Aortic carboxypeptidase-like polypeptide Number:7,094,878 from the United States Patent and Trademark Office (PTO) owispatent The invention features a aortic carboxypeptidase-like polypeptide (ACLP), DNA encoding ACLP, and methods of detecting genetic alterations associated with abdominal wall defects.<H carboxypeptidase, polypeptide, Aortic, carboxyp, 7094878.html, Patent, pto, 7094878

Title: Aortic carboxypeptidase-like polypeptide

Abstract: The invention features a aortic carboxypeptidase-like polypeptide (ACLP), DNA encoding ACLP, and methods of detecting genetic alterations associated with abdominal wall defects.

Patent Number: 7,094,878 Issued on 08/22/2006 to Lee, et al.

Inventors: Lee; Mu-En (Newton, MA), Layne; Matthew D. (Brighton, MA), Yet; Shaw-Fang (Andover, MA)
Assignee: President and Fellows of Harvard College (Cambridge, MA)

Appl. No.: 10/238,876

Filed: September 10, 2002

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09060482	Apr., 1998	6468766
08818009	Mar., 1997
60013439	Mar., 1996

Current U.S. Class: 530/350

Current International Class: C07K 14/47 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


5460951	October 1995	Kawai et al.

Foreign Patent Documents


WO/93/16178	Aug., 1993	WO

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Primary Examiner: Romeo; David
Attorney, Agent or Firm: Mintz,Levin,Cohn,Ferris,Clovsky and Popeo, P.C. Beattie; Ingrid A.

Government Interests

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded in part by the U.S. Government under grant numbers GM53249 awarded by the National Institutes of Health. The Government has certain rights in the invention.

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application U.S. Ser. No. 09/060,482, filed on Apr. 16, 1998, which issued as U. S. Pat. No. 6,468,766, which is a continuation-in-part of U.S. Ser. No. 08/818,009 ABN, filed on Mar. 14, 1997, which in turn claims priority to provisional application U.S. Ser. No. 60/013,439, filed on Mar. 15, 1996, all of which are hereby incorporated by reference.

Claims

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence which is at least 87% identical to SEQ ID NO:2 over the entire length of SEQ ID NO: 2 and which amino acid sequence comprises (a) a discoidin domain comprising amino acids 385 540 of SEQ ID NO:2; (b) a lysine-rich/proline-rich domain comprising amino acids 117 164 of SEQ ID NO: 2; and (c) a carboxypeptidase domain comprising amino acids 562 969 of SEQ ID NO:2, wherein an amino acid substitution in said polypeptide is conservative with respect to SEQ ID NO:2 and wherein the absence of said polypeptide is associated with gastroschisis.

2. The isolated polypeptide of claim 1, wherein said amino acid sequence is at least 90% identical to the sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.

3. The isolated polypeptide of claim 1, wherein said amino acid sequence is at least 95% identical to the sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.

4. The isolated polypeptide of claim 1, wherein said amino acid sequence is at least 99% identical to the sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.

5. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2.

Description

BACKGROUND OF THE INVENTION

The invention relates to gastrointestinal abnormalities.

Gastroschisis is a life-threatening abdominal wall defect that occurs in approximately 1 7 of every 10,000 human births. The defect is thought to originate on the right side of the umbilical cord and may involve the formation of the omphalomesenteric artery. Infants with gastroschisis can be born with abdominal organs outside the body cavity, i.e., protruding through the defect. Factors associated with an increased risk for gastroschisis include a maternal age below 20 years, ingestion of aspirin, and ingestion of pseudoephedrine. The cause of gastroschisis has not been identified.

SUMMARY OF THE INVENTION

A novel human gene encoding aortic carboxypeptidase-like polypeptide (ACLP) has been discovered. A mutation in an ACLP gene has now been shown to be associated with the development of gastroschisis. Thus, a mutation in an ACLP gene is indicative of gastroschisis or a predisposition to develop the condition. Accordingly, the invention provides an isolated nucleic acid (e.g., genomic DNA, cDNA, or synthetic DNA) encoding an ACLP. By the term "human ACLP" is meant a polypeptide having the amino acid sequence of a naturally-occurring human ACLP. For example, the invention encompasses an ACLP with the amino acid sequence of SEQ ID NO:2 as well as naturally-occurring variants thereof such as mutant forms associated with gastroschisis or isoforms resulting from alternative splicing of exons of the ACLP gene.

The invention includes a nucleic acid molecule which contains the nucleotide sequence of human ACLP cDNA (SEQ ID NO:1). A nucleic acid molecule which contains nucleotides 140 3613 (ACLP coding sequence), inclusive, of SEQ ID NO:1 or a degenerate variant thereof, is also within the invention. Nucleotides 214 3613 encode an ACLP which lacks the first 25 residues (a putative signal peptide). Preferably, the nucleic acid molecule contains a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 87% identical to the sequence of SEQ ID NO:2. More preferably, the sequence is at least 90% identical to SEQ ID NO:2, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably, the nucleotide sequence encodes a polypeptide the amino acid sequence of which is SEQ ID NO:2.

An isolated nucleic acid molecule containing a strand which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:1, or the complement thereof is also within the invention. The nucleic acid molecule may be a primer useful to amplify ACLP DNA in a polymerase chain reaction (PCR). For example, the nucleic acid is at least 5 nucleotides but less than 50 nucleotides in length. Alternatively, the nucleic acid molecule may encompass the entire coding sequence of ACLP cDNA, i.e., nucleotides 140 3613, inclusive, of SEQ ID NO:1. Preferably, the nucleic acid molecule spans a gastroschisis-associated mutation in an ACLP gene. Such a molecule is useful as a hybridization probe to identify a genetic alteration, e.g., a deletion, duplication, point mutation, or translocation, that indicates that an individual has gastroschisis, is predisposed to developing gastroschisis, or is a heterozygous carrier of a genetic alteration associated with gastroschisis.

By "isolated nucleic acid molecule" is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism, flank an ACLP gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a procaryote or eucaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term excludes large segments of genomic DNA, e.g., such as those present in cosmid clones, which contain an ACLP gene flanked by one or more other genes which naturally flank it in a naturally-occurring genome.

Nucleic acid molecules include both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a procaryote or eucaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

Hybridization is carried out using standard techniques such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, (1989). "High stringency" refers to DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., hybridization and wash conditions of 65.degree. C. at a salt concentration of 0.1.times. SSC. "Low" to "moderate" stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60.degree. C. at a salt concentration of at least 1.0.times. SSC. For example, high stringency conditions may include hybridization at 42.degree. C. in a solution containing 50% formamide; a first wash at 65.degree. C. using a solution of 2.times. SSC and 1% SDS; followed by a second wash at 65.degree. C. using a solution of 0.1%.times. SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an ACLP gene are detected by, for example, hybridization at 42.degree. C. in the absence of formamide; a first wash at 2.degree. C. in a solution of 6.times. SSC and 1% SDS; and a second wash at 50.degree. C. in a solution of 6.times. SSC and 1% SDS.

TABLE-US-00001 TABLE 1 Human ACLP cDNA (SEQ ID NO:1) 1 tccctcgctc accccatcct ctctcccgcc ccttcctgga ttccctcacc cgtctcgatc 61 ccctctccgc cctttcccag agacccagag cccctgaccc cccgcgccct ccccggagcc 121 ccccgcgcgt gccgcggcca tggcggccgt gcgcggggcg cccctgctca gctgcctcct 181 ggcgttgctg gccctgtgcc ctggagggcg cccgcagacg gtgctgaccg acgacgagat 241 cgaggagttc ctcgagggct tcctgtcaga gctagaacct gagccccggg aggacgacgt 301 ggaggccccg ccgcctcccg agcccacccc gcgggtccga aaagcccagg cggggggcaa 361 gccagggaag cggccaggga cggccgcaga agtgcctccg gaaaagacca aagacaaagg 421 gaagaaaggc aagaaagaca aaggccccaa ggtgcccaag gagtccttgg aggggtcccc 481 caggccgccc aagaagggga aggagaagcc acccaaggcc accaagaagc ccaaggagaa 541 gccacctaag gccaccaaga agcccaagga ggagccaccc aaggccacca agaagcccaa 601 agagaagcca cccaaggcca ccaagaagcc cccgtcaggg aagaggcccc ccattctggc 661 tccctcagaa accctggagt ggccactgcc cccacccccc agccctggcc ccgaggagct 721 accccaggag ggaggggcgc ccctctcaaa taactggcag aatccaggag aggagaccca 781 tgtggaggca caggagcacc agcctgagcc ggaggaggag accgagcaac ccacactgga 841 ctacaatgac cagatcgaga gggaggacta tgaggacttt gagtacattc ggcgccagaa 901 gcaacccagg ccacccccaa gcagaaggag gaggcccgag cgggtctggc cagagccccc 961 tgaggagaag gccccggccc cagccccgga ggagaggatt gagcctcctg tgaagcctct 1021 gctgcccccg ctgccccctg actatggtga tggttacgtg atccccaact acgatgacat 1081 ggactattac tttgggcctc ctccgcccca gaagcccgat gctgagcgcc agacggacga 1141 agagaaggag gagctgaaga aacccaaaaa ggaggacagc agccccaagg aggagaccga 1201 caagtgggca gtggagaagg gcaaggacca caaagagccc cgaaagggcg aggagttgga 1261 ggaggagtgg acgcctacgg agaaagtcaa gtgtcccccc attgggatgg agtcacaccg 1321 tattgaggac aaccagatcc gagcctcctc catgctgcgc cacggcctgg gggcacagcg 1381 cggccggctc aacatgcaga ccggtgccac tgaggacgac tactatgatg gtgcgtggtg 1441 tgccgaggac gatgccagga cccagtggat agaggtggac accaggagga ctacccggtt 1501 cacaggcgtc atcacccagg gcagagactc cagcatccat gacgattttg tgaccacctt 1561 cttcgtgggc ttcagcaatg acagccagac atgggtgatg tacaccaacg gctatgagga 1621 aatgaccttt catgggaacg tggacaagga cacacccgtg ctgagtgagc tcccagagcc 1681 ggtggtggct cgtttcatcc gcatctaccc actcacctgg aatggcagcc tgtgcatgcg 1741 cctggaggtg ctggggtgct ctgtggcccc tgtctacagc tactacgcac agaatgaggt 1801 ggtggccacc gatgacctgg atttccggca ccacagctac aaggacatgc gccagctcat 1861 gaaggtggtg aacgaggagt gccccaccat cacccgcact tacagcctgg gcaagagctc 1921 acgaggcctc aagatctatg ccatggagat ctcagacaac cctggggagc atgaactggg 1981 ggagcccgag ttccgctaca ctgctgggat ccatggcaac gaggtgctgg gccgagagct 2041 gttgctgctg ctcatgcagt acctgtgccg agagtaccgc gatgggaacc cacgtgtgcg 2101 cagcctggtg caggacacac gcatccacct ggtgccctca ctgaaccctg atggctacga 2161 ggtggcagcg cagatgggct cagagtttgg gaactgggcg ctgggactgt ggactgagga 2221 gggctttgac atctttgaag atttcccgga tctcaactct gtgctctggg gagctgagga 2281 gaggaaatgg gtcccctacc gggtccccaa caataacttg cccatccctg aacgctacct 2341 ttcgccagat gccacggtat ccacggaggt ccgggccatc attgcctgga tggagaagaa 2401 ccccttcgtg ctgggagcaa atctgaacgg cggcgagcgg ctagtatcct acccctacga 2461 tatggcccgc acgcctaccc aggagcagct gctggccgca gccatggcag cagcccgggg 2521 ggaggatgag gacgaggtct ccgaggccca ggagactcca gaccacgcca tcttccggtg 2581 gcttgccatc tccttcgcct ccgcacacct caccttgacc gagccctacc gcggaggctg 2641 ccaagcccag gactacaccg gcggcatggg catcgtcaac ggggccaagt ggaacccccg 2701 gaccgggact atcaatgact tcagttacct gcataccaac tgcctggagc tctccttcta 2761 cctgggctgt gacaagttcc ctcatgagag tgagctgccc cgcgagtggg agaacaacaa 2821 ggaggcgctg ctcaccttca tggagcaggt gcaccgcggc attaaggggg tggtgacgga 2881 cgagcaaggc atccccattg ccaacgccac catctctgtg agtggcatta atcacggcgt 2941 gaagacagcc agtggtggtg attactggcg aatcttgaac ccgggtgagt accgcgtgac 3001 agcccacgcg gagggctaca ccccgagcgc caagacctgc aatgttgact atgacatcgg 3061 ggccactcag tgcaacttca tcctggctcg ctccaactgg aagcgcatcc gggagatcat 3121 ggccatgaac gggaaccggc ctatcccaca catagaccca tcgcgcccta tgacccccca 3181 acagcgacgc ctgcagcagc gacgcctaca acaccgcctg cggcttcggg cacagatgcg 3241 gctgcggcgc ctcaacgcca ccaccaccct aggcccccac actgtgcctc ccacgctgcc 3301 ccctgcccct gccaccaccc tgagcactac catagagccc tggggcctca taccgccaac 3361 caccgctggc tgggaggagt cggagactga gacctacaca gaggtggtga cagagtttgg 3421 gaccgaggtg gagcccgagt ttgggaccaa ggtggagccc gagtttgaga cccagttgga 3481 gcctgagttc gagacccagc tggaacccga gtttgaggaa gaggaggagg aggagaaaga 3541 ggaggagata gccactggcc aggcattccc cttcacaaca gtagagacct acacagtgaa 3601 ctttggggac ttctgagatc agcgtcctac caagacccca gcccaactca agctacagca 3661 gcagcacttc ccaagcctgc tgaccacagt cacatcaccc atcagcacat ggaaggcccc 3721 tggtatggac actgaaagga agggctggtc ctgccccttt gagggggtgc aaacatgact 3782 gggacctaag agccagaggc tgtgtagagg ctcctgctcc acctgccagt ctcgtaagag 3841 atggggttgc tgcagtgttg gagtaggggc agagggaggg agccaaggtc actccaataa 3901 aacaagctca tggcaaaaaa aaaaaaaaaa aaaaa

The invention also includes a substantially pure human ACLP polypeptide. A substantially pure ACLP polypeptide may be obtained, for example, by extraction from a natural source (e.g., a vascular smooth muscle cell); by expression of a recombinant nucleic acid encoding an ACLP; or by chemically synthesizing the protein. A polypeptide or protein is substantially pure when it is separated from those contaminants which accompany it in its natural state (proteins and other naturally-occurring organic molecules). Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, ACLP. A substantially pure ACLP may be obtained, for example, by extraction from a natural source (e.g., a vascular smooth muscle cell); by expression of a recombinant nucleic acid encoding an ACLP; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eucaryote but produced in E. coli or another procaryote, or in a eucaryote other than that from which the polypeptide was originally derived.

For expression of recombinant ACLP, an ACLP-encoding nucleic acidc is operably linked to a regulatory sequence, e.g., a promoter. By "promoter" is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible, and may be coupled to other regulatory sequences or "elements" which render promoter-dependent gene expression cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5' or 3' region of the native gene, or within an intron. DNA encoding an ACLP may be operably linked to such regulatory sequences for expression of the polypeptide in procaryotic or eucaryotic cells. By "operably linked" is meant that a coding sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).

To produce recombinant ACLP, a cell containing an ACLP-encoding sequence operably linked to appropriate regulatory sequences is cultured under conditions permitting expression of a nucleic acid molecule. The cell may be a procaryotic cell or a eucaryotic cell. To obtain post-translationally modified, e.g., glycosylated recombinant ACLP, the recombinant polypeptide is produced in a eucaryotic cell, e.g., a yeast or mammalian cell.

An ACLP preferably contains an amino acid sequence that is at least 87% identical to the amino acid sequence of SEQ ID NO:2. More preferably, the amino acid sequence is at least 90% (more preferably at least 95%, more preferably at least 98%, more preferably at least 99%) identical to SEQ ID NO:2. Most preferably, the polypeptide contains the amino acid sequence of SEQ ID NO:2.

The invention also includes polypeptides which contain a portion of naturally-occurring ACLP, e.g., an ACLP fragment containing a lysine-rich/proline rich domain (amino acids 117 164 of SEQ ID NO:2), an ACLP fragment containing a discoidin-like domain (amino acids 385 540 of SEQ ID NO:2), or an ACLP fragment containing a carboxypeptidase-like domain (amino acids 562 969 of SEQ ID NO:2).

Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.

For polypeptides, the length of the reference polypeptide sequence will generally be at least 10 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 25 nucleotides, preferably at least 50 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides or 300 nucleotides.

In the case of polypeptide sequences which are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

Sequence identity can be measured using sequence analysis software (for example, the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters as specified therein.

TABLE-US-00002 TABLE 2 Human ACLP amino acid sequence (SEQ ID NO:2) MAAVRGAPLLSCLLALLALCPGGRPQTVLTDDEIEEFLEGFLSELEPEPR EDDVEAPPPPEPTPRVRKAQAGGKPGKRPGTAAEVPPEKTKDKGKKGKKD KGPKVPKESLEGSPRPPKKGKEKPPKATKKPKEKPPKATKKPKEEPPKAT KKPKEKPPKATKKPPSGKRPPILAPSETLEWPLPPPPSPGPEELPQEGGA PLSNNWQNPGEETHVEAQEHQPEPEEETEQPTLDYNDQIEREDYEDFEYI RRQKQPRPPPSRRRRPERVWPEPPEEKAPAPAPEERIEPPVKPLLPPLPP DYGDGYVIPNYDDMDYYFGPPPPQKPDAERQTDEEKEELKKPKKEDSSPK EETDKWAVEKGKDHKEPRKGEELEEEWTPTEKVKCPPIGMESHRIEDNQI RASSMLRHGLGAQRGRLNMQTGATEDDYYDGAWCAEDDARTQWIEVDTRR TTRFTGVITQGRDSSIHDDFVTTFFVGFSNDSQTWVMYTNGYEEMTFHGN VDKDTPVLSELPEPVVARFIRIYPLTWNGSLCMRLEVLGCSVAPVYSYYA QNEVVATDDLDFRHHSYKDMRQLMKVVNEECPTITRTYSLGKSSRGLKIY AMEISDNPGEHELGEPEFRYTAGIHGNEVLGRELLLLLMQYLCREYRDGN PRVRSLVQDTRIHLVPSLNPDGYEVAAQMGSEFGNWALGLWTEEGFDIFE DFPDLNSVLWGAEERKWVPYRVPNNNLPIPERYLSPDATVSTEVRAIIAW MEKNPFVLGANLNGGERLVSYPYDMARTPTQEQLLAAAMAAARGEDEDEV SEAQETPDHAIFRWLAISFASAHLTLTEPYRGGCQAQDYTGGMGIVNGAK WNPRTGTINDFSYLHTNCLELSFYLGCDKFPHESELPREWENNKEALLTF MEQVHRGIKGVVTDEQGIPIANATISVSGINHGVKTASGGDYWRILNPGE YRVTAHAEGYTPSAKTCNVDYDIGATQCNFILARSNWKRIREIMAMNGNR PIPHIDPSRPMTPQQRRLQQRRLQHRLRLRAQMRLRRLNATTTLGPHTVP PTLPPAPATTLSTTIEPWGLIPPTTAGWEESETETYTEVVTEFGTEVEPE FGTKVEPEFETQLEPEFETQLEPEFEEEEEEEKEEEIATGQAFPFTTVET YTVNFGDF

A substantially pure DNA containing an ACLP promoter/enhancer sequence (SEQ ID NO:3) is useful for directing transcription of DNA encoding all or part of ACLP or of DNA encoding a heterologous polypeptide (e.g., a polypeptide other than ACLP or an ACLP the sequence of which corresponds to a naturally-occurring ACLP of a species other than the species from which the promoter/enhancer sequence is derived). For example, a murine ACLP promoter/enhancer sequence may be operably linked to DNA encoding human ACLP for therapeutic expression of ACLP in human patients. To regulate transcription of the polypeptide-encoding sequence (e.g., developmental stage-specific transcription), the promoter/enhancer sequence is operably linked to a polypeptide-encoding sequence. The ACLP promoter/enhancer sequence directs transcription of a polypeptide-encoding sequence.

By "promoter/enhancer sequence" is meant a DNA sequence located 5' to the transcriptional start site of the ACLP gene and which contains one or more cis-acting elements which regulate transcription, e.g., cell specific transcription. The elements may be contiguous or separated by DNA not involved in the regulation of transcription, e.g., an enhancer element may be in a position immediately adjacent to the promoter element or up to several kilobases upstream or downstream of the transcriptional start site. The promoter/enhancer DNA is preferably derived from the 5' region of a mammalian ACLP gene, such as that of the mouse (SEQ ID NO:3), and regulates expression of a polypeptide-encoding DNA to which it is operably linked. The promoter/enhancer sequence regulates developmental stage-specific expression, e.g., expression in embryonic cells, of a polypeptide-encoding sequence.

TABLE-US-00003 TABLE 3 Mouse ACLP promoter/enhancer (SEQ ID NO:3) AAGCTTAGTCTCCCTCTCTCCTGGCTCCTCTCCTGGGGCTTCCCTATGGA GGTAGCACTTACAGAAGATGCTTGTTCCAAACCTTCAGGGGTACAAACTA CACAGATATACTGAAGGACAGGAGGCTGGGGCCTCCCCCCACCCCCAACA GCCACTGTTCTCTCAGGAGCTCTGCTTCTGCTCTGCAGCATTGAAAACAA AACTGAAGGACACCTTCCTTCTCTCAGGCCAGCCCAGTGCTGTTGTGTGA TCCCTCGGGAAGACTCTAACGCATTCACAGGGACAACAGGAGTTGGGAGG GAGAGGAGTTACAGAACTTTCCAGCAGGACCTCAGGAGAACGCCTGGACA CGGACAGGAACCCCCAACCCCTCAGGGACCCCCTTGGACCCTTTGAGTGC TCCTGATCATGGAAGCCACCAGCCTCCCGATTCCTCAGCTGTGGCCTTGG CAGTGCCCTCTGGACATTTGACTTAAACGCTATGCTCTTCAGCAGAGTGG AGAGCTCTCCTCACAGGCTCTGGCTTCTGGTTGTCCTCTTGCCCCAGCGC TGTGGGCCCAGGTTAGAAAGACTTCCTGAGGACAGGCTCCCTCAGGAGGA TCCCCAGCGTACGACTGTGCTCCCACGCACCTTTCCGGATTTTCTGTGTG GAGGCCTCAACCCCTCAGGCCTCCTGGGCCAGCTCCTCTGCTCGAATTCC TGTCCGTGACTCATTGAGGCTCAGGAAAAGGCTTTCTAGACCTTAGGTTT CTTTGTTTTCCATTTTTGAAATGGCTTCTGTTTTCCCTGGCAGAGAATAT CCAACCCAAATTCAGTCCAAGTATGACCCATGCCTAGGGAAGTGACATCC ATGTCCCCTCATGCACCCTGTGGCATACCCAGCATGACACACTGGACCAG ACTGGGGGCACGGAAGCCAATTCCCAGAACTGACTTTGAGCACAATGATT CAGAGGGTGACCATGAGTGAGACTTGCTTTACTCTTGCTCTGCGACCAGG TTGAAGTCTCTCATGGGGAGGCCTAGCTGTGAGAGGATTGTCCTGGGATG GGGGAAGGGGGAGCAAAGTGGATGAGGACCAACAGCCTGTGGGATGCAAG GGCTGATCGTGTGTGCTAGGCACAGCACAAAGTGGTCCATTTAGCCGGGC AGTGGTGGTGCACACCTTTAATCCCAGCACTTGGGAGGCATCAGCAGGTG GGTTTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGCAAGTTCCAGGACAG CCAGAGCTACACAGAGAAACTCTGTCTCAAAAAAATCGAATAAACCAGAA AGGTGGTCCATTTAATATGCGTATAGTAAGTTGTGGACACGGGAGTTCCC CTGCTGAGTCAGACAGCTAGGAGGGCTAAGATGGGTTAGACCCTCCCCCC CCCCACACACACACACACACACTCACACACACATCAGTTCTTGGCATAGT CTCCATGCTTCCTCAAGGAGAGCCAGAAAGGAGACTGCCGGGAGGAGCTT GCCTACTCCCTGAGAGCAGTGGGTTACAGAGCCCAGTGCCCGAAAATTTC CCCTTTTTCTCCCTGCTCATGCTGGACAGAGAGGGTGAGGGTGAGGGTGA AAGACTGAGGAGGTGGCATCGTGTTGGTGTTTCTTGACCTGCTTTTTCTT TTTTCTCTTCCAGCTGAGATGTAAACTTTCCCATGTCAATCATCTGGGGG TCGCTATTCTTTTTTATCAGAGTGCCTCCCCACCTTGGTTGAAAGCTGCC TGCCACTACCCTGGACCTATGGCTGCTACAAGCCCACGTTCACATCTTTA ATCCTTCATGGGTAAATGCTCTGGCATTCCTGGGCTTAGCTATGATGGCC ATTATGAGCCAGCCAACGTTTGTATTCTAGAAGCCATAGCTGAAGCTGTT GTAAACAATTTGTTGTTTTAACCGCTTCTGGTCAGAGGAAGGAGAGAATA GCTATTACTCCACATTGGGACCTGAGCCCTGAGCTCTGAAGTGGGGCTCC TATCTCCATAAGGACAGCAGCTTGCTGAGAACAGCTTTTCACAGCCTTCC TCGCAAAAATTGGCTCCAAAGACCTGGGATGTTGGTGATAACTGGACAAA GGTGACACCTGTGCAAGCACACAGCAGGTGACACTTTGAAGAGCTAACCT CCAGAAAGTGGAAAGGAGGTGATCGCCAGTACCCTCGAGGGCCCTACTCC CTCCCTCCCCTAGCAATCTCCCTGGGCTCAGAGCAAAGGGCACAGCGGGT TAGAGCACAGGTCTCCTTAGACTCCCCACACTCCCTTCCCCATAACTGTT GCATTCTTTTCTCCCAGGCCTTCCTCCCCGCTAGGCGCCCTGCACCCACA CCCTCTAAACTGGCGCGTGACGCTGCTATTAGTCTGGGCTCCGTGCTGTC CGCCTCCCTCCCCCGCAGCCCCCGGTCCAAGGCCGGCTCCTCCTCCTCCC CCTCCGGAAACCCGAAGCCCCCGCCCCGGCCAGGCCGTCGCAAGCGCTCT GGAGGGCGGTCCGCGTGAGAGCCAGCCACGCGGGGCAGGAGCGCCCAGTT GCTGCCGGAGCTGGGCCCGCCAGAACCTCTCCTGGAGCCCCTTGCTCTCC TTGAATCTCCCTTTCCCACCGCTTTCTGGATACCCTTGACGCCCACGTTC CTCGCGCCCTTTCCCGCCCCTACGCGGGGCGCTGCCCCTGCCACCCAAGT CCCTGCTCAAGCCCGCCCGGTCCCGCGCGTGCCCAGAGCCATG

The invention also includes a vector containing the promoter/enhancer DNA of the invention (operably linked to a polypeptide-encoding DNA sequence), and a vascular smooth muscle cell containing the vector. Also within the invention is a method of directing vascular smooth cell-specific expression of the polypeptide by introducing the vector into a vascular smooth muscle cell and maintaining the cell under conditions which permit expression of the polypeptide, e.g., introducing the vector into a human patient for gene therapy.

A method of detecting a gastroschisis-associated genetic alteration is carried out by providing a sample of DNA or RNA from a patient or fetus, and determining whether the DNA or RNA contains a mutation in a gene encoding an ACLP. Detection of such an ACLP mutation indicates that the patient or fetus has a genetic alteration that is associated with the development of gastroschisis. The presence of a gastroschisis-associated genetic alteration is diagnostic of gastroschisis or a predisposition to developing gastroschisis. The method can also be used to identify heterozygous carriers of a mutation associated with gastroschisis. Such individuals may be asymptomatic but are at risk of having children which are homozygous for an ACLP mutation (and therefore, likely to develop clinical gastroschisis). Tissue samples from adult patients are obtained by conventional means, e.g., biopsy or venipuncture. Prenatal testing is carried out by obtaining fetal tissue samples, e.g., by amniocentesis or chorionic villi sampling.

Patient-derived DNA is examined for genetic abnormalities in the ACLP gene, e.g., by detecting restriction fragment length polymorphisms (RFLPs), deletions, point mutations, or other defects. The diagnostic method includes the step of subjecting the sample to polymerase chain reaction (PCR), using a forward PCR primer complementary to a portion of the antisense strand of the gene, the portion being within (a) a first intron of the gene, or (b) the 5' untranslated region adjacent to the start codon of the gene; and a reverse PCR primer complementary to a fragment of the sense strand of the gene, this fragment being within (a) a second intron of the gene, or (b) the 3' untranslated region adjacent to the termination codon of the gene. PCR can also be used to detect mutations in an ACLP promoter or other regulatory sequences using primers that flank the mutation. ACLP mutations and/or aberrant ACLP expression can also be detected using standard hybridization techniques, such as Northern blotting.

Fragments of ACLP are useful to raise ACLP-specific antibodies. Accordingly, the invention includes an antibody, e.g., a polyclonal antisera or a monoclonal antibody preparation, that selectively binds to an ACLP. ACLP-specific antibodies are used to diagnose gastroschisis or a predisposition thereto. For example, a diagnostic method is carried out by providing a tissue sample from a patient or fetus, and detecting expression of an ACLP gene in the tissue sample. Expression is measured by detecting the amount of ACLP-specific antibody that binds to the tissue sample, e.g., by ELISA assay, Western blot assay, or immunohistochemical staining of tissue sections. Expression of ACLP is also measured by detecting the level of ACLP transcript in the tissue sample. Regardless of the method of detection of ACLP expression, a reduction in the amount of expression in the patient-derived tissue sample compared to the level of expression in a normal control tissue sample indicates that the patient or fetus from which the sample was obtained has or is predisposed to developing gastroschisis.

Methods of treating or preventing the development of gastroschisis are also within the invention. For example, one treatment regimen includes the steps of identifying a patient with or at risk of developing gastroschisis, and introducing into cells of the patient an isolated nucleic acid encoding ACLP, e.g., a nucleic acid which contains the nucleotide sequence of 140 to 3613 of SEQ ID NO:1. The cells into which the DNA was introduced produce the recombinant ACLP to compensate for a gastroschisis-associated genetic alteration, e.g., a mutation resulting in reduced production of ACLP or a mutation resulting in the production of a defective ACLP. Rather than administering ACLP-encoding DNA to the patient, an ACLP (e.g., a polypeptide having the sequence of SEQ ID NO:2) or a fragment thereof may be introduced into the patient.

An animal model for gastroschisis is useful to study the development of the condition as well as to evaluate therapeutic approaches to treatment or prevention of gastroschisis. A genetically-altered non-human mammal, all diploid cells of which contain a mutation in an endogenous gene encoding an ACLP, is included in the invention. For example, a mammal with a homozygous null mutation in its ACLP gene(s) develops gastroschisis. Preferably, the mammal is a rodent such as a mouse. The genetically altered non-human mammal produces altered levels of ACLP or mutant forms of ACLP. The levels of ACLP gene product in the genetically altered mammal can be increased or decreased at different time periods during development. By "genetically altered mammal" is meant a mammal in which the genomic DNA sequence has been manipulated in some way. The genetically altered mammal may be a knockout in which the endogenous ACLP sequences have been deleted or otherwise altered to decrease or change the pattern of expression. Alternatively, the genetically altered mammal may be transgenic. For example, the transgenic mammal may express ACLP sequences from another species, may overexpress ACLP gene product, or may express ACLP in tissues and at developmental stages other than those in which ACLP is expressed in a wild type animal.

The nucleated cells of a genetically altered mammal not producing a functional endogenous ACLP may be engineered to encode a human ACLP, and to express functional human ACLP, or, alternatively, ACLP from another heterologous species.

Preferably, the genetically altered non-human mammal is a rodent such as a mouse or a rat, the germ cells and somatic cells of which contain a mutation in DNA encoding ACLP. All diploid cells of such an animal contain a mutation in one or both alleles of the endogenous ACLP gene. The mutation can, for example, be a deletion, an insertion, or a nucleotide substitution. The mutation could be in the ACLP regulatory regions or in the coding sequence. It can, e.g., introduce a stop codon that results in production of a truncated, inactive gene product or it can be a deletion of all or a substantial portion of the coding sequence. For example, one or more exons, e.g., exons 7 15, of an ACLP gene may be deleted. By the term "null mutation" is meant a mutation that reduces the expression or activity level of the protein encoded by the mutated gene by more than 80% relative to the unmutated gene. A mouse harboring such a null mutation is a knockout mouse. An ACLP knockout mouse, i.e., one that harbors a homozygous ACLP null mutation, has been found to have an abdominal defect with an extrusion of abdominal organs i.e., gastroschisis.

The invention also includes a mammalian cell line, e.g., immortalized ACLP deficient cells, the genomic DNA of which contains a null mutation in DNA encoding ACLP. Such cells lack the ability to synthesize full length functional ACLP. The cells harboring the null mutation may be derived from a cell obtained from a ACLP deficient mammal, e.g., an ACLP knockout mouse.

Compounds capable of promoting expression or function of an ACLP may be therapeutically useful to treat gastroschisis. Accordingly, the invention includes a method of screening a candidate compound to identify a compound capable of stimulating expression of an ACLP, e.g., human ACLP, by (a) providing a cell or tissue expressing capable of expressing a ACLP, (b) contacting the cell or tissue with the candidate compound, and (c) determining the amount of expression of the ACLP by the cell. An increase in the amount of ACLP expression in the presence of the candidate compound compared to that in the absence of the candidate compound indicates that the compound stimulates expression of the ACLP.

In addition to diagnostic methods, such as described above, the present invention encompasses methods and compositions for evaluating appropriate treatment, and treatment effectiveness of pathological conditions associated with aberrant expression of ACLP. For example, the ACLP gene can be used as a probe to classify cells in terms of their level of ACLP expression, or as a source of primers for diagnostic PCR analysis in which mutations and allelic variation of ACLP can be detected.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is a diagram showing a comparison of deduced open reading frames of human ACLP and mouse ACLP. The human and mouse proteins contain 1158 and 1128 amino acids, respectively. Highlighted motifs include a signal peptide (bold, underline), a 4-fold lysine- and proline-rich repeating motif (bold, italic), a discoidin-like domain (bold, italic, underline), and a region with homology to the carboxypeptidases (bold).

FIG. 1B is a diagram showing the location of peptide domains of human ACLP. The signal peptide sequence at the N-terminus is designated "Signal"; the 4-fold repeating motif is designated "Repeat"; the discoidin-like domain is designated "DLD"; and the region with homology to carboxypeptidases is designated "CLD".

FIG. 2A is a diagram of mouse genomic DNA showing a map of the mouse ACLP gene and neighboring DNA polymerase delta small subunit gene.

FIG. 2B is a diagram of a restriction map of genomic DNA containing the mouse ACLP gene and neighboring DNA polymerase delta small subunit gene.

FIG. 3 is a diagram showing a map of the targeting construct used to make an ACLP knockout mouse.

DETAILED DESCRIPTION

A mutation in an ACLP-encoding nucleic acid resulting in a decrease in production of an ACLP compared to the level of ACLP production in an animal lacking the mutation has now been shown to result in the development of gastroschisis in newborn mice. The following examples describe the cloning and characterization of human ACLP and methods of diagnosing and treating gastroschisis the underlying defect of which is a genetic alteration in the ACLP genes.

EXAMPLE 1

Cloning of ACLP Genes

ACLP was identified in a screen for proteins interacting with the E47 product of the E2A gene. A recombinant E47 fusion protein (N3-SH[ALA]), containing the basic helix loop helix domain of hamster shPan-1 (amino acids 509 646, with mutations R551A, V552L, and R553A) with a heart muscle kinase recognition sequence and the FLAG epitope, was expressed and purified as described (Blanar et al., 1995, Proc. Natl. Acad. Sci. USA 92:5870 4; Blanar and Rutter, 1992, Science 256:1014 8). N3-SH[ALA] was phosphorylated by heart muscle kinase in the presence of .gamma.-.sup.32P-ATP and used to screen a human aorta .lamda.gt11 cDNA expression library (Clonetech) by interaction cloning (Blanar et al., 1995, Proc. Natl. Acad. Sci. USA 92:5870 4; Blanar and Rutter, 1992, Science 256:1014 8). A 1450-bp cDNA clone (.DELTA.E2A-BP) obtained from interaction cloning was radiolabeled by random priming and used to isolate a 2786 bp cDNA clone from the same human aorta .lamda.gt11 cDNA library. Data from Northern blotting experiments revealed that the ACLP-1 RNA was about 3.9 kb in size and suggested that the 2786 bp cDNA clone was a partial cDNA clone. Additional 5' sequences of the ACLP cDNA were isolated by 5' rapid amplification of cDNA ends from human aortic smooth muscle cell RNA (Gibco-BRL). The full length sequence of the human ACLP cDNA was found to be 3935 bp and is shown in Table 1 (SEQ ID NO:1). The full length human ACLP cDNA contains an open reading frame (nucleotides 140 3613 of SEQ ID NO:1) encoding a polypeptide of 1158 amino acids. The open reading frame is preceded by a Kozak consensus translation initiation sequence, which in turn is preceded by an in frame stop codon.

The human ACLP protein has a calculated molecular mass of 130 kDa, an estimated pI of 4.8, and contains a putative signal peptide sequence. In addition, it contains an 11 amino acid lysine- and proline-rich motif repeated four times at the N-terminus, a domain with 30% amino acid identity to the slime mold adhesion protein discoidin I, and a C-terminal domain with 39% identity to carboxypeptidase E. The human ACLP gene maps to the short arm of chromosome 7 (between D7S478 amd D7S519).

The sequence of the human ACLP cDNA (GENBANK.TM. accession number AF053944) was compared to sequences present in GENBANK.TM. databases. A 3' portion of ACLP cDNA was found to share homology with the sequence of a cDNA encoding mouse adipocyte enhancer binding protein 1 (AEBP1; He et al., 1995, Nature 378:92). AEBP1 was originally identified as a 2.5 kb cDNA that hybridized to a 4 kb band on Northern blot analysis, and was predicted to encode a 719 amino acid, 79 kDa protein.

To isolate mouse ACLP cDNA (GENBANK.TM. accession number AF053943), first strand cDNA from C2C12 mouse myoblast total RNA was synthesized by reverse transcription with the primer 5' ATCTGGTTGTCCTCAAT 3' (SEQ ID NO:4). The nested primer 5' TGACTCCATCCCAATAG 3' (SEQ ID NO:5) and the anchor primer included in the kit for 5' rapid amplification of cDNA ends was then amplified to produce a product of approximately 1400 bp in size. This product was sequenced using standard methods.

The entire open reading frame of mouse ACLP was then amplified from C2C12 RNA by reverse transcription PCR (EXPANDLONG.TM. Template PCR System, Boehringer Mannheim, Indianapolis, Ind.). The human and mouse clones were sequenced by the dideoxy nucleotide chain termination method using a combination of Sequenase Version 2.0 (Amersham, Arlington Heights, Ill.), the Thermo Sequenase .sup.33P terminator cycle sequencing kit (Amersham), and the Thermo Sequenase fluorescent-labeled cycle sequencing kit with 7-deaza-GTP (Amersham) on a Licor (Lincoln, Nebr.) apparatus.

Sequencing of the 3633 bp mouse ACLP cDNA fragment, revealed that it encoded an open reading frame (1128 amino acids) similar to that of the full-length human ACLP cDNA, indicating that it is the mouse ACLP homologue. A comparison of the human and mouse ACLP amino acid sequences is shown in FIG. 1A. Overall, the two proteins are 85% identical and 90% similar.

EXAMPLE 2

Identification of a Promoter-Enhancer Sequence Associated with the ACLP Gene

To identify genomic sequences that mediate tissue specific and developmental expression pattern of the ACLP gene, a region of genomic DNA adjoining the 5' end of the mouse ACLP coding sequences was isolated (FIGS. 2A and 2B). Portions of this genomic DNA were then used in reporter transfection assays to determine their ability to direct expression of a reporter gene in transfection assays. ACLP promoter/enhancer DNA was cloned into the pGL2 Basic vector (Stratagene) and transfected into rat aortic smooth muscle cells (RASMC) to measure promoter activity. Using this assay, a region containing an ACLP promoter/enhancer sequence was identified and is shown in Table 3 (SEQ ID NO:3). ACLP promoter/enhancer DNA was found to have transcriptional activity both in vitro (using cultured cells) and in vivo (in a transgenic mouse).

EXAMPLE 3

Generation and Characterization of Antibodies to ACLP Peptides

A carboxy terminal fragment of mouse ACLP was expressed in bacteria, purified, and used as an immunogen to raise antibodies in rabbits.

To produce a polyclonal anti-ACLP antibody, a BamHI-EcoRI fragment of mouse ACLP (encoding amino acids 615 1128) was subcloned into the pRSET C bacterial expression vector (Invitrogen), and the resulting plasmid was transformed into BL21(DE3)pLysS-competent bacteria (Stratagene). Protein expression was induced with 1 mM isopropyl .beta.-D-thiogalactopyranoside for 3 h. Bacteria were sonicated in lysis buffer (50 mM NaH.sub.2PO.sub.4, 10 mM Tris, pH 8, 100 mM NaCl) containing the protease inhibitors aprotinin, leupeptin, and phenylmethylsulfonyl fluoride. Lysates were clarified by centrifugation at 10,000 g for 15 min, and the pellet was resuspended in lysis buffer supplemented with 8 M urea. His-tagged proteins were purified with Talon resin (Clontech) and eluted in lysis buffer containing 8 M urea and 100 mM ethylene diamine tetraacetic acid. Proteins were dialyzed against water and measured with the Bio-Rad (Hercules, Calif.) protein assay reagent. 100 .mu.g of the purified protein was used to immunize New Zealand white rabbits. Antiserum was collected, titered against the recombinant protein, and used for immunoblot analysis. Specificity of the antiserum was determined by using preimmune serum and by competition with a recombinant protein. The same methods are used to raise antibodies to human ACLP. The rabbit antisera raised against a portion of mouse ACLP was found to crossreact with human ACLP.

Protein extracts from cultured cells were prepared for Western blotting in extraction buffer (25 mM Tris, pH 7.4, 50 mM NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40, and 0.2% sodium dodecyl sulfate) containing the protease inhibitors aprotinin, leupeptin, and phenylmethylsulfonyl fluoride. To obtain proteins from mouse tissues, individual organs were homogenized in 25 mM Tris, pH 7.5, 50 mM NaCl, and 10 mM ethylene diamine tetraacetic acid containing protease inhibitors (Complete, Boehringer Mannheim). Proteins were measured with the BCA protein assay kit (Pierce, Rockford, Ill.). After 50 .mu.g aliquots had been resolved on 6% sodium dodecyl sulfate-polyacrylamide gels (18), proteins were transferred electrophoretically to nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.) in 48 mM Tris, pH 8.3, 39 mM glycine, 0.037% sodium dodecyl sulfate, and 20% methanol transfer buffer. Blots were equilibrated with 25 mM Tris, pH 8, 125 mM NaCl, and 0.1% Tween 20 and blocked in the same solution containing 4% nonfat dry milk. Blots were incubated with anti-ACLP serum diluted 1:1000 and then horseradish peroxidase-conjugated goat anti-rabbit serum diluted 1:4000. Membranes were processed with an enhanced chemiluminescence reagent (ECL reagent, NEN, Boston, Mass.) and exposed to film.

By Western blot analysis, this antibody detected a single band corresponding to a protein with an apparent mobility of approximately 175 kDa in mouse aortic smooth muscle cells (MASMC) extracts. This protein showed a similar migration to a protein generated by transcription and translation in vitro of a mouse ACLP cDNA clone, providing additional evidence that the isolated human and mouse cDNA clones encode full-length ACLP.

Monoclonal antibodies can be obtained using full-length human or mouse ACLP or fragments thereof using standard methods, e.g., the process described by Milstein and Kohler, 1975, Nature 256:495 97, or as modified by Gerhard, 1980, Monoclonal Antibodies, Plenum Press, pages 370 371. Hybridomas are screened to identify those producing antibodies that are specific for an ACLP. Preferably, the antibody will have an affinity of at least about 10.sup.8 liters/mole and more preferably, an affinity of at least about 10.sup.9 liters/mole.

EXAMPLE 4

Subcellular Localization and Tissue Localization of ACLP Proteins

To assess the subcellular localization of ACLP, a mouse ACLP expression construct was generated with a c-myc epitope at the C-terminus. The myc epitope was placed at the C-terminus to avoid interference with signal peptide-mediated processes, e.g., ACLP secretion mechanisms. To construct a c-myc-tagged ACLP expression plasmid (pcDNA3.1/ACLP-Myc-His), the open reading frame of mouse ACLP was amplified with the Expand Long Template PCR System (Boehringer Mannheim). A 5' primer containing an EcoRI site (5' CGGAATTCAGTCCCTGCTCAAGCCCG 3'; SEQ ID NO:6) and a 3' primer containing a HindIII site (5' CGAAGCTTGAAGTCCCCAAAGTTCACTG 3'; SEQ ID NO:7) was used, which resulted in the deletion of the endogenous termination codon in the PCR product. The PCR product was then digested with EcoRI and HindIII restriction enzymes and ligated into the EcoRI and HindIII sites of pcDNA3.1(-)/Myc-His A (Invitrogen). Cells were transfected transiently with pcDNA3.1/ACLP-Myc-His by the DEAE-dextran method with minor modifications (Tan et al., Kidney International 46:690, 1994). Twenty-four hours after transfection, cells were trypsinized and plated onto chamber slides (Nunc, Naperville, Ill.) and grown for an additional 24 h. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline and immunostained using standard methods. Amonoclonal anti-c-myc primary antibody (9E10 Ab-1, Oncogene Research Products, Cambridge, Mass.) and a rhodamine-conjugated goat anti-mouse IgG secondary antibody were used to immunostain the cells. Nuclei were counterstained with Hoechst 33258 (1 .mu.g/ml) and visualized with a fluorescence microscope.

RASMC and A7r5 cells both exhibited strong membrane-associated or cytoplasmic staining. Staining was most intense in the perinuclear region and was not observed in the nucleus. Various other tissues were examined for the presence of ACLP mRNA and protein. Gene expression studies confirmed expression in aortic smooth muscle cells, and levels of ACLP mRNA were found to be high in the whole aorta (including adventitia) compared to most other tissue types tested, e.g., heart, brain, stomach, thymus, and liver. ACLP message was also detectable in colon and kidney tissue.

To examine expression of ACLP, extracts from mouse tissues were subjected to Western blot analysis using anti-ACLP sera. ACLP was strongly expressed in the mouse aorta (without adventitia) but not in the adventitia, heart, liver, skeletal muscle, or kidney. The presence of ACLP mRNA in the kidney (but absence of protein) indicates that the level of ACLP in the cells is regulated at the level of translation of ACLP mRNA into polypeptide.

To identify cell types expressing ACLP in an adult animal, in situ hybridization was performed on adult rat aorta and skeletal muscle using known methods. Adult male Sprague-Dawley rats were perfused with 4% paraformaldehyde and their organs were removed and sectioned. ACLP mRNA was detected with a [.sup.35S]UTP-labeled antisense riboprobe synthesized with SP6 RNA polymerase from a linearized 0.7 kb fragment of ACLP cDNA. As a control, a sense RNA probe was synthesized with T7 RNA polymerase from a linearized ACLP cDNA fragment. The antisense riboprobe detected specific ACLP expression in the smooth muscle cells of the aorta, whereas the control (sense) probe did not. Neither the sense nor the antisense probe hybridized to skeletal muscle cells.

EXAMPLE 5

ACLP Expression in Smooth Muscle Cell Differentiation

ACLP protein expression was examined during vascular smooth muscle cell growth and differentiation. RASMC and MASMC were isolated from the thoracic aortas of adult male Sprague-Dawley rats and C57Bl/6 mice using standard methods. Human aortic smooth muscle cells (HASMC) were purchased from Clonetics (San Diego, Calif.), and rat A7r5 smooth muscle cells and C2C12 mouse myoblasts were purchased from the American Type Culture Collection (Rockville, Md.). Mouse neural crest cells (Monc-1 cells) were cultured on fibronectin-coated plates. RASMC, MASMC, and A7r5 cells were cultured in Dulbecco's modified Eagle's medium with 3.7 g/liter glucose (Gibco-BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), 4 mML-glutamine, 100 .mu.g/ml streptomycin, 100 units/ml penicillin, and 10 mM HEPES (pH 7.4). C2C12 cells were grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum, 4 mM L-glutamine, 100 .mu.g/ml streptomycin, and 100 units/ml penicillin. HASMC were cultured in M199 medium (Gibco) supplemented with 20% fetal bovine serum, 4 mM L-glutamine, 100 .mu.g/ml streptomycin, and 100 units/ml penicillin. Cells were grown at 37.degree. C. in a humidified incubator containing 5% CO.sub.2. MASMC were cultured for 3 days in 0.4% calf serum containing medium that induces quiescence. RNA and protein extracts were then prepared from the cells and analyzed.

The amount of ACLP mRNA was higher (about 2-fold) in serum-starved (quiescent) MASMC than in control cells (normal proliferating MASMC). In RASMC, ACLP mRNA was approximately 3-fold more abundant in quiescent cells than in their actively proliferating counterparts. ACLP protein was also elevated in quiescent MASMC.

ACLP expression was examined in an in vitro system for differentiating smooth muscle cells from a Monc-1 cell line, a mouse line derived from the neural crest. Monc-1 cells differentiate into smooth muscle cells when tissue culture medium supplemented with chick embryo extract is replaced with differentiation medium. To examine ACLP expression during the transition of undifferentiated Monc-1 cells to smooth muscle, the time course of ACLP expression was measured. ACLP mRNA was nearly undetectable in undifferentiated Monc-1 cells. As the cells differentiated, however, ACLP expression increased until it became marked at days 4 and 6 after the start of differentiation. Under these conditions, induction of ACLP appeared to lag behind that of smooth muscle a-actin, a marker for smooth muscle cells. To compare the level of ACLP protein in cells treated similarly, protein extracts were prepared from undifferentiated Monc-1 cells and from cells allowed to differentiate for 6 days. ACLP protein was not detectable in undifferentiated Monc-1 cells but was expressed highly (day 6) under conditions that promote Monc-1 cell differentiation into smooth muscle cells. The abundance of ACLP protein in these cells was similar to that in MASMC.

As is described below, the Mon