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M Ptashne

Publications and source records attributed to M Ptashne.

At least 109 records · Page 6Linked to original sources
PubMed1984

Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG.

The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.

Base Sequence↗
PubMed1983

Repressor structure and the mechanism of positive control.

It has been suggested that the lambda repressor stimulates transcription of its own gene by binding to the lambda operator and contacting RNA polymerase bound to the adjacent promoter. We describe three different mutants (called pc) of the lambda phage repressor that are specifically deficient in the positive control function. We show that the amino acid residues altered in the pc mutants lie on the surface of the DNA-bound repressor that we predict, based on structural and other evidence, would most closely approach DNA-bound polymerase. Furthermore, we describe a pc mutant of the P22 repressor. We argue that in both the lambda and P22 repressors a structure comprised of two alpha helices has two functions: to bind DNA and to contact RNA polymerase. In the two cases, however, different regions of this structure contact polymerase to mediate positive control.

Bacteriophage lambda↗
PubMed1983

Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli.

A strong promoter has been cloned on a series of plasmid vectors that facilitate the expression of cloned genes. This promoter, named tac [first described by DeBoer et al. (in Rodriguez, R.L. and Chamberlin, M.J. (Eds.),Promoters, Structure and Function. Praeger, New York, 1982, pp. pp. 462-481)] contains the -10 region of the lacUV5 promoter and the -35 region of the trp promoter. Our vectors contain various cloning sites followed by transcription termination signals. In addition, we describe plasmids that facilitate the conversion of the lac promoter to the stronger tac promoter. Thus, preexisting gene fusions using the lac or the lacUV5 promoter can be readily converted to tac promoter gene fusions without changing the ribosome-binding site (RBS). The tac promoter is repressed in lacIQ strains and can be induced by isopropylthio-beta-D-galactoside (IPTG). Studies of expression of the cI repressor of bacteriophage lambda show that the tac promoter is at least five times more efficient than the lacUV5 promoter. Under optimal conditions lambda repressor constitutes up to 30% of the total cellular protein.

Bacterial Proteins↗
PubMed1982

Cleavage of the lambda and P22 repressors by recA protein.

The site of recA cleavage of the phage lambda and P22 repressors has been determined. Each repressor is cut once by the recA enzyme, lambda repressor between residues 111 and 112, and P22 repressor between residues 94 and 95. recA cleavage occurs at identical alanyl-glycyl sequences in both repressors, and in both repressors, the cleavage separates the repressor's NH2-terminal DNA-binding domain from its COOH-terminal oligomerization domain. A papain-generated proteolytic fragment of lambda repressor consisting of repressor residues 93-236 is also efficiently cleaved by recA protein. Moreover, the recA cleavage of radioactive lambda repressor can be inhibited by certain COOH-terminal proteolytic fragments of lambda repressor which do not contain the alanyl-glycyl cleavage sequence. These facts suggest that recA cleavage of lambda repressor requires an intact COOH-terminal domain but not an intact NH2-terminal domain.

Adenosine Triphosphatases↗
PubMed1982

Regulated expression of an extrachromosomal human beta-interferon gene in mouse cells.

Beta(fibroblast)-interferon mRNA and protein are induced by the synthetic double-stranded RNA poly(I) X poly(C) in cultured human fibroblasts. To study the mechanism of this induction, we have isolated a human beta-interferon gene and inserted it in a vector plasmid containing DNA of the bovine papilloma virus. After removal of bacterial plasmid sequences, the bovine papilloma virus-beta-interferon recombinant was used to morphologically transform mouse fibroblasts. Analysis of DNA from the transformed cell lines indicated that this recombinant is propagated as a stable multicopy extrachromosomal element. Human beta-interferon mRNA and protein are inducible by poly(I) X poly(C) in all of these cell lines, and the mRNA is indistinguishable from beta-interferon mRNA synthesized by induced human cells.

Animals↗
PubMed1982

Mutant lambda phage repressor with a specific defect in its positive control function.

The lambda phage repressor is both a positive and a negative regulator of gene transcription. We describe a mutant lambda phage repressor that has specifically lost its activator function. The mutant binds to the lambda phage operator sites and represses the lambda phage promoters PR and PL. However, it fails to stimulate transcription from the promoter PRM. The mutation lies in that portion of repressor--namely, the amino-terminal domain--that has been shown [Sauer, R. T., Pabo, C. O., Meyer, B. J., Ptashne, M. & Backman, K. C. (1979) Nature (London) 279, 396-400] to mediate stimulation of PRM. We suggest that the mutation has altered that region of repressor which, in the wild-type, contacts RNA polymerase to activate transcription from PRM.

Bacteriophage lambda↗
PubMed1981

lambda Repressor and cro--components of an efficient molecular switch.

In a lysogen, most genes of phage lambda are repressed; in response to a transient induction signal, they are efficiently switched on. The switch, which consists in part of a tripartite operator to which two regulatory proteins bind, depends not only on DNA--protein interactions, but also on effects transmitted from one DNA-bound protein to another. lambda Exemplifies a strategy that facilitates efficient switching between two physiological states in response to a transient signal.

Animals↗
PubMed1981

Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae.

Hybrid genes between the Escherichia coli lacZ gene and the iso-1-cytochrome c (CYC1) gene of Saccharomyces cerevisiae were constructed by recombination in vitro. Each of the hybrid genes encodes a chimeric protein with a cytochrome c moiety at the amino terminus and an active beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) moiety at the carboxy terminus. When these hybrids are introduced into S. cerevisiae on plasmid vectors, they direct synthesis of beta-galactosidase. beta-Galactosidase levels directed by one such plasmid display the pattern of regulation normally seen for cytochrome c (i.e., a reduction of synthesis in cells grown in glucose). This plasmid contains one codon of CYC1 fused to lacZ, and the fused gene is preceded by the 1100 nucleotides that lie upstream from CYC1. An analysis of deletions in the upstream DNA suggests that sequences required for efficient transcription initiation of CYC1 lie within the DNA segment 250--700 base pairs upstream from the start of the CYC1 coding sequence. This region is at least 130 base pairs upstream from the "Hogness box" sequence that precedes the CYC1 coding sequence.

Animals↗
PubMed1981

Mechanism of action of the lexA gene product.

Genetic experiments have suggested that the lexA gene product of Escherichia coli represses a number of genes involved in the response to DNA damage, including recA and lexA. We purified the lexA gene product from bacterial strains that bear plasmids that direct the synthesis of large amounts of the protein. Purified lexA protein bound to two symmetrical DNA sequences in front of lexA and one in front of recA, protecting them from digestion with DNase I and blocking methylation of purines in the major groove. lexA protein repressed transcription of both genes in vitro. lexA protein binds to the two sites in front of the lexA gene with approximately the same affinity and with greater affinity to the single site in front of the recA gene. The affinity of lexA protein for its operator sites was measured under conditions that mimic conditions in vivo. Differences in the affinity with which lexA protein binds to the operators of genes it represses may account for the differences in the timing and extent of their induction after DNA damage.

Bacterial Proteins↗
PubMed1980

A technique for expressing eukaryotic genes in bacteria.

Methods are described that allow efficient expression in Escherichia coli of cloned eukaryotic genes. The methods require that the coding sequence of the gene in question be available in a form uninterrupted by intervening sequences (for example, as a complementary DNA clone). The gene products are synthesized unfused to other amino acid sequences. The genetic manipulations are simple, and require the plasmids described and commercially available enzymes.

Cloning, Molecular↗