BACKGROUND OF THE INVENTION Fatty acid CoA ligase enzymes catalyze the activation of short-and medium-chain fatty acids and xenobiotic carboxylic acids. Vessey & Hu, R Bzochem. Toxicol. 1 D, 329-37,1995 ; Vessey et al., J. Biochem. Toxicol. 11, 73-78,1996 ; Vessey & Kelley, Biochim. Biophys. Acta 1346, 231-36,1997; Vessey et al., R Biochem. Mol. Toxicol.

12, 151-55,1998; Vessey & Kelley, Biochim. Biophys. Acta 1382, 243-48,1998; Vessey & Lau, J. Biochem. Mol. Toxicol. 12, 275-79,1998; Vessey et al., Biochim.

Biophys. Acta 1428, 455-62,1999; Vessey et al., J. Biochem. Mol. Toxicol. 14, 11- 19,2000. Various forms of the enzyme exist. Id. Because of the importance of fatty acid CoA ligases in metabolism, there is a need in the art to identify additional such enzymes which can be regulated to provide therapeutic effects.

SUMMARY OF THE INVENTION It is an object of the invention to provide reagents and methods of regulating a human fatty acid CoA ligase. This and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a fatty acid CoA ligase polypeptide comprising an amino acid sequence selected from the group consisting of : amino acid sequences which are at least about 23% identical to the amino acid sequence shown in SEQ ID NO: 2 ; and

the amino acid sequence shown in SEQ ID NO: 2.

Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a fatty acid CoA ligase polypeptide comprising an amino acid sequence selected from the group consisting of : amino acid sequences which are at least about 23% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.

Binding between the test compound and the fatty acid CoA ligase polypeptide is detected. A test compound which binds to the fatty acid CoA ligase polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.

The agent can work by decreasing the activity of the fatty acid CoA ligase.

Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a poly- nucleotide encoding a fatty acid CoA ligase polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of : nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 ; and the nucleotide sequence shown in SEQ ID NO: 1.

Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing

extracellular matrix degradation. The agent can work by decreasing the amount of the fatty acid CoA ligase through interacting with the fatty acid CoA ligase mRNA.

Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a fatty acid CoA ligase polypeptide comprising an amino acid sequence selected from the group consisting of : amino acid sequences which are at least about 23% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.

A fatty acid CoA ligase activity of the polypeptide is detected. A test compound which increases fatty acid CoA ligase activity of the polypeptide relative to fatty acid CoA ligase activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation. A test compound which decreases fatty acid CoA ligase activity of the polypeptide relative to fatty acid CoA ligase activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.

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

Binding of the test compound to the fatty acid CoA ligase product is detected. A test compound which binds to the fatty acid CoA ligase product is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a fatty acid CoA ligase polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of : nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.

Fatty acid CoA ligase activity in the cell is thereby decreased.

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

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the DNA-sequence encoding a fatty acid CoA ligase Polypeptide (SEQ ID NO: 1).

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

Fig. 3 shows the amino acid sequence of the bacillus subtillis protein identified by Accession No. P93062 (SEQ ID NO: 3).

Fig. 4 shows the BLASTP-alignment of SEQ ID NO: 2 against pdbllLCItlLCI luciferase.

Fig. 5 shows the HMMPFAM-alignment of SEQ ID NO: 2 against pfamlhinmlAMP-binding AMP-binding enzyme.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to an isolated polynucleotide encoding a fatty acid CoA ligase polypeptide and being selected from the group consisting of : a) a polynucleotide encoding a fatty acid CoA ligase polypeptide comprising an amino acid sequence selected from the group consisting of : amino acid sequences which are at least about 23% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2. b) a polynucleotide comprising the sequence of SEQ ID NO: 1 ; c) a polynucleotide which hybridizes under stringent conditions to a poly- nucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and

e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

Furthermore, it has been discovered by the present applicant that a novel fatty acid CoA ligase, particularly a human fatty acid CoA ligase, can be used in therapeutic methods to treat cancer, cardiovascular disorders or CNS disorders. Human fatty acid CoA ligase comprises the amino acid sequence shown in SEQ ID NO: 2. A coding sequence for human fatty acid CoA ligase is shown in SEQ ID NO : 1. Related sequences. are expressed in uterus, liver, tumors, and kidney.

Human fatty acid CoA ligase is 29% identical over 286 amino acids to pdbllLCIllLCI luciferase (Fig. 4). Human fatty acid CoA ligase also is similar to pfamlhmmlAMP-binding AMP-binding enzyme (Fig. 5). Human fatty acid CoA ligase has 99% identity to the SA gene, which is a gene identified in screening for genes with increased expression in the kidney of the spontaneously hypertensive rat (SHR) compared with the kidney of the normotensive Wistar-Kyoto rat at the early hypertensive stage.. In addition to this identity, this protein shares close similarity to the xenobiotic-metabolizing medium-chain fatty acid: CoA ligase that is involved in catalyzing the activation of short-and medium-chain fatty acids and xenobiotic carboxylic acids. Additionally, human fatty acid CoA ligase is 54% identical over 522 amino acids to the product of the KS gene, which is linked to blood pressure levels in a number of crosses involving the spontaneously hypertensive rat and other strains of genetically hypertensive rats.

Human fatty acid CoA ligase of the invention is expected to be useful for the same purposes as previously identified fatty acid CoA ligase enzymes. Human fatty acid CoA ligase is believed to be useful in therapeutic methods to treat disorders such as CNS disorders, cardiovascular disorders, and cancer. Human fatty acid CoA ligase also can be used to screen for human fatty acid CoA ligase activators and inhibitors.

Polypeptides Human fatty acid CoA ligase polypeptides according to the invention comprise at least 6,10,15,20,25,50,75,100,125,150,175,200,225,250,275,300,325, 350, 375,400,425,450,475,500,525,550, or 578 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 2 a biologically active variant thereof, as defined below. A fatty acid CoA ligase polypeptide of the invention therefore can be a portion of a fatty acid CoA ligase protein, a full-length fatty acid CoA ligase. protein, or a fusion protein comprising all or a portion of a fatty acid CoA ligase protein.

Biologically Active Variants Human fatty acid CoA ligase polypeptide variants which are biologically active, e. g., retain a fatty acid CoA ligase activity, also are fatty acid CoA ligase polypeptides.

Preferably, naturally or non-naturally occurring fatty acid CoA ligase polypeptide variants have amino acid sequences which are at least about 23,25,30,35,40,45, 50,55,60,65, or 70, preferably about 75,80,85,90,96,96, or 98% identical to the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof. Percent identity between a putative fatty acid CoA ligase polypeptide variant and an amino acid sequence of SEQ ID NO: 2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence.

They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a fatty acid CoA ligase polypeptide can be found using computer programs well known in the art, such as DNASTAR software.

Whether an amino acid change results in a biologically active fatty acid CoA ligase polypeptide can readily be determined by assaying for fatty acid CoA ligase activity, as described for example, in Vessey et al., J Biochem. Mol. Toxicol. 12, 151-55, 1998.

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

A fatty acid CoA ligase polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6,10,15,20,25,50,75,100,125,150,175,200,225,250,275, 300,325,350,375,400,425,450,475,500,525,550, or 578 contiguous amino acids of SEQ ID NO: 2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length fatty acid CoA ligase protein.

The second polypeptide segment can be a full-length protein or a protein fragment.

Proteins commonly used in fusion protein construction include ß-galactosidase, ß- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including

blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horse- radish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Addition- ally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the fatty acid CoA ligase polypeptide-encoding sequence and the heterologous protein sequence, so that the fatty acid CoA ligase polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from the complement of SEQ ID NO: 1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada ; 1-888-DNA-KITS).

Identification of Species Homologs Species homologs of human fatty acid CoA ligase polypeptide can be obtained using fatty acid CoA ligase polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such

as mice, monkeys, or yeast, identifying cDNAs which encode homologs of fatty acid CoA ligase polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides A fatty acid CoA ligase polynucleotide can be single-or double-stranded and comprises a coding sequence or the complement of a coding sequence for a fatty acid CoA ligase polypeptide. A coding sequence for human fatty acid CoA ligase is shown in SEQ ID NO: 1.

Degenerate nucleotide sequences encoding human fatty acid CoA ligase poly- peptides, as well as homologous nucleotide sequences which are at least about 50, 55,60,65,70, preferably about 75,90,96, or 98% identical to the nucleotide se- quence shown in SEQ ID NO: 1 or its complement also are fatty acid CoA ligase polynucleotides. Percent sequence identity between the sequences of two poly- nucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of-12 and a gap extension penalty of-2. Complementary DNA (cDNA) molecules, species homologs, and variants of fatty acid CoA ligase polynucleotides which encode biologically active fatty acid CoA ligase polypeptides also are fatty acid CoA ligase polynucleotides. Polynucleotides comprising at least 6,7,8,9,10,11,12,15,25,50, 75,100,200,300,400,500,600,700,800,900,1000,1100,1200,1300,1 400,1500, 1600, or 1656 contiguous nucleotides of SEQ ID NO: 1 or its complement also are fatty acid CoA ligase polynucleotides.

Identi, fcation ofPolvnucleotide V'ariants ad Homologs Variants and homologs of the fatty acid CoA ligase polynucleotides described above also are fatty acid CoA ligase polynucleotides. Typically, homologous fatty acid CoA ligase polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known fatty acid CoA ligase polynucleotides under stringent

conditions, as is known in the art. For example, using the following wash conditions--2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each ; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each--homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the fatty acid CoA ligase polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of fatty acid CoA ligase polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5°C with every 1% decrease in homology (Bonner et al., J : Mol. Biol. 81, 123 (1973). Variants of human fatty acid CoA ligase polynucleotides or fatty acid CoA ligase polynucleotides of other species can therefore be identified by hybridizing a putative homologous fatty acid CoA ligase polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising poly- nucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

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

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the

calculated Tm of the hybrid under study. The Tm of a hybrid between a fatty acid CoA ligase polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75,90,96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc.

Natl. Acad. Sci. U. SA. 48, 1390 (1962): Tm = 81.5°C-16.6 (logio [Na]) + 0.41 (% G + C)-0.63 (% formamide)-600/1), where/= the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide, 4X SSC at 42°C, or 0. 5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.

Preparation ofpol le A fatty acid CoA ligase polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification tech- niques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating poly- nucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated fatty acid CoA ligase polynucleotides.

For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises fatty acid CoA ligase nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70,80, or 90% free of other molecules.

Human fatty acid CoA ligase cDNA molecules can be made with standard molecular biology techniques, using fatty acid CoA ligase mRNA as a template. Human fatty acid CoA ligase cDNA molecules can thereafter be replicated using molecular

biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesizes fatty acid CoA ligase polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a fatty acid CoA ligase polypeptide having, for example, an amino acid sequence shown in SEQ ID NO: 1 or a biologically active variant thereof.

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

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

enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

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

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

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

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

display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

Obtaining Polypeptides Human fatty acid CoA ligase polypeptides can be obtained, for example, by purification from human cells, by expression of fatty acid CoA ligase poly- nucleotides, or by direct chemical synthesis.

Protein Purification Human fatty acid CoA ligase polypeptides can be purified from any cell which expresses the enzyme, including host cells which have been transfected with fatty acid CoA ligase expression constructs. A purified fatty acid CoA ligase polypeptide is separated from other compounds which normally associate with the fatty acid CoA ligase polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chroma- tography, affinity chromatography, and preparative gel electrophoresis. A prepa- ration of purified fatty acid CoA ligase polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electro- phoresis.

Expression o f Polvnucleotides To express a fatty acid CoA ligase polynucleotide, the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing

sequences encoding fatty acid CoA ligase polypeptides and appropriate transcrip- tional and translational control elements. These methods include in vitro recom- binant DNA techniques, synthetic techniques, and in vivo genetic recombination.

Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N. Y., 1989.

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

The control elements or regulatory sequences are those non-translated regions of the vector--enhancers, promoters, 5'and 3'untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTI plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e. g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e. g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a

nucleotide sequence encoding a fatty acid CoA ligase polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the fatty acid CoA ligase polypeptide. For example, when a large quantity of a fatty acid CoA ligase polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multi- functional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).

In a BLUESCRIPT vector, a sequence encoding the fatty acid CoA ligase poly- peptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of 8-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

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

Plant and Insect Expression Systems If plant expression vectors are used, the expression of sequences encoding fatty acid CoA ligase polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311,1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO R 3, 1671-1680, 1984; Broglie et al., Science 224, 838-843, 1984 ; Winter et al., Results Probl. Cell Differ. 17, 85-105,1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e. g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N. Y., pp. 191-196,1992).

An insect system also can be used to express a fatty acid CoA ligase polypeptide.

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

Mammalian Expression Systems A number of viral-based expression systems can be used to express fatty acid CoA ligase polypeptides in mammalian host cells. For example, if an adenovirus is used

as an expression vector, sequences encoding fatty acid CoA ligase polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a fatty acid CoA ligase polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659,1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

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

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding fatty acid CoA ligase polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a fatty acid CoA ligase polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert.

Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Diffier. 20,125-162,1994).

Host Cells A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed fatty acid CoA ligase polypeptide. in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a"prepro"form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.

Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e. g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

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

Freshney, ed., 1986.

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

These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32,1977) and adenine phosphoribosyltransferase (Lowy

et al., Cell 22, 817-23, 1980) genes which can be employed in tk or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77,3567-70,1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J : Mol. Biol. 150, 1-14,1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD which allows cells to utilize. histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Scad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, ß-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes ef al., Methods Mol. Biol. 55, 121-131,1995).

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

Alternatively, host cells which contain a fatty acid CoA ligase polynucleotide and which express a fatty acid CoA ligase polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or

immunoassay techniques which include membrane, solution, or chip-based technolo- gies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a fatty acid CoA ligase poly- peptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a fatty acid CoA ligase polypeptide. Nucleic acid amplification-based assays involve the use of oligo- nucleotides selected from sequences encoding a fatty acid CoA ligase polypeptide to detect transformants which contain a fatty acid CoA ligase polynucleotide.

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

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding fatty acid CoA ligase polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Alternatively, sequences encoding a fatty acid CoA ligase polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).

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

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

As discussed above, other constructions can be used to join a sequence encoding a fatty acid CoA ligase polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the fatty acid CoA ligase polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a fatty acid CoA ligase polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp. Purif 3,263-281,1992), while the enterokinase cleavage

site provides a means for purifying the fatty acid CoA ligase polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al., DNA Cell Biol. 12, 441-453,1993.

Chemical S Sequences encoding a fatty acid CoA ligase polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al., Nucl.

Acids Res. Symp. Ser., 215-223,1980 ; Hom et al. NucL 4cids Res. Synip. Ser.

225-232,1980). Alternatively, a fatty acid CoA ligase polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc.

85, 2149-2154,1963; Roberge et al., Science 269, 202-204,1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of fatty acid CoA ligase polypeptides can be separately synthesized and combined using chemical methods to produce a full- length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e. g., Creighton, PROTEINS : STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N. Y., 1983). The composition of a synthetic fatty acid CoA ligase polypeptide can be confirmed by amino acid analysis or sequencing (e. g., the Edman degradation procedure ; see Creighton, supra). Additionally, any portion of the amino acid sequence of the fatty acid CoA ligase polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

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

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

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

An antibody which specifically binds to an epitope of a fatty acid CoA ligase poly- peptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immuno- precipitations, or other immunochemical assays known in the art. Various immuno- assays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex forma- tion between an immunogen and an antibody which specifically binds to the im- munogen.

Typically, an antibody which specifically binds to a fatty acid CoA ligase polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to fatty acid CoA ligase polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a fatty acid CoA ligase polypeptide from solution.

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

Monoclonal antibodies which specifically bind to a fatty acid CoA ligase polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not

limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al., Nature 256, 495-497,1985; Kozbor et al., S : Immunol. Methods 81, 31-42,1985 ; Cote et al., Proc. Natl. Acad. Soi 80, 2026-2030,1983; Cole etal., Mol. Cell Biol. 62, 109-120,1984).

In addition, techniques developed for the production of"chimeric antibodies,"the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454,1985). Monoclonal and other antibodies also can be"humanized"to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to a fatty acid CoA ligase polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U. S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to fatty acid CoA ligase polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated. by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci.

SS, 11120-23,1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996, Eur. J

Cancer Prev. 5, 507-11). Single-chain antibodies can be mono-or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. BiotechnoL 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J : Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995, Int.

R Cancer 61, 497-501; Nicholls et al., 1993, J : Immunol. Meth. 165, 81-91).

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

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

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a fatty acid CoA ligase polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

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

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

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

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

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

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

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

215, 3539-3542,1987.

Ribozymes Ribozymes are RNA molecules with catalytic activity. See, e. g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568 ; 1990, Cech, Curr. Opin.

Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e. g., Haseloff et al. U. S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a fatty acid CoA ligase polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the fatty acid CoA ligase polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591,1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete"hybridization"region into the ribozyme.

The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201).

Specific ribozyme cleavage sites within a fatty acid CoA ligase RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate fatty acid CoA ligase RNA targets also can be evaluated by testing accessibility to

hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease fatty acid CoA ligase expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

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

Differentially Expressed Genes Described herein are methods for the identification of genes whose products interact with human fatty acid CoA ligase. Such genes may represent genes which are differentially expressed in disorders including, but not limited to, CNS disorders, cardiovascular disorders, and cancer. Further, such genes may represent genes which are differentially regulated in response to manipulations relevant to the progression or

treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modu- lated under control versus experimental conditions. In addition, the human fatty acid CoA ligase gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentiallv Expressed Genes To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects.

Any RNA isolation technique which does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al., ed."CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.

New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U. S. Patent 4,843,155.

Transcripts within the collected RNA samples which represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al., Proc.

Natl. Acad. Sci. U. SA. 85, 208-12,1988), subtractive hybridization (Hedrick et al., Nature 308, 149-53; Lee et al., Proc. Natl. Acad. Sci. US. A. 88, 2825,1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71,1992 ; U. S.

Patent 5,262,311).

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

Screening Methods The invention provides assays for screening test compounds which bind to or modulate the activity of a fatty acid CoA ligase polypeptide or a fatty acid CoA ligase polynucleotide. A test compound preferably binds to a fatty acid CoA ligase poly- peptide or polynucleotide. More preferably, a test compound decreases or increases fatty acid CoA ligase activity by at least about 10, preferably about 50, more preferably about 75,90, or 100% relative to the absence of the test compound.

Test Compounds Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The com- pounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the"one-bead one-compound"library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide,

non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145,1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al., Proc. Natl. Acad. Sci. U. A. 90, 6909,1993 ; Erb et al. Proc.

Natl. Acad. Sci. U. SA. 91, 11422,1994 ; Zuckerinann et al., J Med. Chem. 3 7,2678, 1994; Cho et al., Science 261, 1303,1993; Carell et al., Angew. Chem. Int. Ed. Engl.

33,2059,1994 ; Carell et al., Ange. Chem. Int. Ed. Engl. 33,2061; Gallop et al., J : Med. Chem. 37,1233,1994). Libraries of compounds can be presented in solution (see, e. g., Houghten, BioTechniques 13, 412-421,1992), or on beads (Lam, Nature 354, 82-84,1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U. S. Patent 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. US. A.

89, 1865-1869,1992), or phage (Scott & Smith, Science 249, 386-390,1990; Devlin, Science 249, 404-406,1990); Cwirla et al., Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J ; Mol. Biol. 222,301-310,1991; and Ladner, U. S. Patent 5,223,409).

High Throughput Screening Test compounds can be screened for the ability to bind to fatty acid CoA ligase polypeptides or polynucleotides or to affect fatty acid CoA ligase activity or fatty acid CoA ligase gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 u. l. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively,"free format assays,"or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by

Jayawickreme et al., Proc. Natl. Acad. Sci. U. SA. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

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

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

Another high throughput screening method is described in Beutel et al., U. S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.

When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

BindingAssavs For binding assays, the test compound is preferably a small molecule which binds to and occupies, for example, the active site of the fatty acid CoA ligase polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the fatty acid CoA ligase polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemilumi- nescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the fatty acid CoA ligase polypeptide can then be accomplished, for example, by direct counting of radio- emission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a fatty acid CoA ligase polypeptide can be determined without labeling either of the interactants. For example, a micro- physiometer can be used to detect binding of a test compound with a fatty acid CoA ligase polypeptide. A microphysiometer (e. g, Cytosensor) is an analytical instru- ment that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a fatty acid CoA ligase polypeptide (McConnell et al., Science 257, 1906-1912,1992).

Determining the ability of a test compound to bind to a fatty acid CoA ligase poly- peptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5, 699-705,1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e. g., BIAcoreTM). Changes in the optical phenomenon surface

plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, a fatty acid CoA ligase polypeptide can be used as a"bait protein"in a two-hybrid assay or three-hybrid assay (see, e. g., U. S.

Patent 5,283,317; Zervos et al., Cell 72,223-232,1993 ; Madura et al., J BioL Chem.

268, 12046-12054,1993 ; Bartel et al., BioTechniques 14, 920-924,1993; Iwabuchi et al., Oncogene 8, 1693-1696,1993; and Brent W094/10300), to identify other proteins which bind to or interact with the fatty acid CoA ligase polypeptide and modulate its activity.

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

Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the fatty acid CoA ligase polypeptide.

It may be desirable to immobilize either the fatty acid CoA ligase polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the fatty acid CoA ligase polypeptide (or polynucleotide) or the

test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, poly- styrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked.

Binding of a test compound to a fatty acid CoA ligase polypeptide (or poly- nucleotide) can be accomplished in any vessel suitable for containing the reactants.

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

In one embodiment, the fatty acid CoA ligase polypeptide is a fusion protein comprising a domain that allows the fatty acid CoA ligase polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed fatty acid CoA ligase polypeptide; the mixture is then incubated under conditions conducive to complex formation (e. g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components.

Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a fatty acid CoA ligase polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated fatty acid CoA ligase

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

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

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

Enzvme 4sues Test compounds can be tested for the ability to increase or decrease the fatty acid CoA ligase activity of a human fatty acid CoA ligase polypeptide. Fatty acid CoA ligase activity can be measured, for example, as described in Vessey et al., J Biochem. Mol. Toxicol. 12, 151-55,1998.

Enzyme assays can be carried out after contacting either a purified fatty acid CoA ligase polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which decreases a fatty acid CoA ligase activity of a fatty acid CoA ligase polypeptide by at least about 10, preferably about 50, more preferably about 75,90, or 100% is identified as a potential therapeutic agent for decreasing fatty acid CoA ligase activity. A test compound which increases a fatty acid CoA ligase activity of a human fatty acid CoA ligase polypeptide by at least about 10, preferably about 50, more preferably about 75,90, or 100% is identified as a potential therapeutic agent for increasing human fatty acid CoA ligase activity.

Gene Expression In another embodiment, test compounds which increase or decrease fatty acid CoA ligase gene expression are identified. A fatty acid CoA ligase polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the fatty acid CoA ligase polynucleotide is determined. The level-of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of fatty acid CoA ligase mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide.

Either qualitative or quantitative methods can be used. The presence of polypeptide products of a fatty acid CoA ligase polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods

such as radioimmunoassay, Western blotting, and immunohistochemistry. Alterna- tively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a fatty acid CoA ligase polypeptide.

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

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

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous,

intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

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

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

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be

dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, sus- pensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

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

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co.,

Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of admini- stration.

Therapeutic Indications and Methods Human fatty acid CoA ligase can be regulated to treat CNS disorders, cardiovascular disorders, and cancer.

Cancer Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.

Most standard cancer therapies target cellular proliferation and rely on the differential proliferative capacities between transformed and normal cells for their efficacy. This approach is hindered by the facts that several important normal cell types are also highly proliferative and that cancer cells frequently become resistant to these agents.

Thus, the therapeutic indices for traditional anti-cancer therapies rarely exceed 2.0.

The advent of genomics-driven molecular target identification has opened up the possibility of identifying new cancer-specific targets for therapeutic intervention that

will provide safer, more effective treatments for cancer patients. Thus, newly discovered tumor-associated genes and their products can be tested for their role (s) in disease and used as tools to discover and develop innovative therapies. Genes playing important roles in any of the physiological processes outlined above can be characterized as cancer targets.

Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins.

These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Agonists and/or antagonists of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.

CNS Disorders CNS disorders which may be treated include brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease.

Dementias, such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld- Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff s psychosis also can be treated. Similarly, it may be possible to treat cognitive-related disorders, such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit

disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities, by regulating the activity of human fatty acid CoA ligase polynucleotides.

Pain associated with CNS disorders also can be treated by regulating the activity of human Patched-like protein. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e. g., infarct, hemorrhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e. g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.

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

Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms

of pumping failure, such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.

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

Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen. This group of diseases includes stable angina, unstable angina, and asymptomatic ischemia.

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

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

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

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a

test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e. g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a fatty acid CoA ligase polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects fatty acid CoA ligase activity can be administered to a human cell, either in vitro or in vivo, to reduce fatty acid CoA ligase activity. The reagent preferably binds to an expression product of a human fatty acid CoA ligase gene. If the expression product is a protein, the reagent is preferably an antibody.

For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

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

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

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

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

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu & Wu, R Biol. Chem. 263, 621-24 (1988); Wu et al., R Biol. Chem. 269, 542-46 (1994) ; Zenke et al., Proc. Natl. Acad. Sci.

U. S. A. 87, 3655-59 (1990); Wu et al., J : Biol. Chem. 266, 338-42 (1991).

Determination of a Therapeuticallv Effective Dose The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases fatty acid CoA ligase activity relative to the fatty acid CoA ligase activity which occurs in the absence of the therapeu- tically effective dose.

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

Therapeutic efficacy and toxicity, e. g., EDso (the dose therapeutically effective in 50% of the population) and LDso (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LDso/EDso.

Pharmaceutical compositions which exhibit large therapeutic indices are preferred.

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

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

Factors which can be taken into account include the severity of the disease state,

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

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

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

Effective in vivo dosages of an antibody are in the range of about 5 gg to about 50 llg/kg, about 50 jig to about 5 mg/kg, about 100 jig to about 500 llg/kg of patient body weight, and about 200 to about 250 pLg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 llg to about 2 mg, about 5 pg to about 500 llg, and about 20 ug to about 100 Fg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligo- nucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

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

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

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

Diagnostic Methods Human fatty acid CoA ligase also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme.

For example, differences can be determined between the cDNA or genomic sequence encoding fatty acid CoA ligase in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

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

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e. g., Myers et al., Science 230,1242,1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e. g., Cotton et al., Proc. Natl. Acad. Sci. USA 85, 4397-4401,1985). Thus, the detection of a specific DNA sequence can be performed

by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.

In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of a fatty acid CoA ligase also can be detected in various tissues.

Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays ; competitive binding assays, Western blot analysis, and ELISA assays.

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

EXAMPLE 1 Detection offatty acid CoA ligase activity The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-fatty acid CoA ligase polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and fatty acid CoA ligase activity is determined in the following assay: The cell extracts are added to reaction mixtures containing 200 mM Tris HC1, pH 7.5,50 uM ATP, 8 mM MgC12, 2 mM EDTA, 20 mM NaF, 0.1% Triton X-100, varying concentrations of [3H] oleate, 0.5 mM coenzyme A and incubated 10 min at 37°C. Reactions are initiated by the addition of CoA and terminated by the addition of isopropyl, n-heptane, 1 M H2S04 (40: 10: 1). The aqueous phase, containing oleoyl-CoA formed during the reaction, is extracted three times with 2.5 ml of n-

heptane and subjected to scintillation counting. It is shown that the polypeptide of SEQ ID NO: 2 has a fatty acid CoA ligase activity.

EXAMPLE 2 Expression of recombinant human fatty acid CoA ligase The Pichia pastors expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human fatty acid CoA ligase poly- nucleotides polypeptides in yeast. The fatty acid CoA ligase-encoding DNA sequence is derived from SEQ ID NO: 1. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5'-end an initiation codon and at its 3'-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastors, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

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

EXAMPLE 3 Identification of test compounds that bind to fatty acid CoA ligasepolypeptides Purified fatty acid CoA ligase polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human fatty acid CoA ligase polypeptides comprise the amino acid sequence shown in SEQ ID NO: 2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a fatty acid CoA ligase polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a fatty acid CoA ligase polypeptide.

EXAMPLE 4 Identification of a test compound which decreases fatty acid CoA ligase geze expression A test compound is administered to a culture of human cells transfected with a fatty acid CoA ligase expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al., Biochem. 18, 5294-99,1979). Northern blots are prepared using 20 to 30 ig total RNA and

hybridized with a 32P-labeled fatty acid CoA ligase-specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1. A test compound which decreases the fatty acid CoA ligase-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of fatty acid CoA ligase gene expression.

EXAMPLE 5 Identification of a test compound which decreases fatty acid CoA ligase activity A test compound is administered to a culture of human cells transfected with a fatty acid CoA ligase expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control. Fatty acid CoA ligase activity is measured using the method of Vessey et al., J. Biochem. Mol.

Toxicol. 12, 151-55,1998.

A test compound which decreases the fatty acid CoA ligase activity of the fatty acid CoA ligase relative to the fatty acid CoA ligase activity in the absence of the test compound is identified as an inhibitor of fatty acid CoA ligase activity.

EXAMPLE 6 Tissue-specific expression offatty acid CoA ligase To demonstrate that fatty acid CoA ligase is involved in CNS disorders, the following tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus,

cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.

To demonstrate that fatty acid CoA ligase is involved in cancer, expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes.

Expression in the following cancer cell lines also is determined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205 (colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87.

Matched pairs of malignant and normal tissue from the same patient also are tested.

To demonstrate that fatty acid CoA ligase is involved in cardiovascular diseases, expression is determined in the following tissues: heart, coronary artery, aorta, small arteries, veins, muscle, liver, kidney, lung, brain, spinal cord, thymus, testis, bladder, prostate colon, small intestine, placenta, pancreas, adrenal gland, bone marrow, corpus cavernosum, fetal liver, CD34+ cells, Hep3B cells, macrophages, smooth muscle cells, and endothelial cells.

Quantitative expression profiling. Quantitative expression profiling is performed by the form of quantitative PCR analysis called"kinetic analysis"firstly described in Higuchi et al., BioTechnology 10, 413-17,1992, and Higuchi et al., BioTechnology 11, 1026-30,1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.

If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5'-3'endonuclease activity of Taq DNA polymerase and a

fluorescent dye released in the medium (Holland et al., Proc. Natl. Acad. Sci. U. S. A.

88, 7276-80,1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al., Genome Res. 6, 986-94,1996, and Gibson et al., Genome Res. 6, 995-1001, 1996).

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

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

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

Fifty llg of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/lll RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/lll RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; 10 mM MgCl2 ; 50 mM NaCl ; and 1 mM DTT.

After incubation, RNA is extracted once with 1 volume of phenol: chloroform :- isoamyl alcohol (24: 24: 1) and once with chloroform, and precipitated with 1/10 volume of 3 M NaAcetate, pH 5. 2, and 2 volumes of ethanol.

Fifty gg of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectro-

photometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200 ng/pL. Reverse transcription is carried out with 2.5, uM of random hexamer primers.

TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems and are listed below : forward primer: 5'- (gene specific sequence)-3' reverse primer: 5'- (gene specific sequence)-3' probe: 5'- (FAM)- (gene specific sequence) (TAMRA)-3' where FAM = 6-carboxy-fluorescein and TAMRA = 6-carboxy-tetramethyl-rhodamine.

The expected length of the PCR product is- (gene specific length) bp.

Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: 1X final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); 1X PDAR control-18S RNA (from 20X stock) ; 300 nM forward primer; 900 nM reverse primer ; 200 nM probe ; 10 ng cDNA ; and water to 25 ml.

Each of the following steps are carried out once: pre PCR, 2 minutes at 50°C, and 10 minutes at 95°C. The following steps are carried out 40 times: denaturation, 15

seconds at 95°C, annealing/extension, 1 minute at 60°C.

The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.