Smooth muscle cell LIM promoter

Details Smooth muscle cell LIM promoter Smooth muscle cell LIM promoter

1997-03-14

2001-07-10

Martin, Jill D. (Department: 1632)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C435S455000, C435S006120, C536S023100, C536S023500, C536S024310, C514S04400A

Reexamination Certificate

active

06258557

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to diagnosis and treatment of vascular injury.
In their normal state, vascular smooth muscle cells regulate vessel tone and blood pressure. Unlike skeletal muscle and cardiac muscle cells, these cells are not terminally differentiated. In response to mechanical, chemical, or immunologic injury (Libby et al., 1991, Lab Invest. 64:5-15; Munro et al., 1988, Lab Invest. 58:249-261; Ross, R., 1993, Nature 362:801-809; Tsai et al., 1994, Proc. Natl. Acad. Sci. USA 91:6369-6373; and Tsai et al., 1996, Clin. Invest. 97:146-153), the phenotype of these cells changes rapidly from that of a differentiated, quiescent cell to that of a dedifferentiated, proliferating cell. Although vascular smooth muscle cell proliferation is a hallmark of arteriosclerosis, the leading cause of death in developed countries, little is known about the molecular mechanisms regulating this phenotypic change.
SUMMARY OF THE INVENTION
The invention is based on the identification and characterization of a smooth muscle cell LIM (SmLIM/CRP2) polypeptide which is expressed preferentially in arterial smooth muscle cells. SmLIM/CRP2 expression was found to decrease as vascular smooth muscle cells changed from a quiescent, differentiated phenotype to a proliferative phenotype in response to vascular injury.
The invention features a substantially pure DNA containing a sequence which encodes a SmLIM/CRP2 polypeptide. By the term “SmLIM/CRP2” is meant a polypeptide that contains at least two LIM domains, lacks a homeobox domain and a protein kinase domain, and inhibits proliferation of vascular smooth muscle cells. By “substantially pure DNA” is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the SmLIM/CRP2 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 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 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. A “LIM domain” is defined by the amino acid consensus sequence CX
2
CX
17±1
HX
2
CX
2
CX
2
CX
17±1
CX
2
C/D/H (SEQ ID NO:18).
The SmLIM/CRP2 polypeptide of the invention preferably has at least 85% sequence identity with SEQ ID NO:1, and more preferably at least 90% (e.g., at least 95%). The DNA may encode a naturally occurring mammalian SmLIM/CRP2 polypeptide such as a human, rat, mouse, guinea pig, hamster, dog, cat, pig, cow, goat, sheep, horse, monkey, or ape SmLIM/CRP2. For example, the SmLIM/CRP2 polypeptide may have the amino acid sequence of the naturally-occurring human polypeptide, e.g., a polypeptide which includes the amino acid sequence of SEQ ID NO:1. Preferably, the DNA includes the nucleotide sequence of SEQ ID NO:2. The DNA may contain a strand which hybridizes at high stringency to a DNA probe having a portion or all of the nucleotide sequence of SEQ ID NO:2, or the complement thereof. The probe to which the DNA of the invention hybridizes preferably consists of at least 20 nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 100 nucleotides or more (up to 100%) of the nucleotide sequence of SEQ ID NO:2, or the complement thereof. Such a probe is useful for detecting expression of a SmLIM/CRP2 transcript in a cell by a method which includes the steps of (a) contacting mRNA obtained from the cell with the labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA transcript. The invention also includes a substantially pure strand of DNA containing at least 15 nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of SEQ ID NO:2.
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., wash conditions of 65° C. at a salt concentration of approximately 0.1×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° C. at a salt concentration of at least 1.0×SSC. For example, high stringency conditions may include hybridization at about 42° C., and about 50% formamide; a first wash at about 65° C., about 2×SSC, and 1% SDS; followed by a second wash at about 650° C. and about 0.1%×SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to a SmLIM/CRP2 gene are detected by, for example, hybridization at about 42° C. in the absence of formamide; a first wash at about 42° C., about 6×SSC, and about 1% SDS; and a second wash at about 50° C., about 6×SSC, and about 1% SDS.
The invention also includes a substantially pure DNA encoding a SmLIM/CRP2 polypeptide, which DNA includes a nucleotide sequence having at least 50% sequence identity to SEQ ID NO:2. Preferably the DNA has at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 99% identity to SEQ ID NO:2. The percent sequence identity of one DNA to another is determined by standard means, e.g., by the Sequence Analysis Software Package developed by the Genetics Computer Group (University of Wisconsin Biotechnology Center, Madison, Wis.) (or an equivalent program), employing the default parameters thereof.
The DNA may be operably linked to regulatory sequences, e.g., a promoter, for expression of the polypeptide. Preferably, the promoter is vascular cell-specific, more preferably, it is vascular smooth muscle cell-specific, and most preferably, it is arterial smooth muscle cell-specific. 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). By “promoter” is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible.
The invention includes a substantially pure DNA containing a sequence at least 50% identical to SEQ ID NO:3 or SEQ ID NO:16, which regulates arterial smooth muscle cell-specific transcription of a polypeptide-encoding sequence to which it is operably linked. Preferably, the DNA is at least 75% identical, more preferably at least 90% identical, more preferably at least 95%, and most preferably 100% identical to SEQ ID NO:3 or SEQ ID NO:16. The DNA may be operably linked to a heterologous polypeptide-encoding sequence and may be used in a method of directing arterial smooth muscle cell-specific expression of the polypeptide, e.g., by introducing the DNA linked to the coding sequence into an arterial cell. By the term “heterologous polypeptide” is meant a polypeptide other than a SmLIM/CRP2 polypeptide.
The invention also includes a substantially pure DNA comprising a first DNA sequence containing a SmLIM/CRP2-derived promoter sequence, e.g., one which is at least 50% identical to SEQ ID NO:3 or 16, operably linked to a second DNA sequence encoding a polypeptide other than SmLIM/CRP2, i.e., a heterlogous peptide, wherein the first DNA sequence directs transcription of the second DNA sequence preferentially in an a vascular smooth muscle cell, e.g., an arterial smooth muscle cell, compared to in a non-vascular smooth muscle cell. Preferably, the second DNA sequence does not encode SmLIM/CRP2. Vascular smooth muscle cell-specific expression of a polypeptide is accomplished by introducing into a

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