30° before loading onto TLC plates.

Figure 1 shows the activity level of Sf9 cells transfected with hPLDl (Hammond, et al, J. Biol. Chem. 270: 29640-29643, 1995) and hPLD2.1, also referred as hPCPLD, as evidenced by the fluorescent intensity of the products corresponding to NBD-PBt and NBD- PA on the TLC plate. Lane 1 refers to NBD-PC digested with cabbage PCPLD (Sigma, St. Louis, MO) for the generation of certain lipid standards. Lanes 2 to 4 refer to NBD-PC treated with cell lysates transfected with Baculoviral constructs expressing hPCPLD, hPLDl, and 3-glucuronidase as a negative control. Lane 5 refers to NBD-PC treated with B. cereus PCPLC (Sigma, St. Louis, MO) for the generation of NBD-DAG standard. Lane 6 refers to the starting substrate, NBD-PC, by itself.

Figure 2 demonstrates the PCPLD activity in cell extracts transfected with hPLD2.1, and its isoform, hPLD2.2. Lane 1 refers to the starting substrate, NBD-PC, by itself. Lanes 2 and 3 refer to duplicate samples of NBD-PC treated with cell lysates transfected with Baculoviral constructs expressing /3-glucuronidase as a negative control. Lanes 4 and 5 refer to duplicate samples of NBD-PC treated with cell lysates transfected with Baculoviral constructs expressing hPLDl .4. Lanes 6 and 7 refer to duplicate samples of NBD-PC treated with cell lysates transfected with Baculoviral constructs expressing hPLD2.1. Lanes 8 and 9 refer to duplicate samples of NBD-PC treated with cell lysates transfected with Baculoviral constructs expressing hPLD2.2. The result displays that Sf9 cells transfected with hPLD1.4, hPLD2.1, or hPLD2.2 contain approximately 12-fold, 1.5-fold, and 4-fold, respectively, higher activities of PCPLD, as evidenced by the increased fluorescent intensity of the products corresponding to NBD-PBt and NBD-PA on the TLC plate (lanes 4 to 9) when compared to controls (lanes 2 and 3). hPLD2.2 was found to have higher PCPLD activity than hPLD2.1, suggesting that some of the minor changes in amino acid sequence can affect the enzymatic activity.

Example 2

This example illustrates how recombinant hPLDl, as representative of other PLD isoforms, could be used in a screening assay for compounds that modulate PCPLD activity. The results of this assay are shown in figure 3.

Figure 3 illustrates an example of CT-2584 on recombinant hPLDl activity. In this particular example, cell lysate was prepared from insect cell extracts transfected with Baculoviral vector expressing hPLDl. The samples were incubated with NBD-PC and butanol along with various concentrations of an anti-tumor compound, CT-2584 for 30 min before loading onto TLC plates (Lanes 3 to 9). Lane 10 refers to NBD-PC, a PC with a NBD-group at the SNl acyl chain treated with cabbage PLD. Lanes 3 to 9 refer to cell extract treated with various concentrations of CT-2584. Lanes 10 and 11 refer to NBD-PC substrate with no enzyme treatment. Lanes 2 and 12 refer to NBD-PC treated with SF9 lysate overexpressing hPLDl. Lane 13 refers to NBD-PC treated with B. cereus PCPLC (Sigma, St. Louis, MO) for the generation of NBD-DAG standard. Lane 14 refers to NBD- PAF treated with hPLDl and lane 15 refers to the mobility of the marker NBD-FA. Figure 3 shows that increasing concentration of CT-2584 led to increased activity of PCPLD and PCPLC, as evidenced by the increased flourescent intensity of the products corresponding to NBD-Pa-Bt, NBD-LPA-Bt, and NBD-PA on the TLC plate. On the other hand, CT-2584 has little effect on PLA1 and PLA2 activity, as evidenced by the even flourescent intensity of the products corresponding to NBD-free fatty acid (NBD-FFA) and NBD-lysophosphatidic acid (NBD-LPC) across the TLC plate. This type of assays is useful to screen for agonists and antagonists of PCPLD, as PCPLD has been found to be activated in response to treatment of cells with various hormones and growth factors (Exton, Biochim Biophys Ada 1212: 26-42, 1994).

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Leung, David . Tompkins, Christopher

(ii) TITLE OF INVENTION: PHOSPHATIDYLCHOLINE PHOSPHOLIPASE D

(ϋi) NUMBER OF SEQUENCES: 16 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Cell Therapeutics, Inc.

(B) STREET: 200 Elliott Avenue West, Suite 400

(C) CITY: Seattle

(D) STATE: Washington

(E) COUNTRY: U.S.A. (F) ZIP 98119

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" disk, 1.44Mb, double side, high density

(B) COMPUTER: Hewlett Packard-IBM Compatible (C) OPERATING SYSTEM: MS-DOS Version 6

(D) SOFTWARE: WORD for WINDOWS (vi) CURRENT APPLICATION DATA :

(A) APPLICATION NUMBER:

(B) FILING DATE: 16-Dec-1996 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: Provisional US60008768

(B) FILING DATE: 15-Dec-1995 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Faciszewski, Stephen

(B) REGISTRATION NUMBER: 36,131,

(C) REFERENCE/DOCKET NUMBER: 1802A (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206)282-7100 (B) TELEFAX: (206)284-6206

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3525 (B) TYPE: nucleic acid

(C) STRANDEDNESS : single stranded

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (V) FRAGMENT TYPE: (Vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapien

(B) STRAIN: (C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE: (G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE: (vii) IMMEDIATE SOURCE: (A) LIBRARY:

(B) CLONE: (viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT:

(B) MAP POSITION: (C) UNITS:

(ix) FEATURE:

(A) NAME/KEY: hPCPLD

(B) LOCATION:

(C) IDENTIFICATION METHOD: (D) OTHER INFORMATION:

(X) PUBLICATION INFORMATION:

(A) AUTHORS:

(B) TITLE:

(C) JOURNAL: (D) VOLUME:

(E) ISSUE:

(F) PAGES:

(G) DATE:

(H) DOCUMENT NUMBER: (I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES IN SEQ ID NO:l: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TGCAGCTCCGGTCTGCTCTCTTGGCTCGGGAACCCCCGCGGGCGCTGGCTCCGTCTGCCA 60

GGG ATG ACG GCG ACC CCT GAG AGC CTC TTC CCC ACT GGG GAC GAA 105 Met Thr Ala Thr Pro Glu Ser Leu Phe Pro Thr Gly Asp Glu

5 10

CTG GAC TCC AGC CAG CTC CAG ATG GAG TCC GAT GAG GTG GAC ACC 150 Leu Asp Ser Ser Gin Leu Gin Met Glu Ser Asp Glu Val Asp Thr 15 20 25

CTG AAG GAG GGA GAG GAC CCA GCC GAC CGG ATG CAC CCG TTT CTG 195 Leu Lys Glu Gly Glu Asp Pro Ala Asp Arg Met His Pro Phe Leu 30 35 40 GCC ATC TAT GAG CTT CAG TCT CTG AAA GTG CAC CCC TTG GTG TTC 240 Ala lie Tyr Glu Leu Gin Ser Leu Lys Val His Pro Leu Val Phe

45 50 55

GCA CCT GGG GTC CCT GTC ACA GCC CAG GTG GTG GGC ACC GAA AGA 285 Ala Pro Gly Val Pro Val Thr Ala Gin Val Val Gly Thr Glu Arg 60 65 70

TAT ACC AGC GGA TCC AAG GTG GGA ACC TGC ACT CTG TAT TCT GTC 330 Tyr Thr Ser Gly Ser Lys Val Gly Thr Cys Thr Leu Tyr Ser Val

75 80 85

CGC TTG ACT CAC GGC GAC TTT TCC TGG ACA ACC AAG AAG AAA TAC 375 Arg Leu Thr His Gly Asp Phe Ser Trp Thr Thr Lys Lys Lys Tyr 90 95 100

CGT CAT TTT CAG GAG CTG CAT CGG GAC CTC CTG AGA CAC AAA GTC 420 Arg His Phe Gin Glu Leu His Arg Asp Leu Leu Arg His Lys Val 105 110 115 TTG ATG AGT CTG CTC CCT CTG GCT CGA TTT GCC GTT GCC TAT TCT 465

Leu Met Ser Leu Leu Pro Leu Ala Arg Phe Ala Val Ala Tyr Ser

120 125 130

CCA GCC CGA GAT GCA GGC AAC AGA GAG ATG CCC TCT CTA CCC CGG 510

Pro Ala Arg Asp Ala Gly Asn Arg Glu Met Pro Ser Leu Pro Arg

135 140 145

GCA GGT CCT GAG GGC CC ACC AGA CAT GCA GCC AGC AAA CAG AAA 555

Ala Gly Pro Glu Gly Ser Thr Arg His Ala Ala Ser Lys Gin Lys

150 155 160

TAC CTG GAG AAT TAC CTC AAC CGT CTC TTG ACC ATG TCT TTC TAT 600

Tyr Leu Glu Asn Tyr Leu Asn Arg Leu Leu Thr Met Ser Phe Tyr

165 170 175

CGC AAC TAC CAT GCC ATG ACA GAG TTC CTG GAA GTC AGT CAG CTG 645

Arg Asn Tyr His Ala Met Thr Glu Phe Leu Glu Val Ser Gin Leu

180 185 190

TCC TTT ATC CCG GAA TTG GGC CGC AAA GGA CTG GAG GGG ATG ATC 690

Ser Phe He Pro Glu Leu Gly Arg Lys Gly Leu Glu Gly Met He

195 200 205

CGG AAG CGC TCA GGT GGC CAC CGT GTT TCT GGC CTC ACC TGC TGT 735

Arg Lys Arg Ser Gly Gly His Arg Val Ser Gly Leu Thr Cys Cys

210 215 220

GGC CGA GAC CAA GTT TGT TAT CGC TGG TCC AAG AGG TGG CTG GTG 780

Gly Arg Asp Gin Val Cys Tyr Arg Trp Ser Lys Arg Trp Leu Val

225 230 235

GTG AAG GAC TCC TTC CTG CTG TAC ATG TGC CTC GAG ACA GGT GCC 825

Val Lys Asp Ser Phe Leu Leu Tyr Met Cys Leu Glu Thr Gly Ala

240 245 250

ATC TCA TTT GTT CAG CTC TTT GAC CCT GGC TTT GAG GTG CAA GTG 870

He Ser Phe Val Gin Leu Phe Asp Pro Gly Phe Glu Val Gin Val

255 260 265

GGG AAA AGG AGC ACG GAG GCA CGG CAC GGC GTG CGG ATC GAT ACC 915

Gly Lys Arg Ser Thr Glu Ala Arg His Gly Val Arg He Asp Thr

270 275 280

TCC CAC AGG TCC TTG ATT CTC AAG TGC AGC AGC TAC CGG CAG GCA 960

Ser His Arg Ser Leu He Leu Lys Cys Ser Ser Tyr Arg Gin Ala

285 290 295

CGG TGG TGG GCC CAA GAG ATC ACT GAG CTG GCA CAG GGC CCA GGC 1005

Arg Trp Trp Ala Gin Glu He Thr Glu Leu Ala Gin Gly Pro Gly

300 305 310

AGA GAC TTC CTA CAG CTG CAC CGG CAT GAC AGC TAC GCC CCA CCC 1050

Arg Asp Phe Leu Gin Leu His Arg His Asp Ser Tyr Ala Pro Pro

315 320 325

CGG CCT GGG ACC TTG GCC CGG TGG TTT GTG AAT GGG GCA GGT TAC 1095

Arg Pro Gly Thr Leu Ala Arg Trp Phe Val Asn Gly Ala Gly Tyr

330 335 340

TTT GCT GCT GTG GCA GAT GCC ATC CTT CGA GCT CAA GAG GAG ATT 1140

Phe Ala Ala Val Ala Asp Ala He Leu Arg Ala Gin Glu Glu He

345 350 355

TTC ATC ACA GAC TGG TGG TTG AGT CCT GAG GTT TAC CTG AAG CGT 1185

Phe He Thr Asp Trp Trp Leu Ser Pro Glu Val Tyr Leu Lys Arg

360 365 370

CCG GCC CAT TCA GAT GAC TGG AGA CTG GAC ATT ATG CTC AAG AGG 1230

Pro Ala His Ser Asp Asp Trp Arg Leu Asp He Met Leu Lys Arg

375 380 385

AAG GCG GAG GAG GGT GTC CGT GTG TCT ATT CTG CTG TTT AAA GAA 1275 Lys Ala Glu Glu Gly Val Arg Val Ser He Leu Leu Phe Lys Glu

390 395 400

GTG GAA TTG GCC TTG GGC ATC AAC AGT GGC TAT AGC AAG AAG GCG 1320

Val Glu Leu Ala Leu Gly He Asn Ser Gly Tyr Ser Lys Lys Ala 405 410 415

CTG ATG CTG CTG CAC CCC AAC ATA AAG GTG ATG CGT CAC CCA GAC 1365

Leu Met Leu Leu His Pro Asn He Lys Val Met Arg His Pro Asp

420 425 430

CAA GTG ACG TTG TGG GCC CAT CAT GAG AAG CTC CTG GTG GTG GAC 1410 Gin Val Thr Leu Trp Ala His His Glu Lys Leu Leu Val Val Asp

435 440 445

CAA GTG GTA GCA TTC CTG GGG GGA CTG GAC CTT GCC TAT GGC CGC 1455

Gin Val Val Ala Phe Leu Gly Gly Leu Asp Leu Ala Tyr Gly Arg

450 455 460 TGG GAT GAC CTG CAC TAC CGA CTG ACT GAC CTT GGA GAC TCC TCT 1500

Trp Asp Asp Leu His Tyr Arg Leu Thr Asp Leu Gly Asp Ser Ser

465 470 475

GAA TCA GCT GCC TCC CAG CCT CCC ACC CCG CGC CCA GAC TCA CCA 1545

Glu Ser Ala Ala Ser Gin Pro Pro Thr Pro Arg Pro Asp Ser Pro 480 485 490

GCC ACC CCA GAC CTC TCT CAC AAC CAA TTC TTC TGG CTG GGC AAG 1590

Ala Thr Pro Asp Leu Ser His Asn Gin Phe Phe Trp Leu Gly Lys

495 500 505

GAC TAC AGC AAT CTT ATC ACC AAT GAC TGG GTG CAG CTG GAC CGG 1635 Asp Tyr Ser Asn Leu He Thr Asn Asp Trp Val Gin Leu Asp Arg

510 515 520

CCT TTC GAA GAT TTC ATT GAC AGG GAG ACG ACC CCT CGG ATG CCA 1680

Pro Phe Glu Asp Phe He Asp Arg Glu Thr Thr Pro Arg Met Pro

525 530 535 TGG CGG GAC GTT GGG GTG GTC GTC CAT GGC CTA CCG GCC CGG GAC 1725

Trp Arg Asp Val Gly Val Val Val His Gly Leu Pro Ala Arg Asp

540 545 550

CTT GCC CGG CAC TTC ATC CAG CGC TGG AAC TTC ACC AAG ACC ACC 1770

Leu Ala Arg His Phe He Gin Arg Trp Asn Phe Thr Lys Thr Thr 555 560 565

AAG GCC AAG TAC AAG ACT CCC ACA TAC CCC TAC CTG CTT CCC AAG 1815

Lys Ala Lys Tyr Lys Thr Pro Thr Tyr Pro Tyr Leu Leu Pro Lys

570 575 580

TCT ACC AGC ACG GCC AAT CAG CTC CCC TTC ACA CTT CCA GGA GGG 1860 Ser Thr Ser Thr Ala Asn Gin Leu Pro Phe Thr Leu Pro Gly Gly

585 590 595

CAG TGC ACC ACC GTA CAG GTC TTG CGA TCA GTG GAC CGC TGG TCA 1905

Gin Cys Thr Thr Val Gin Val Leu Arg Ser Val Asp Arg Trp Ser

600 605 610 GCA GGG ACT CTG GAG AAC TCC ATC CTC AAT GCC TAC CTG CAC ACC 1950

Ala Gly Thr Leu Glu Asn Ser He Leu Asn Ala Tyr Leu His Thr

615 620 625

ATC AGG GGG AGC CAG CAC TTC CTC TAC ATT GAG AAT CAG TTC TTC 1995

He Arg Gly Ser Gin His Phe Leu Tyr He Glu Asn Gin Phe Phe 630 625 640

ATT AGC TGC TCA GAT GGG CGG ACG GTT CTG AAC AAG GTG GGC GAT 2040

He Ser Cys Ser Asp Gly Arg Thr Val Leu Asn Lys Val Gly Asp

645 650 655

GAG ATT GTG GAC AGA ATC CTG AAG GCC CAC AAA CAG GGG TGG TGT 2085 Glu He Val Asp Arg He Leu Lys Ala His Lys Gin Gly Trp Cys 660 665 670

TAC CGA GTC TAC GTG CTT TTG CCC TTA CTC CCT GGC TTC GAG GGT 2130

Tyr Arg Val Tyr Val Leu Leu Pro Leu Leu Pro Gly Phe Glu Gly

675 680 685

GAC ATC TCC ACG GGC GGT GGC AAG TCC ATC CAG GCC ATT CTG CAC 2175

Asp He Ser Thr Gly Gly Gly Lys Ser He Gin Ala He Leu His

690 695 700

TTT ACT TAC AGG ACC CTG TGT CGT GGG GAG TAT TCA ATC CTG CAT 2220

Phe Thr Tyr Arg Thr Leu Cys Arg Gly Glu Tyr Ser He Leu His

705 710 715

CGC CTT AAA GCA GCC ATG GGG ACA GCA TGG CGG GAC TAT ATT TCC 2265

Arg Leu Lys Ala Ala Met Gly Thr Ala Trp Arg Asp Tyr He Ser

720 725 730

ATC TGC GGG CTT CGT ACA CAC GGA GAG CTG GGC GGG CAC CCC GTC 2310

He Cys Gly Leu Arg Thr His Gly Glu Leu Gly Gly His Pro Val

735 740 745

TCG GAG CTC ATC TAC ATC CAC AGC AAG GTG CTC ATC GCA GAT GAC 2355

Ser Glu Leu He Tyr He His Ser Lys Val Leu He Ala Asp Asp

750 755 760

CGG ACA GTC ATC ATT GAT TCT GCA AAC ATC AAT GAC CGG AGC TTG 2400

Arg Thr Val He He Asp Ser Ala Asn He Asn Asp Arg Ser Leu

765 770 775

CTG GGG AAG CGG GAC AGT GAG CTG GCC GTG CTA ATC GAG GAC ACA 2445

Leu Gly Lys Arg Asp Ser Glu Leu Ala Val Leu He Glu Asp Thr

780 785 790

GAG ACG GAA CCA TCC CTC ATG AAT GGG GCA GAG TAT CAG GCG GGC 2490

Glu Thr Glu Pro Ser Leu Met Asn Gly Ala Glu Tyr Gin Ala Gly

795 800 805

AGG TTT GCC TTG AGT CTG CGG AAG CAC TGC TTC AGT GTG ATT CTT 2535

Arg Phe Ala Leu Ser Leu Arg Lys His Cys Phe Ser Val He Leu

810 815 820

GGA GCA AAT ACC CGG CCA GAC TTG GAT CTC CGA GAC CCC ATC TGT 2580

Gly Ala Asn Thr Arg Pro Asp Leu Asp Leu Arg Asp Pro He Cys

825 830 835

GAT GAC TTC TTC CAG TTG TGG CAA GAC ATG GCT GAG AGC AAC GCC 2625

Asp Asp Phe Phe Gin Leu Trp Gin Asp Met Ala Glu Ser Asn Ala

840 845 850

AAT ATC TAT GAG CAG ATC TTC CGC TGC CTG CCA TCC AAT GCC ACG 2670

Asn He Tyr Glu Gin He Phe Arg Cys Leu Pro Ser Asn Ala Thr

855 860 865

CGT CC CTG CGG ACT CTC CGG GAG TAC GTG GCC GTG GAG CCC TTG 2715

Arg Ser Leu Arg Thr Leu Arg Glu Tyr Val Ala Val Glu Pro Leu

870 875 880

GCC ACG GTC AGT CCC CCC TTG GCT CGG TCT GAG CTC ACC CAG GTC 2760

Ala Thr Val Ser Pro Pro Leu Ala Arg Ser Glu Leu Thr Gin Val

885 890 895

CAG GGC CAC CTG GTC CAC TTC CCC CTC AAG TTC CTA GAG GAT GAG 2805

Gin Gly His Leu Val His Phe Pro Leu Lys Phe Leu Glu Asp Glu

900 905 910

TCT TTG CTG CCC CCG CTG GGT AGC AAG GAG GGC AAG ATC CCC CTA 2850

Ser Leu Leu Pro Pro Leu Gly Ser Lys Glu Gly Lys He Pro Leu

915 920 925

GAA GTG TGG ACA TAG TTGAGGCCCCCGTCAGGGAGAGGTCACCAGCTGCTGTGCC 2905

Glu Val Trp Thr ***

930 CCACCACGTCTGGCTCCCTGCCCCTTAACCCCAAGGACTGAGGGCAGTGCCCTTTGAGAT 2965

CTGGGGAGGCAGGCATTCCTGAAGGGAACTAGAGGTGTTACAGAGGACCCTTACGTGAGA 3025

AATAGCTGAAAAGGGCACTCCCAACCCTGGGCTGGGGAGGAGGAGAGAGTCCCAGAGCTC 3085

ATCCCCCCTGCTGCCCAGTGCAAACCACTTCTCCATGCTGCAAAGGAGAAGCACAGCTCC 3145

TGCCAGGGTGAGCAGGGTCAAGCCTCTTATTCCAGGAGAAGGGGGCTCTGCCCCAGGCCC 3205

TACTACCCATTGTTCCCTTCCTCTTCCTGCCCTTGAACCCCCTCCCTGTCCCAGGGCCCT 3265

CCCAGCCCATTGCTGCCAAGGTGGAGGGAAGGATAAAGCCACTTCTGGCTTCAGCCCCCA 3325

CCAGGGGAAGGAAGGAGGGCACATTAACTCCCTCCACCAGCCTGCTGACAGACACTAACT 3385

TTGTATCCGTTCAATAAGCATTTCATAAAAAAAAAAAAAA 3425

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1074 (B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (V) FRAGMENT TYPE: (Vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapien

(B) STRAIN: (C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE:

(I) ORGANELLE: (vii) IMMEDIATE SOURCE:

(A) LIBRARY:

(B) CLONE: (viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT:

(B) MAP POSITION:

(C) UNITS: (ix) FEATURE: (A) NAME/KEY: hPLDl

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: (x) PUBLICATION INFORMATION:

(A) AUTHORS:

(B) TITLE:

(C) JOURNAL:

(D) VOLUME: (E) ISSUE:

(F) PAGES:

(G) DATE:

(H) DOCUMENT NUMBER:

(I) FILING DATE: (J) PUBLICATION DATE:

(K) RELEVANT RESIDUES IN SEQ ID NO: 2: ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ser Leu Lys Asn Glu Pro Arg Val Asn Thr Ser Ala Leu Gin 5 10 15

Lys lie Ala Ala Asp Met Ser Asn lie lie Glu Asn Leu Asp Thr

20 25 30

Arg Glu Leu His Phe Glu Gly Glu Glu Val Asp Tyr Asp Val Ser

35 40 45 Pro Ser Asp Pro Lys lie Gin Glu Val Tyr lie Pro Phe Ser Ala

50 55 60 lie Tyr Asn Thr Gin Gly Phe Lys Glu Pro Asn lie Gin Thr Tyr

65 70 75

Leu Ser Gly Cys Pro lie Lys Ala Gin Val Leu Glu Val Glu Arg 80 90

Phe Thr Ser Thr Thr Arg Val Pro Ser lie Asn Leu Tyr Thr lie

100 Glu Leu Thr His Gly Glu Phe Lys Trp Gin Val Lys Arg Lys Phe

110 120 Lys His Phe Gin Glu Phe His Arg Glu Leu Leu Lys Tyr Lys Ala

130 Phe lie Arg lie Pro lie Pro Thr Arg Arg His Thr Phe Arg Arg

140 150

Gin Asn Val Arg Glu Glu Pro Arg Glu Met Pro Ser Leu Pro Arg 160

Ser Ser Glu Asn Met lie Arg Glu Glu Gin Phe Leu Gly Arg Arg

170 180

Lys Gin Leu Glu Asp Tyr Leu Thr Lys lie Leu Lys Met Pro Met

190 Tyr Arg Asn Tyr His Ala Thr Thr Glu Phe Leu Asp lie Ser Gin

200 210

Leu Ser Phe lie His Asp Leu Gly Pro Lys Gly lie Glu Gly Met

220 lie Met Lys Arg Ser Gly Gly His Arg lie Pro Gly Leu Asn Cys 230 240

Cys Gly Gin Gly Arg Ala Cys Tyr Arg Trp Ser Lys Arg Trp Leu

250 lie Val Lys Asp Ser Phe Leu Leu Tyr Met Lys Pro Asp Ser Gly

260 270 Ala He Ala Phe Val Leu Leu Val Asp Lys Glu Phe Lys He Lys 280

Val Gly Lys Lys Glu Thr Glu Thr Lys Tyr Gly He Arg He Asp 290 300

Asn Leu Ser Arg Thr Leu He Leu Lys Cys Asn Ser Tyr Arg His 310

Ala Arg Trp Trp Gly Gly Ala He Glu Glu Phe He Gin Lys His 320 330

Gly Thr Asn Phe Leu Lys Asp His Arg Phe Gly Ser Tyr Ala Ala 340

He Gin Glu Asn Ala Leu Ala Lys Trp Tyr Val Asn Ala Lys Gly

350 360

Tyr Phe Glu Asp Val Ala Asn Ala Met Glu Glu Ala Asn Glu Glu 370

He Phe He Thr Asp Trp Trp Leu Ser Pro Glu He Phe Leu Lys 380 390

Arg Pro Val Val Glu Gly Asn Arg Trp Arg Leu Asp Cys He Leu 400

Lys Arg Lys Ala Gin Gin Gly Val Arg He Phe He Met Leu Tyr 410 420

Lys Glu Val Glu Leu Ala Leu Gly He Asn Ser Glu Tyr Thr Lys 430

Arg Thr Leu Met Arg Leu His Pro Asn He Lys Val Met Arg His 440 450

Pro Asp His Val Ser Ser Thr Val Tyr Leu Trp Ala His His Glu 460

Lys Leu Val He He Asp Gin Ser Val Ala Phe Val Gly Gly He 470 480

Asp Leu Ala Tyr Gly Arg Trp Asp Asp Asn Glu His Arg Leu Thr 490

Asp Val Gly Ser Val Lys Arg Val Thr Ser Gly Pro Ser Leu Gly 500 510

Ser Leu Pro Pro Ala Ala Met Glu Ser Met Glu Ser Leu Arg Leu 520

Lys Asp Lys Asn Glu Pro Val Gin Asn Leu Pro He Gin Lys Ser 530 540

He Asp Asp Val Asp Ser Lys Leu Lys Gly He Gly Lys Pro Arg 550

Lys Phe Ser Lys Phe Ser Leu Tyr Lys Gin Leu His Arg His His 560 570

Leu His Asp Ala Asp Ser He Ser Ser He Asp Ser Thr Ser Ser 580

Tyr Phe Asn His Tyr Arg Ser His His Asn Leu He His Gly Leu 590 600

Lys Pro His Phe Lys Leu Phe His Pro Ser Ser Glu Ser Glu Gin 610

Gly Leu Thr Arg Pro His Ala Asp Thr Gly Ser He Arg Ser Leu 620 630

Gin Thr Gly Val Gly Glu Leu His Gly Glu Thr Arg Phe Trp His 640

Gly Lys Asp Tyr Cys Asn Phe Val Phe Lys Asp Trp Val Gin Leu 650 660

Asp Lys Pro Phe Ala Asp Phe He Asp Arg Tyr Ser Thr Pro Arg 670 Met Pro Trp His Asp He Ala Ser Ala Val His Gly Lys Ala Ala

680 690

Arg Asp Val Ala Arg His Phe He Gin Arg Trp Asn Phe Thr Lys

700 He Met Lys Ser Lys Tyr Arg Ser Leu Ser Tyr Pro Phe Leu Leu

710 720

Pro Lys Ser Gin Thr Thr Ala His Glu Leu Arg Tyr Gin Val Pro

730 Gly Ser Val His Ala Asn Val Gin Leu Leu Arg Ser Ala Ala Asp 740 750

Trp Ser Ala Gly He Lys Tyr His Glu Glu Ser He His Ala Ala

760 Tyr Val His Val He Glu Asn Ser Arg His Tyr He Tyr He Glu

770 780 Asn Gin Phe Phe He Ser Cys Ala Asp Asp Lys Val Val Phe Asn

790 Lys He Gly Asp Ala He Ala Gin Arg He Leu Lys Ala His Arg

800 810

Glu Asn Gin Lys Tyr Arg Val Tyr Val Val He Pro Leu Leu Pro 820

Gly Phe Glu Gly Asp He Ser Thr Gly Gly Gly Asn Ala Leu Gin

830 840

Ala He Met His Phe Asn Tyr Arg Thr Met Cys Arg Gly Glu Asn

850 Ser He Leu Gly Gin Leu Lys Ala Glu Leu Gly Asn Gin Trp He

860 870

Asn Tyr He Ser Phe Cys Gly Leu Arg Thr His Ala Glu Leu Glu

880 Gly Asn Leu Val Thr Glu Leu He Tyr Val His Ser Lys Leu Leu 890 900

He Ala Asp Asp Asn Thr Val He He Gly Ser Ala Asn He Asn

910 Asp Arg Ser Met Leu Gly Lys Arg Asp Ser Glu Met Ala Val He

920 930 Val Gin Asp Thr Glu Thr Val Pro Ser Val Met Asp Gly Lys Glu

940 Tyr Gin Ala Gly Arg Phe Ala Arg Gly Leu Arg Leu Gin Cys Phe

950 960

Arg Val Val Leu Gly Tyr Leu Asp Asp Pro Ser Glu Asp He Gin 970

Asp Pro Val Ser Asp Lys Phe Phe Lys Glu Val Trp Val Ser Thr

980 990

Ala Ala Arg Asn Ala Thr He Tyr Asp Lys Val Phe Arg Cys Leu

1000 Pro Asn Asp Glu Val His Asn Leu He Gin Leu Arg Asp Phe He

1010 1020

Asn Lys Pro Val Leu Ala Lys Glu Asp Pro He Arg Ala Glu Glu

1030 Glu Leu Lys Lys He Arg Gly Phe Leu Val Gin Phe Pro Phe Tyr 1040 1050

Phe Leu Ser Glu Glu Ser Leu Leu Pro Ser Val Gly Thr Lys Glu

1060 Ala He Val Pro Met Glu Val Trp Thr ***

1070 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(A) ORGANISM: castor bean

B) STRAIN:

C) INDIVIDUAL ISOLATE:

D) DEVELOPMENTAL STAGE:

E) HAPLOTYPE:

F) TISSUE TYPE:

G) CELL TYPE: H) CELL LINE: I) ORGANELLE:

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: ;B) CLONE:

(viii) POSITION IN GENOME: A) CHROMOSOME/SEGMENT:

[B) MAP POSITION: ;C) UNITS:

(ix) FEATURE:

[A) NAME/KEY: PCPLD fragment

[B) LOCATION:

;C) IDENTIFICATION METHOD: ID) OTHER INFORMATION:

(X) PUBLICATION INFORMATION: (A) AUTHORS:

B) TITLE:

C) JOURNAL:

D) VOLUME:

E) ISSUE:

F) PAGES:

G) DATE:

H) DOCUMENT NUMBER: I) FILING DATE: J) PUBLICATION DATE: K) RELEVANT RESIDUES IN SEQ ID NO: 3;

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gin Arg Ser Met Asp Gly Ala Arg Asp Ser Glu He Ala Met Gly 5 10 15

Ala Tyr Gin Pro ( 2 ) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: polypeptide

(iii) HYPOTHETICAL: no

(iv) ANTI-SENSE: no

(V) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM: yeast

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

(vii) IMMEDIATE SOURCE:

(A) LIBRARY:

(B) CLONE:

(Vϋi) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT:

(B) MAP POSITION:

(C) UNITS:

(ix) FEATURE:

(A) NAME/KEY: PCPLD fragment

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(X) PUBLICATION INFORMATION:

(A) AUTHORS :

(B) TITLE:

(C) JOURNAL:

(D) VOLUME:

(E) ISSUE:

(F) PAGES :

(G) DATE:

(H) DOCUMENT NUMBER:

(I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES IN SEQ ID NO: 4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Glu Arg Ser Gin Leu Gly Asn Arg Asp Ser Glu Val Ala He Leu

5 10 15

He Arg Asp Thr

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(A) ORGANISM: yeast

B) STRAIN:

C) INDIVIDUAL ISOLATE:

D) DEVELOPMENTAL STAGE:

E) HAPLOTYPE:

F) TISSUE TYPE:

G) CELL TYPE: H) CELL LINE: I) ORGANELLE:

(vii) IMMEDIATE SOURCE:

(A) LIBRARY:

;B) CLONE: (viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT:

[B) MAP POSITION: ;c) UNITS:

(ix) FEATURE:

(A) NAME/KEY: polypeptide fragment

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(X) PUBLICATION INFORMATION:

(A) AUTHORS :

(B) TITLE:

(C) JOURNAL:

(D) VOLUME:

(E) ISSUE:

(F) PAGES :

(G) DATE:

(H) DOCUMENT NUMBER:

(I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES IN SEQ ID NO: 5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5i sp Arg Ser Leu Leu Gly Lys Arg Asp Ser Glu Leu Ala Val Leu

5 10 15

He Glu Asp Thr

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24

(B) TYPE: OLIGONUCLEOTIDE

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide fragment (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(Vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE: (D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: o.r83570.1

(B) LOCATION:

(C) IDENTIFICATION METHOD: (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 1 GTATTCAATCCTGCATCGCCTTAA 24

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27

(B) TYPE: OLIGONUCLEOTIDE

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide fragment (iϋ) HYPOTHETICAL: no

(iv) ANTI-SENSE: no

(V) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE: (A) ORGANISM: (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE: (G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE: (ix) FEATURE:

(A) NAME/KEY: O.R83570.1R (B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 1 GTCATCTGCGATGAGCACCTTGCTGTG 27 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: OLIGONUCLEOTIDE (C) STRANDEDNESS: single

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: oligonucleotide fragment (iii) HYPOTHETICAL:no (iv) ANTI-SENSE:no (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN: (C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE:

(I) ORGANELLE: (ix) FEATURE:

(A) NAME/KEY: o. sport. IR

(B) LOCATION: (C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 1 CTAGCTTATAATACGACTCAC 21

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE:OLIGONUCLEOTIDE

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide fragment

(iii) HYPOTHETICAL: no

(iv) ANTI-SENSE: no

(v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE: (A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE: (F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE: (ix) FEATURE: (A) NAME/KEY: o. sport. IR

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 1 GACTCTAGCCTAGGCTTTTGC 21

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: OLIGONUCLEOTIDE

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide fragment (ϋi) HYPOTHETICAL: no (iv) ANTI-SENSE: no (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (A) ORGANISM: (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE: (G) CELL TYPE:

(H) CELL LINE: (I) ORGANELLE: (ix) FEATURE:

(A) NAME/KEY: o.pld3.2R (B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 1 CTCAGGACTCAACCACCAGTC 21 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29

(B) TYPE:OLIGONUCLEOTIDE

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide fragment (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (ix) FEATURE:

(A) NAME/KEY: PCR PRIMER

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 1 GGCTCTAGATATTAATAGTAATCAATTAC 29

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26

(B) TYPE: OLIGONUCLEOTIDE

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide fragment (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE: (F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (ix) FEATURE: (A) NAME/KEY: PCR PRIMER

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 1 CCTCACGCATGCACCATGGTAATAGC 26

(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24

(B) TYPE: OLIGONUCLEOTIDE (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide fragment (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE: (D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: PCR PRIMER

(B) LOCATION:

(C) IDENTIFICATION METHOD: (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 1 GGTGCATGCGTGAGGCTCCGGTGC 24

(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28

(B) TYPE: OLIGONUCLEOTIDE

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide fragment (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (V) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (ix) FEATURE:

(A) NAME/KEY: PCR PRIMER

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 1 GTAGTTTTCACGGTACCTGAAATGGAAG 28

(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3425

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no

(iv) ANTI-SENSE: no

(V) FRAGMENT TYPE:

(Vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapien (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE: (G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE: (vii) IMMEDIATE SOURCE:

(A) LIBRARY: (B) CLONE:

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT:

(B) MAP POSITION:

(C) UNITS: (ix) FEATURE:

(A) NAME/KEY: hPLD2.2

(B) LOCATION:

(C) IDENTIFICATION METHOD: (D) OTHER INFORMATION:

(X) PUBLICATION INFORMATION:

(A) AUTHORS:

(B) TITLE:

(C) JOURNAL: (D) VOLUME:

(E) ISSUE:

(F) PAGES:

(G) DATE:

(H) DOCUMENT NUMBER: (I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES IN SEQ ID NO: 15: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

T TGGCCAi -GGCCTC :CGGI ΓCTGC :τcτc :TTGC ;cτcc 3GGA2 ^CCCC :CGCC 3GGCC ;CTGC 3CTC( :GTC DGCCA 60 GGG ATG ACG GCG ACC CCT GAG AGC CTC TC CCC ACT GGG GAC GAA 105

Met Thr Ala Thr Pro Glu Ser Leu Phe Pro Thr Gly Asp Glu

5 10

CTG GAC TCC AGC CAG CTC CAG ATG GAG TCC GAT GAG GTG GAC ACC 150

Leu Asp Ser Ser Gin Leu Gin Met Glu Ser Asp Glu Val Asp Thr

15 20 25

CTG AAG GAG GGA GAG GAC CCA GCC GAC CGG ATG CAC CCG TTT CTG 195

Leu Lys Glu Gly Glu Asp Pro Ala Asp Arg Met His Pro Phe Leu

30 35 40

GCC ATC TAT GAG CTT CAG TCT CTG AAA GTG CAC CCC TTG GTG TTC 240

Ala He Tyr Glu Leu Gin Ser Leu Lys Val His Pro Leu Val Phe

45 50 55

GCA CCT GGG GTC CCT GTC ACA GCC CAG GTG GTG GGC ACC GAA AGA 285

Ala Pro Gly Val Pro Val Thr Ala Gin Val Val Gly Thr Glu Arg

60 65 70

TAT ACC AGC GGA TCC AAG GTG GGA ACC TGC ACT CTG TAT TCT GTC 330

Tyr Thr Ser Gly Ser Lys Val Gly Thr Cys Thr Leu Tyr Ser Val

75 80 85

CGC TTG ACT CAC GGC GAC TTT TCC TGG ACA ACC AAG AAG AAA TAC 375

Arg Leu Thr His Gly Asp Phe Ser Trp Thr Thr Lys Lys Lys Tyr

90 95 100

CGT CAT TTT CAG GAG CTG CAT CGG GAC CTC CTG AGA CAC AAA GTC 420

Arg His Phe Gin Glu Leu His Arg Asp Leu Leu Arg His Lys Val

105 110 115

TTG ATG AGT CTG CTC CCT CTG GCT CGA TTT GCC GTT GCC TAT TCT 465

Leu Met Ser Leu Leu Pro Leu Ala Arg Phe Ala Val Ala Tyr Ser

120 125 130

CCA GCC CGA GAT GCA GGC AAC AGA GAG ATG CCC TCT CTA CCC CGG 510

Pro Ala Arg Asp Ala Gly Asn Arg Glu Met Pro Ser Leu Pro Arg

135 140 145

GCA GGT CCT GAG GGC TCC ACC AGA CAT GCA GCC AGC AAA CAG AAA 555

Ala Gly Pro Glu Gly Ser Thr Arg His Ala Ala Ser Lys Gin Lys

150 155 160

TAC CTG GAG AAT TAC CTC AAC CGT CTC TTG ACC ATG TCT TTC TAT 600

Tyr Leu Glu Asn Tyr Leu Asn Arg Leu Leu Thr Met Ser Phe Tyr 165 170 175

CGC AAC TAC CAT GCC ATG ACA GAG TTC CTG GAA GTC AGT CAG CTG 645

Arg Asn Tyr His Ala Met Thr Glu Phe Leu Glu Val Ser Gin Leu

180 185 190

TCC TTT ATC CCG GAC TTG GGC CGC AAA GGA CTG GAG GGG ATG ATC 690

Ser Phe He Pro Asp Leu Gly Arg Lys Gly Leu Glu Gly Met He

195 200 205

CGG AAG CGC TCA GGT GGC CAC CGT GTT CCT GGC CTC ACC TGC TGT 735

Arg Lys Arg Ser Gly Gly His Arg Val Pro Gly Leu Thr Cys Cys

210 215 220

GGC CGA GAC CAA GTT TGT TAT CGC TGG TCC AAG AGG TGG CTG GTG 780

Gly Arg Asp Gin Val Cys Tyr Arg Trp Ser Lys Arg Trp Leu Val

225 230 235

GTG AAG GAC TCC TTC CTG CTG TAC ATG TGC CTC GAG ACA GGT GCC 825

Val Lys Asp Ser Phe Leu Leu Tyr Met Cys Leu Glu Thr Gly Ala

240 245 250

ATC TCA TTT GTT CAG CTC TTT GAC CCT GGC TTT GAG GTG CAA GTG 870

He Ser Phe Val Gin Leu Phe Asp Pro Gly Phe Glu Val Gin Val

255 260 265

GGG AAA AGG AGC ACG GAG GCA CGG CAC GGC GTG CGG ATC GAT ACC 915

Gly Lys Arg Ser Thr Glu Ala Arg His Gly Val Arg He Asp Thr

270 275 280 CC CAC AGG TCC TTG ATT CTC AAG TGC AGC AGC TAC CGG CAG GCA 960

Ser His Arg Ser Leu He Leu Lys Cys Ser Ser Tyr Arg Gin Ala

285 290 295

CGG TGG TGG GCC CAA GAG ATC ACT GAG CTG GCA CAG GGC CCA GGC 1005

Arg Trp Trp Ala Gin Glu He Thr Glu Leu Ala Gin Gly Pro Gly

300 305 310

AGA GAC TTC CTA CAG CTG CAC CGG CAT GAC AGC TAC GCC CCA CCC 1050

Arg Asp Phe Leu Gin Leu His Arg His Asp Ser Tyr Ala Pro Pro

315 320 325

CGG CCT GGG ACC TTG GCC CGG TGG TTT GTG AAT GGG GCA GGT TAC 1095

Arg Pro Gly Thr Leu Ala Arg Trp Phe Val Asn Gly Ala Gly Tyr

330 335 340

TTT GCT GCT GTG GCA GAT GCC ATC CTT CGA GCT CAA GAG GAG ATT 1140

Phe Ala Ala Val Ala Asp Ala He Leu Arg Ala Gin Glu Glu He

345 350 355

TTC ATC ACA GAC TGG TGG TTG AGT CCT GAG GTT TAC CTG AAG CGT 1185

Phe He Thr Asp Trp Trp Leu Ser Pro Glu Val Tyr Leu Lys Arg

360 365 370

CCG GCC CAT TCA GAT GAC TGG AGA CTG GAC ATT ATG CTC AAG AGG 1230

Pro Ala His Ser Asp Asp Trp Arg Leu Asp He Met Leu Lys Arg

375 380 385

AAG GCG GAG GAG GGT GTC CGT GTG TCT ATT CTG CTG TTT AAA GAA 1275

Lys Ala Glu Glu Gly Val Arg Val Ser He Leu Leu Phe Lys Glu

390 395 400

GTG GAA TTG GCC TTG GGC ATC AAC AGT GGC TAT AGC AAG AGG GCG 1320

Val Glu Leu Ala Leu Gly He Asn Ser Gly Tyr Ser Lys Arg Ala

405 410 415

CTG ATG CTG CTG CAC CCC AAC ATA AAG GTG ATG CGT CAC CCA GAC 1365

Leu Met Leu Leu His Pro Asn He Lys Val Met Arg His Pro Asp

420 425 430

CAA GTG ACG TTG TGG GCC CAT CAT GAG AAG CTC CTG GTG GTG GAC 1410

Gin Val Thr Leu Trp Ala His His Glu Lys Leu Leu Val Val Asp

435 440 445 CAA GTG GTA GCA TTC CTG GGG GGA CTG GAC CTT GCC TAT GGC CGC 1455

Gin Val Val Ala Phe Leu Gly Gly Leu Asp Leu Ala Tyr Gly Arg

450 455 460

TGG GAT GAC CTG CAC TAC CGA CTG ACT GAC CTT GGA GAC TCC TCT 1500 Trp Asp Asp Leu His Tyr Arg Leu Thr Asp Leu Gly Asp Ser Ser

465 470 475

GAA TCA GCT GCC TCC CAG CCT CCC ACC CCG CGC CCA GAC TCA CCA 1545

Glu Ser Ala Ala Ser Gin Pro Pro Thr Pro Arg Pro Asp Ser Pro

480 485 490 GCC ACC CCA GAC CTC TCT CAC AAC CAA TTC TTC TGG CTG GGC AAG 1590

Ala Thr Pro Asp Leu Ser His Asn Gin Phe Phe Trp Leu Gly Lys

495 500 505

GAC TAC AGC AAT CTT ATC ACC AAG GAC TGG GTG CAG CTG GAC CGG 1635

Asp Tyr Ser Asn Leu He Thr Lys Asp Trp Val Gin Leu Asp Arg 510 515 520

CCT TTC GAA GAT TTC ATT GAC AGG GAG ACG ACC CCT CGG ATG CCA 1680

Pro Phe Glu Asp Phe He Asp Arg Glu Thr Thr Pro Arg Met Pro

525 530 535

TGG CGG GAC GTT GGG GTG GTC GTC CAT GGC CTA CCG GCC CGG GAC 1725 Trp Arg Asp Val Gly Val Val Val His Gly Leu Pro Ala Arg Asp

540 545 550

CTT GCC CGG CAC TTC ATC CAG CGC TGG AAC TTC ACC AAG ACC ACC 1770

Leu Ala Arg His Phe He Gin Arg Trp Asn Phe Thr Lys Thr Thr

555 560 565 AAG GCC AAG TAC AAG ACT CCC ACA TAC CCC TAC CTG CTT CCC AAG 1815

Lys Ala Lys Tyr Lys Thr Pro Thr Tyr Pro Tyr Leu Leu Pro Lys

570 575 580

TCT ACC AGC ACG GCC AAT CAG CTC CCC TTC ACA CTT CCA GGA GGG 1860

Ser Thr Ser Thr Ala Asn Gin Leu Pro Phe Thr Leu Pro Gly Gly 585 590 595

CAG TGC ACC ACC GTA CAG GTC TTG CGA TCA GTG GAC CGC TGG TCA 1905

Gin Cys Thr Thr Val Gin Val Leu Arg Ser Val Asp Arg Trp Ser

600 605 610

GCA GGG ACT CTG GAG AAC TCC ATC CTC AAT GCC TAC CTG CAC ACC 1950 Ala Gly Thr Leu Glu Asn Ser He Leu Asn Ala Tyr Leu His Thr

615 620 625

ATC AGG GGG AGC CAG CAC TTC CTC TAC ATT GAG AAT CAG TTC TTC 1995

He Arg Gly Ser Gin His Phe Leu Tyr He Glu Asn Gin Phe Phe

630 625 640 ATT AGC TGC TCA GAT GGG CGG ACG GTT CTG AAC AAG GTG GGC GAT 2040

He Ser Cys Ser Asp Gly Arg Thr Val Leu Asn Lys Val Gly Asp

645 650 655

GAG ATT GTG GAC AGA ATC CTG AAG GCC CAC AAA CAG GGG TGG TGT 2085

Glu He Val Asp Arg He Leu Lys Ala His Lys Gin Gly Trp Cys 660 665 670

TAC CGA GTC TAC GTG CTT TTG CCC TTA CTC CCT GGC TTC GAG GGT 2130

Tyr Arg Val Tyr Val Leu Leu Pro Leu Leu Pro Gly Phe Glu Gly

675 680 685

GAC ATC TCC ACG GGC GGT GGC AAC TCC ATC CAG GCC ATT CTG CAC 2175 Asp He Ser Thr Gly Gly Gly Asn Ser He Gin Ala He Leu His

690 695 700

TTT ACT TAC AGG ACC CTG TGT CGT GGG GAG TAT TCA ATC CTG CAT 2220

Phe Thr Tyr Arg Thr Leu Cys Arg Gly Glu Tyr Ser He Leu His

705 710 715 CGC CTT AAA GCA GCC ATG GGG ACA GCA TGG CGG GAC TAT ATT TCC 2265 Arg Leu Lys Ala Ala Met Gly Thr Ala Trp Arg Asp Tyr He Ser

720 725 730

ATC TGC GGG CTT CGT ACA CAC GGA GAG CTG GGC GGG CAC CCC GTC 2310

He Cys Gly Leu Arg Thr His Gly Glu Leu Gly Gly His Pro Val 735 740 745

TCG GAG CTC ATC TAC ATC CAC AGC AAG GTG CTC ATC GCA GAT GAC 2355

Ser Glu Leu He Tyr He His Ser Lys Val Leu He Ala Asp Asp

750 755 760

CGG ACA GTC ATC ATT GGT TCT GCA AAC ATC AAT GAC CGG AGC TTG 2400 Arg Thr Val He He Gly Ser Ala Asn He Asn Asp Arg Ser Leu

765 770 775

CTG GGG AAG CGG GAC AGT GAG CTG GCC GTG CTA ATC GAG GAC ACA 2445

Leu Gly Lys Arg Asp Ser Glu Leu Ala Val Leu He Glu Asp Thr

780 785 790 GAG ACG GAA CCA TCC CTC ATG AAT GGG GCA GAG TAT CAG GCG GGC 2490

Glu Thr Glu Pro Ser Leu Met Asn Gly Ala Glu Tyr Gin Ala Gly

795 800 805

AGG TTT GCC TTG AGT CTG CGG AAG CAC TGC TTC GGT GTG ATT CTT 2535

Arg Phe Ala Leu Ser Leu Arg Lys His Cys Phe Gly Val He Leu 810 815 820

GGA GCA AAT ACC CGG CCA GAC TTG GAT CTC CGA GAC CCC ATC TGT 2580

Gly Ala Asn Thr Arg Pro Asp Leu Asp Leu Arg Asp Pro He Cys

825 830 835

GAT GAC TTC TTC CAG TTG TGG CAA GAC ATG GCT GAG AGC AAC GCC 2625 Asp Asp Phe Phe Gin Leu Trp Gin Asp Met Ala Glu Ser Asn Ala

840 845 850

AAT ATC TAT GAG CAG ATC TTC CGC TGC CTG CCA TCC AAT GCC ACG 2670

Asn He Tyr Glu Gin He Phe Arg Cys Leu Pro Ser Asn Ala Thr

855 860 865 CGT TCC CTG CGG ACT CTC CGG GAG TAC GTG GCC GTG GAG CCC TTG 2715

Arg Ser Leu Arg Thr Leu Arg Glu Tyr Val Ala Val Glu Pro Leu

870 875 880

GCC ACG GTC AGT CCC CCC TTG GCT CGG TCT GAG CTC ACC CAG GTC 2760

Ala Thr Val Ser Pro Pro Leu Ala Arg Ser Glu Leu Thr Gin Val 885 890 895

CAG GGC CAC CTG GTC CAC TTC CCC CTC AAG TTC CTA GAG GAT GAG 2805

Gin Gly His Leu Val His Phe Pro Leu Lys Phe Leu Glu Asp Glu

900 905 910

TCT TTG CTG CCC CCG CTG GGT AGC AAG GAG GGC ATG ATC CCC CTA 2850 Ser Leu Leu Pro Pro Leu Gly Ser Lys Glu Gly Met He Pro Leu

915 920 925

GAA GTG TGG ACA TAG TTGAGGCCCCCGTCAGGGAGAGGTCACCAGCTGCTGTGCC 2905

Glu Val Trp Thr ***

930 CCACCACGTCTGGCTCCCTGCCCCTTAACCCCAAGGACTGAGGGCAGTGCCCTTTGAGAT 2965

CTGGGGAGGCAGGCATTCCTGAAGGGAACTAGAGGTGTTACAGAGGACCCTTACGTGAGA 3025

AATAGCTGAAAAGGGCACTCCCAACCCTGGGCTGGGGAGGAGGAGAGAGTCCCAGAGCTC 3085

ATCCCCCCTGCTGCCCAGTGCAAACCACTTCTCCATGCTGCAAAGGAGAAGCACAGCTCC 3145

TGCCAGGGTGAGCAGGGTCAAGCCTCTTATTCCAGGAGAAGGGGGCTCTGCCCCAGGCCC 3205 TACTACCCATTGTTCCCTTCCTCTTCCTGCCCTTGAACCCCCTCCCTGTCCCAGGGCCCT 3265

CCCAGCCCATTGCTGCCAAGGTGGAGGGAAGGATAAAGCCACTTCTGGCTTCAGCCCCCA 3325

CCAGGGGAAGGAAGGAGGGCACATTAACTCCCTCCACCAGCCTGCTGACAGACACTAACT 3385

TTGTATCCGTTCAATAAGCATTTCATAAAAAAAAAAAAAA 3425 (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3118

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) AN I-SENSE: no (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapien

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:

(vii) IMMEDIATE SOURCE:

(A) LIBRARY:

(B) CLONE:

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT:

(B) MAP POSITION:

(C) UNITS: (ix) FEATURE:

(A) NAME/KEY: hPLDl .5

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: (x) PUBLICATION INFORMATION:

(A) AUTHORS:

(B) TITLE:

(C) JOURNAL:

(D) VOLUME:

(E) ISSUE:

(F) PAGES:

(G) DATE:

(H) DOCUMENT NUMBER:

(I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES IN SEQ ID NO: 16: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

5 GCGGCCCCTTCGCCCTGCAGCCCCTTTGCTTTTACTCTGTCCAAAGTTAAC ATG TCA 61 1 Met Ser 2

62 CTG AAA AAC GAG CCA CGG GTA AAT ACC TCT GCA CTG CAG AAA ATT 106 2 Leu Lys Asn Glu Pro Arg Val Asn Thr Ser Ala Leu Gin Lys He 17

107 GCT GCT GAC ATG AGT AAT ATC ATA GAA AAT CTG GAC ACG CGG GAA 151 17 Ala Ala Asp Met Ser Asn He He Glu Asn Leu Asp Thr Arg Glu 32

152 CTC CAC TTT GAG GGA GAG GAG GTA GAC TAC GAC GTG TCT CCC AGC 196 32 Leu His Phe Glu Gly Glu Glu Val Asp Tyr Asp Val Ser Pro Ser 47

197 GAT CCC AAG ATA CAA GAA GTG TAT ATC CCT TTC TCT GCT ATT TAT 241 47 Asp Pro Lys He Gin Glu Val Tyr He Pro Phe Ser Ala He Tyr 62

242 AAC ACT CAA GGA TTT AAG GAG CCT AAT ATA CAG ACG TAT CTC TCC 286 62 Asn Thr Gin Gly Phe Lys Glu Pro Asn He Gin Thr Tyr Leu Ser 77 287 GGC TGT CCA ATA AAA GCA CAA GTT CTG GAA GTG GAA CGC TTC ACA 331 77 Gly Cys Pro He Lys Ala Gin Val Leu Glu Val Glu Arg Phe Thr 92

332 TCT ACA ACA AGG GTA CCA AGT ATT AAT CTT TAC ACT ATT GAA TTA 376 92 Ser Thr Thr Arg Val Pro Ser He Asn Leu Tyr Thr He Glu Leu 107

377 ACA CAT GGG GAA TTT AAA TGG CAA GTT AAG AGG AAA TTC AAG CAT 421

107 Thr His Gly Glu Phe Lys Trp Gin Val Lys Arg Lys Phe Lys His 122

422 TTT CAA GAA TTT CAC AGA GAG CTG CTC AAG TAC AAA GCC TTT ATC 466

122 Phe Gin Glu Phe His Arg Glu Leu Leu Lys Tyr Lys Ala Phe He 137

467 CGC ATC CCC ATT CCC ACT AGA AGA CAC ACG TTT AGG AGG CAA AAC 511

137 Arg He Pro He Pro Thr Arg Arg His Thr Phe Arg Arg Gin Asn 152

512 GTC AGA GAG GAG CCT CGA GAG ATG CCC AGT TTG CCC CGT TCA TCT 556

152 Val Arg Glu Glu Pro Arg Glu Met Pro Ser Leu Pro Arg Ser Ser 167

557 GAA AAC ATG ATA AGA GAA GAA CAA TTC CTT GGT AGA AGA AAA CAA 601

167 Glu Asn Met He Arg Glu Glu Gin Phe Leu Gly Arg Arg Lys Gin 182

602 CTG GAA GAT TAC TTG ACA AAG ATA CTA AAA ATG CCC ATG TAT AGA 646

182 Leu Glu Asp Tyr Leu Thr Lys He Leu Lys Met Pro Met Tyr Arg 197

647 AAC TAT CAT GCC ACA ACA GAG TTT CTT GAT ATA AGC CAG CTG TCT 691

197 Asn Tyr His Ala Thr Thr Glu Phe Leu Asp He Ser Gin Leu Ser 212

692 TTC ATC CAT GAT TTG GGA CCA AAG GGC ATA GAA GGT ATG ATA ATG 736

212 Phe He His Asp Leu Gly Pro Lys Gly He Glu Gly Met He Met 227

737 AAA AGA TCT GGA GGA CAC AGA ATA CCA GGC TTG AAT TGC TGT GGT 781

227 Lys Arg Ser Gly Gly His Arg He Pro Gly Leu Asn Cys Cys Gly 242

782 CAG GGA AGA GCC TGC TAC AGA TGG TCA AAA AGA TGG TTA ATA GTG 826

242 Gin Gly Arg Ala Cys Tyr Arg Trp Ser Lys Arg Trp Leu He Val 257

827 AAA GAT TCC TTT TTA TTG TAT ATG AAA CCA GAC AGC GGT GCC ATT 871

257 Lys Asp Ser Phe Leu Leu Tyr Met Lys Pro Asp Ser Gly Ala He 272

872 GCC TTC GTC CTG CTG GTA GAC AAA GAA TTC AAA ATT AAG GTG GGG 916

272 Ala Phe Val Leu Leu Val Asp Lys Glu Phe Lys He Lys Val Gly 287

917 AAG AAG GAG ACA GAA ACG AAA TAT GGA ATC CGA ATT GAT AAT CTT 961

287 Lys Lys Glu Thr Glu Thr Lys Tyr Gly He Arg He Asp Asn Leu 302

962 TCA AGG ACA CTT ATT TTA AAA TGC AAC AGC TAT AGA CAT GCT CGG 1006

302 Ser Arg Thr Leu He Leu Lys Cys Asn Ser Tyr Arg His Ala Arg 317

1007 TGG TGG GGA GGG GCT ATA GAA GAA TTC ATC CAG AAA CAT GGC ACC 1051

317 Trp Trp Gly Gly Ala He Glu Glu Phe He Gin Lys His Gly Thr 332

1052 AAC TTT CTC AAA GAT CAT CGA TTT GGG TCA TAT GCT GCT ATC CAA 1096

332 Asn Phe Leu Lys Asp His Arg Phe Gly Ser Tyr Ala Ala He Gin 347

1097 GAG AAT GCT TTA GCT AAA TGG TAT GTT AAT GCC AAA GGA TAT TTT 1141

347 Glu Asn Ala Leu Ala Lys Trp Tyr Val Asn Ala Lys Gly Tyr Phe 362

1142 GAA GAT GTG GCA AAT GCA ATG GAA GAG GCA AAT GAA GAG ATT TTT 1186

362 Glu Asp Val Ala Asn Ala Met Glu Glu Ala Asn Glu Glu He Phe 377

1187 ATC ACA GAC TGG TGG CTG AGT CCA GAA ATC TTC CTG AAA CGC CCA 1231

377 He Thr Asp Trp Trp Leu Ser Pro Glu He Phe Leu Lys Arg Pro 392

1232 GTG GTT GAG GGA AAT CGT TGG AGG TTG GAC TGC ATT CTT AAA CGA 1276

392 Val Val Glu Gly Asn Arg Trp Arg Leu Asp Cys He Leu Lys Arg 407 1277 AAA GCA CAA CAA GGA GTG AGG ATC TTC ATA ATG CTC TAC AAA GAG 1321 407 Lys Ala Gin Gin Gly Val Arg He Phe He Met Leu Tyr Lys Glu 422

1322 GTG GAA CTC GCT CTT GGC ATC AAT AGT GAA TAC ACC AAG AGG ACT 1366 422 Val Glu Leu Ala Leu Gly He Asn Ser Glu Tyr Thr Lys Arg Thr 437

1367 TTG ATG CGT CTA CAT CCC AAC ATA AAG GTG ATG AGA CAC CCG GAT 1411 437 Leu Met Arg Leu His Pro Asn He Lys Val Met Arg His Pro Asp 452

1412 CAT GTG TCA TCC ACC GTC TAT TTG TGG GCT CAC CAT GAG AAG CTT 1456 452 His Val Ser Ser Thr Val Tyr Leu Trp Ala His His Glu Lys Leu 467

1457 GTC ATC ATT GAC CAA TCG GTG GCC TTT GTG GGA GGG ATT GAC CTG 1501 467 Val He He Asp Gin Ser Val Ala Phe Val Gly Gly He Asp Leu 482

1502 GCC TAT GGA AGG TGG GAC GAC AAT GAG CAC AGA CTC ACA GAC GTG 1546 482 Ala Tyr Gly Arg Trp Asp Asp Asn Glu His Arg Leu Thr Asp Val 497

1547 GGC AGT GTG AAG CGG GTC ACT TCA GGA CCG TCT CTG GGT TCC CTC 1591 497 Gly Ser Val Lys Arg Val Thr Ser Gly Pro Ser Leu Gly Ser Leu 512

1592 CCA CCT GCC GCA ATG GAG TCT ATG GAA TCC TTA AGA CTC AAA GAT 1636 512 Pro Pro Ala Ala Met Glu Ser Met Glu Ser Leu Arg Leu Lys Asp 527

1637 AAA AAT GAG CCT GTT CAA AAC CTA CCC ATC CAG AAG AGT ATT GAT 1681 527 Lys Asn Glu Pro Val Gin Asn Leu Pro He Gin Lys Ser He Asp 542

1682 GAT GTG GAT TCA AAA CTG AAA GGA ATA GGA AAG CCA AGA AAG TTC 17 6 542 Asp Val Asp Ser Lys Leu Lys Gly He Gly Lys Pro Arg Lys Phe 557

1727 TCC AAA TTT AGT CTC TAC AAG CAG CTC CAC AGG CAC CAC CTG CAC 1771 557 Ser Lys Phe Ser Leu Tyr Lys Gin Leu His Arg His His Leu His 572

1772 GAC GCA GAT AGC ATC AGC AGC ATT GAC AGC ACC TCC AGT TAT TTT 1816 572 Asp Ala Asp Ser He Ser Ser He Asp Ser Thr Ser Ser Tyr Phe 587

1817 AAT CAC TAT AGA AGT CAT CAC AAT TTA ATC CAT GGT TTA AAA CCC 1861 587 Asn His Tyr Arg Ser His His Asn Leu He His Gly Leu Lys Pro 602

1862 CAC TTC AAA CTC TTT CAC CCG TCC AGT GAG TCT GAG CAA GGA CTC 1906 602 His Phe Lys Leu Phe His Pro Ser Ser Glu Ser Glu Gin Gly Leu 617

1907 ACT AGA CCT CAT GCT GAT ACC GGG TCC ATC CGT AGT TTA CAG ACA 1951

617 Thr Arg Pro His Ala Asp Thr Gly Ser He Arg Ser Leu Gin Thr 632

1952 GGT GTG GGA GAG CTG CAT GGG GAA ACC AGA TTC TGG CAT GGA AAG 1996

632 Gly Val Gly Glu Leu His Gly Glu Thr Arg Phe Trp His Gly Lys 647

1997 GAC TAC TGC AAT TTC GTC TTC AAA GAC TGG GTT CAA CTT GAT AAA 2041

647 Asp Tyr Cys Asn Phe Val Phe Lys Asp Trp Val Gin Leu Asp Lys 662

2042 CCT TTT GCT GAT TTC ATT GAC AGG TAC TCC ACG CCC CGG ATG CCC 2086

662 Pro Phe Ala Asp Phe He Asp Arg Tyr Ser Thr Pro Arg Met Pro 677

2087 TGG CAT GAC ATT GCC TCT GCA GTC CAC GGG AAG GCG GCT CGT GAT 2131

677 Trp His Asp He Ala Ser Ala Val His Gly Lys Ala Ala Arg Asp 692

2132 GTG GCA CGT CAC TTC ATC CAG CGC TGG AAC TTC ACA AAA ATT ATG 2176

692 Val Ala Arg His Phe He Gin Arg Trp Asn Phe Thr Lys He Met 707

2177 AAA TCA AAA TAT CGG TCC CTT TCT TAT CCT TTT CTG CTT CCA AAG 2221

707 Lys Ser Lys Tyr Arg Ser Leu Ser Tyr Pro Phe Leu Leu Pro Lys 722

2222 TCT CAA ACA ACA GCC CAT GAG TTG AGA TAT CAA GTG CCT GGG TCT 2266

722 Ser Gin Thr Thr Ala His Glu Leu Arg Tyr Gin Val Pro Gly Ser 737 2267 GTC CAT GCT AAC GTA CAG TTG CTC CGC TCT GCT GCT GAT TGG TCT 2311

737 Val His Ala Asn Val Gin Leu Leu Arg Ser Ala Ala Asp Trp Ser 752

2312 GCT GGT ATA AAG TAC CAT GAA GAG TCC ATC CAC GCC GCT TAC GTC 2356

752 Ala Gly He Lys Tyr His Glu Glu Ser He His Ala Ala Tyr Val 767

2357 CAT GTG ATA GAG AAC AGC AGG CAC TAT ATC TAT ATC GAA AAC CAG 2401

767 His Val He Glu Asn Ser Arg His Tyr He Tyr He Glu Asn Gin 782

2402 TTT TTC ATA AGC TGT GCT GAT GAC AAA GTT GTG TTC AAC AAG ATA 2446

782 Phe Phe He Ser Cys Ala Asp Asp Lys Val Val Phe Asn Lys He 797

2447 GGC GAT GCC ATT GCC CAG AGG ATC CTG AAA GCT CAC AGG GAA AAC 2491

797 Gly Asp Ala He Ala Gin Arg He Leu Lys Ala His Arg Glu Asn 812

2492 CAG AAA TAC CGG GTA TAT GTC GTG ATA CCA CTT CTG CCA GGG TTC 2536

812 Gin Lys Tyr Arg Val Tyr Val Val He Pro Leu Leu Pro Gly Phe 827

2537 GAA GGA GAC ATT TCA ACC GGC GGA GGA AAT GCT CTA CAG GCA ATC 2581

827 Glu Gly Asp He Ser Thr Gly Gly Gly Asn Ala Leu Gin Ala He 842

2582 ATG CAC TTC AAC TAC AGA ACC ATG TGC AGA GGA GAA AAT TCC ATC 2626

842 Met His Phe Asn Tyr Arg Thr Met Cys Arg Gly Glu Asn Ser He 857

2627 CTT GGA CAG TTA AAA GCA GAG CTT GGT AAT CAG TGG ATA AAT TAC 2671

857 Leu Gly Gin Leu Lys Ala Glu Leu Gly Asn Gin Trp He Asn Tyr 872

2672 ATA TCA TTC TGT GGT CTT AGA ACA CAT GCA GAG CTC GAA GGA AAC 2716

872 He Ser Phe Cys Gly Leu Arg Thr His Ala Glu Leu Glu Gly Asn 887

2717 CTA GTC ACT GAG CTT ATC TAT GTC CAC AGC AAG TTG TTA ATT GCT 2761

887 Leu Val Thr Glu Leu He Tyr Val His Ser Lys Leu Leu He Ala 902

2762 GAT GAT AAC ACT GTT ATT ATT GGC TCT GCC AAC ATA AAT GAC CGC 2806

902 Asp Asp Asn Thr Val He He Gly Ser Ala Asn He Asn Asp Arg 917

2807 AGC ATG CTG GGA AAG CGT GAC AGT GAA ATG GCT GTC ATT GTG CAA 2851

917 Ser Met Leu Gly Lys Arg Asp Ser Glu Met Ala Val He Val Gin 932

2852 GAT ACA GAG ACT GTT CCT TCA GTA ATG GAT GGA AAA GAG TAC CAA 2896

932 Asp Thr Glu Thr Val Pro Ser Val Met Asp Gly Lys Glu Tyr Gin 947

2897 GCT GGC CGG TTT GCC CGA GGA CTT CGG CTA CAG TGC TTT AGG TCT 2941

947 Ala Gly Arg Phe Ala Arg Gly Leu Arg Leu Gin Cys Phe Arg Ser 962

2942 AAA ATG ACT CCA GGT GTC GAA GAT CCC TGA TCTTTGGCAAGAAGATGCAA 2991

962 Lys Met Thr Pro Gly Val Glu Asp Pro *** 972

2992 ATTTTAAACTAATCTGTGGTGAAGCAGAGAGAATACTGGGCTAGGAAGCTGGGCTCGTTT

3051 3052 CAGCTGTGCGATCCTAAATAAGTCCATTCAATAAAGTGTTATTTAGAACTTTCAAAAAAA

3111

3112 AAAAAAA

3118

Claims

We claim:

1. A polynucleotide (i) that codes for a PCPLD isoform selected from group consisting of hPLD2.1, hPLD2.2, and hPLD1.5 or (ii) that hybridizes to a polynucleotide encoding said isoform.

2. A polynucleotide according to claim 1, comprising the nucleotide sequence of SEQ ID No. 1.

3. A polynucleotide according to claim 1, comprising the nucleotide sequence of SEQ ID No. 15.

4. A polynucleotide according to claim 1, comprising the nucleotide sequence of SEQ ID No. 16.

5. An isolated PCPLD isoform selected from group consisting of hPLD2.1 , 2.2, and hPLD1.5

6. An isolated PCPLD isoform according to claim 5, said isoform comprising the amino acid sequence of SEQ ID No. 1 or an enzymatically active fragment thereof.

7. An isolated PCPLD isoform according to claim 5, said isoform comprising the amino acid sequence of SEQ ID NO. 15 or an enzymatically active fragment thereof.

8. An isolated PCPLD isoform according to claim 5, said isoform comprising the amino acid sequence of SEQ ID NO. 16.

9. A method for screening a drug candidate, comprising:

(a) providing a PCPLD isoform according to claim 6 or claim 7;

(b) contacting said isoform with said drug candidate; and then

(c) determining whether said drug candidate affects PCPLD activity of said isoform.

10. A method according to claim 9, wherein step (c) comprises measuring the PCPLD activity of said isoform against a control sample.

11. A method according to claim 10, wherein said control sample contains a PCPLD isoform comprising the amino acid sequence of SEQ ID NO. 16.

12. The method according to claim 9, wherein said drug candidate is a pool of compounds from combinatorial library expression.