Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1993-12-21
2002-11-05
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C536S023100, C536S023500, C536S023510, C435S320100, C435S091100, C435S091330, C435S091400, C435S091410, C435S091420, C435S091500, C435S235100, C435S252300, C435S254110, C424S093100, C424S093200, C424S093210
Reexamination Certificate
active
06475781
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to trans-dominant suppressor genes.
Naturally occurring dominant suppressor genes are known that trigger substantial phenotypic alterations in eucaryotic cells and tissues. Herskowitz (Nature 329:219-222, 1987) has proposed that the natural process can be mimicked by altering a cloned gene so that it encodes a mutant product, capable of inhibiting the wildtype gene product in a cell, thus causing the cell to be deficient in the function of that gene product. Malin et al. (Cell 58:205-214, 1989) and Green et al. (Cell 58:215-223, 1989) performed mutational analyses of two HIV-1 trans-activators essential for viral replication and obtained results which they attributed to effects of the sort described by Herskowitz.
One common, naturally occurring dimeric protein is platelet derived growth factor (PDGF), first recognized and purified from human blood platelets. PDGF has been isolated from a wide variety of cells and tissues of numerous eucaryotic species. The two known PDGF subunits, A and B, are expressed from separate genes and are active as either homo or heterodimers. Dimerization occurs through disulfide linkage between cysteine residues on the individual subunits. The positioning of these residues is conserved between the A and B chains, and between species at least as divergent as human and Xenopus. Four of the eight cysteines residues on each subunit chain are believed to be essential for catalytic activity. The initial translation product of the PDGF A and B genes is a “preproPDGF”. A hydrophobic leader sequence (the “pre” sequence) and a substantial length of N-terminal material (the “pro” sequence) are removed by proteolytic cleavage to generate the mature form of a PDGF subunit.
SUMMARY OF THE INVENTION
The invention features two methods for making eucaryotic trans-dominant suppressor genes. A “eucaryotic trans-dominant suppressor gene”, as defined herein, is a gene encoding a polypeptide translation product capable of suppressing the activity of a eucaryotic protein that requires an oligomeric state for function, by forming an inactive oligomer with a wildtype subunit of the protein and thereby preventing that wildtype subunit from forming an active dimer with a second wildtype subunit. The methods are particularly useful for producing a trans-dominant suppressor gene that encodes an inactive subunit of a eucaryotic growth factor and thus suppresses the activity of that growth factor.
The first method involves providing a nucleic acid encoding a subunit of the growth factor, the subunit being one that is initially synthesized containing extraneous peptide material which is subsequently removed by cleavage at a proteolytic cleavage site to generate the mature form of the subunit; modifying the base sequence in the nucleic acid in the region encoding the proteolytic cleavage site, so that proteolytic cleavage of the initial translation product of the nucleic acid to the mature form is prevented; and cloning the modified nucleic acid to form the trans-dominant suppressor gene. The modifying step involves either addition or deletion of a codon for an amino acid essential for proteolytic cleavage, or, preferably, substitution of one such essential codon with a codon for a different amino acid. Either way, the reading frame must be preserved. A “proteolytic cleavage site” is an amino acid sequence in the immediate vicinity of a peptide bond that is cleaved by a protease, which amino acid sequence is required for cleavage by the protease. In a preferred embodiment, the proteolytic cleavage site is defined by the sequence -ArgArgLysArg- (SEQ ID NO: 1).
The second method involves providing a nucleic acid encoding a subunit of the growth factor, the subunit being one that is bonded in the oligomeric state by means of a plurality of cysteine residues, at least one of which is essential for the catalytic activity of the protein; modifying in the nucleic acid the codon for one of the cysteine residues essential for the activity of the growth factor, so that another amino acid is substituted for the one cysteine residue, the codon for at least one cysteine residue remaining unmodified; and cloning the modified nucleic acid to form the trans-dominant suppressor gene. In a preferred embodiment, the cysteine residue modified in this method is the third cysteine residue of the mature form of PDGF A.
In preferred embodiments, the growth factor is a member of the platelet derived growth factor (PDGF) superfamily, e.g. PDGF A or B, or vascular endothelial cell growth factor (VEGF); colony stimulating factor I (CSF-I); or a member of the transforming growth factor beta (TGF-&bgr;) superfamily, e.g. TGF-&bgr;1 or TGF-&bgr;2.
In another aspect, the invention features a eucaryotic trans-dominant suppressor gene. In one embodiment, the eucaryotic trans-dominant suppressor gene encodes a protein translation product that is a mutant form of a PDGF subunit having a modification in the amino acid sequence corresponding to the proteolytic cleavage site of the PDGF subunit which prevents cleavage at the site. In another embodiment, the eucaryotic trans-dominant suppressor gene encodes a mutant form of a PDGF subunit in which one of the cysteine residues essential for the mitogenic activity of PDGF is replaced with a different amino acid, and at least one other cysteine residue remains unmodified. In other embodiments, the growth factor is TGF-&bgr;1, TGF-&bgr;2, CSF-I, or VEGF. The invention also features the protein translation products of the eucaryotic trans-dominant suppressor genes of the invention. These protein translation products also are referred to as “dominant suppressor proteins” or “dominant negative mutants”.
In another aspect, the invention features a vector containing the trans-dominant suppressor gene, which may be operably linked to a functional promoter. A “vector” is defined as a replicable nucleic acid construct. Vectors are used in the invention to amplify and/or express nucleic acid encoding the dominant suppressors. An expression vector is a replicable construct in which a nucleic acid sequence encoding the dominant suppressor protein is operably linked to suitable control sequences capable of effecting expression of the dominant suppressor protein in a cell. The need for such control sequences will vary depending upon the cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter and/or enhancer, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. The only sequences required in an amplification vector are one which confers the ability to replicate in a cell, which is usually an origin of replication, and a selection gene to facilitate recognition of transformants. A gene and a promoter and/or enhancer are defined as being “operably linked” if the promoter and/or enhancer controls the transcription of the gene. A “functional promoter” is defined as a region of DNA involved in binding RNA polymerase to initiate transcription. Vectors of the invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the invention are those derived from retroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses.
The invention also features a cell containing a eucaryotic trans-dominant suppressor gene. In one embodiment, the cell expresses the eucaryotic trans-dominant suppressor gene. The cell can be either a procaryotic cell, e.g. an
Escherichia coli
cell, or a eucaryotic cell. Eucaryotic cells that can be used in the invention include, but are not limited to, Cos, CHO, and Sf9 cells. In the case of a eucaryotic cell, the gene may or may not be integrated into the genome of the cell. Also included in the invention is an essentially homogeneous population of procaryotic or eucaryotic cells, each of which contains a eucaryotic trans-dominant suppressor gene.
In a related a
Deininger Prescott L.
Mercola Mark K.
Stiles Charles D.
Dana-Farber Cancer Institute Inc.
Low Christopher S. F.
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