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Biomedical subjects

M Ptashne

Publications and source records attributed to M Ptashne.

At least 127 records · Page 7Linked to original sources
PubMed1980

Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin.

In this paper we describe a method for constructing E. coli plasmids that direct efficient expression of genes that encode eucaryotic or procaryotic proteins. No functional assays for the proteins are needed, and they are produced in their native, unfused state. The only requirement is that the genes be isolable without intervening sequences. We describe as an example the construction of a plasmid that directs the synthesis of about 10,000-15,000 monomers per cell of rabbit beta-globin. The essential steps in a typical construction are as follows. --A region of the gene encoding the amino-terminal portion of the protein is fused to DNA encoding an enzymatically active carboxy terminal fragment of beta-galactosidase. The latter is carried on one of three plasmids designed to facilitate the fusion (the construction of these three plasmids is described in the Appendix). --A "portable promoter" of the lac operon is placed at many positions in front of the fused gene using nucleases in vitro. Those promoter placements that elicit efficient expression of the fused gene are identified by the beta-galactosidase activity that they express. (In the special case we describe, plasmids identified as directing efficient expression of beta-globin were found to bear "hybrid" ribosome binding sites consisting of the Shine-Dalgarno sequence carried on the promoter fragment and the ATG of the beta-globin gene.) --The gene of interest is reconstituted intact, with the portable promoter in place, by recombination in vitro or in vivo.

Animals↗
PubMed1980

The lexA gene product represses its own promoter.

The products of the lexA and recA genes of Escherichia coli regulate the cellular response to DNA damage (the SOS response). Here we describe the cloning of the wild-type lexA gene and the identification of its 24,000-dalton protein product. We also describe construction, by recombination in vitro, of a phage that bears the lexA promoter fused to the lacZ gene. Experiments with this fusion phage and with multicopy plasmids that carry the lexA gene showed that the lexA gene product represses of its own promoter. This repression occurs even if the cell has no recA gene, showing that the lexA protein need not be complexed to the recA protein for activity. Moreover, the presence of multicopy plasmids that carry the lexA gene blocks expression of all SOS responses tested. This presumably results from two effects: (i) repression of the recA gene, the product of which is required to activate many of these responses; and (ii) direct repression of other functions involved in the SOS response.

Bacterial Proteins↗
PubMed1980

Expression of the human fibroblast interferon gene in Escherichia coli.

We applied the method of Guarente et al. [Guarente, L., Lauer, G., Roberts, T.M. & Ptashne, M. (1980) Cell 20, 543-553] to construct plasmids that direct expression in Escherichia coli of the human fibroblast interferon (F-IF) gene. Two plasmids were recovered. One directs efficient synthesis of a protein whose primary sequence is that of pre-F-IF and the other, that of mature F-IF. Extracts of bacteria synthesizing mature F-IF display antiviral activity characteristic of human F-IF. This activity is lower than that expected from the differential rate of synthesis of the protein. We have detected no such activity in extracts of bacteria synthesizing pre-F-IF.

Base Sequence↗
PubMed1979

Interactions between DNA-bound repressors govern regulation by the lambda phage repressor.

The lambda phage repressor binds cooperatively to the three sites in the right operator (O(R)) according to the following pattern. If the DNA is wild type, O(R)1 and O(R)2 are filled coordinately because of interactions between repressor dimers bound to these two sites. Site O(R)3 is filled only at higher repressor concentrations. In contrast, if O(R)1 is mutant, O(R)2 and O(R)3 are filled coordinately because of interactions between repressors bound to these sites. In this case, the affinity of O(R)3 is increased and that of O(R)2 is decreased relative to the wild type. We infer that a repressor dimer bound to the middle site O(R)2 can interact either with another repressor dimer bound to O(R)1 (wild-type case) or, alternatively, with one bound to O(R)3 (mutant O(R)1 case). We argue that these repressor interactions are mediated by protein-protein contacts between adjacent repressor dimers, because the isolated amino-terminal domains of repressor bind to the operator sites noncooperatively. The cro protein of phage lambda, a second regulatory protein, which recognizes the same three sites in O(R) as does repressor, binds non-cooperatively. Experiments performed in vivo show that regulation of gene expression by repressor can be influenced critically by cooperative interactions. We demonstrate that the effect of repressor in a lysogen on the activity of the promoter P(RM) can be changed from activation to repression by deletion of O(R)1. We explain this effect in terms of the alternative cooperative interactions described above.

Bacteriophage lambda↗
PubMed1979

Synthesis of simian virus 40 t antigen in Escherichia coli.

Plasmids are constructed by using recombination in vitro according to Roberts, T.M., Kacich, R. & Ptashne, M. (1979) Proc. Natl. Acad. Sci. USA 76, 760-764 in which the t antigen gene of simian virus 40 is fused to a promoter of the Escherichia coli lac operon. In the fusions, transcription commences at the lac promoter, and, in some of the fusions, translation begins at the ATG initiator codon of the t gene. This translation is directed most efficiently by those plasmids in which the lac sequences abut the t gene such that a hybrid ribosome binding is encoded. In this case, the Shine-Dalgarno sequence is of lac origin but the ATG derives from the t gene. translation from this initiator codon is greatly decreased if the lac sequences are separated from the ATG by 17 base pairs and is abolished if the AT of this triplet is deleted. Cells bearing the productive fusions synthesize a 20,000-dalton protein with t antigenic determinants. This protein has an isoelectric point(s) indistinguishable from that of t antigen isolated from simian virus 40-transformed cells. Moreover, a partial sequence of the amino-terminal region of the bacterial product is that predicted for authentic t antigen. We conclude that these bacteria are for authentic t antigen. We conclude that these bacteria are producing a protein, the sequence of which is identical to that of authentic t antigen unfused to other polypeptides.

Amino Acid Sequence↗
PubMed1979

A general method for maximizing the expression of a cloned gene.

We present a method, utilizing a combination of restriction endonuclease cleavage and digestion with Escherichia coli exonuclease III and Aspergillus orizae nuclease S1, that allows us to position a restriction fragment bearing the promoter of the lacZ gene of E. coli at virtually any distance in front of any cloned gene. In particular, we have used this method to examine the effect on protein production of gene-promoter separation for the cro gene of phage lambda and to produce plasmids that, upon transformation into appropriate E. coli hosts, direct the synthesis of up to 190,000 cro protein monomers per cell.

Base Sequence↗
PubMed1979

The lambda repressor contains two domains.

Papain digestion of the lambda phage repressor produces two fragments that are relatively resistant to further digestion. One includes the amino terminus (residues 1-92) and the other the carboxyl terminus (residues 132-236). Calorimetry shows that the amino-terminal fragment denatures near 50 degrees C and that the carboxyl-terminal fragment denatures near 70 degrees C. Intact repressor undergoes two denaturations, one near 50 degrees C and another near 70 degrees C. These and other data show that lambda repressor consists of two domains joined by a "connector" 40 amino acids long that is sensitive to proteases. The amino-terminal domain binds DNA, and the carboxyl-terminal domain oligomerizes.

Amino Acid Sequence↗
PubMed1978

Maximizing gene expression on a plasmid using recombination in vitro.

Recombination in vitro has been used to place one or more copies of a strong promoter, the lac promoter, at varying distances from the cl (repressor) gene of bacteriophage lambda on the E. coli plasmid pMB9. In all constructions, lambda repressor synthesis is driven wholly or predominantly by the inserted lac promoter. One of our fusions directs the synthesis of very high levels of lambda repressor. In this case, the fused DNA encodes a ribosome binding site which is a "hybrid" of lambda and lac sequences. In principle, this method of construction should elicit high levels of expression in E. coli of any gene, whatever its source. We also described strains with different sequence arrangements that, for reasons not completely understood, produce less repressor.

Binding Sites↗
PubMed1978

Mechanism of action of the cro protein of bacteriophage lambda.

The mechanism of action of cro protein was probed by measuring its ability to protect DNA against methylation by dimethyl sulfate and its effect on transcription in vitro. The cro protein binds to the same three sites in the right operator (OR) of bacteriophage lambda DNA as does the lambda repressor. Dimethyl sulfate protection experiments reveal major groove contacts for both proteins, and cro protein protects from methylation a subset of those purines protected by lambda repressor. These experiments also show that the relative affinity of these two proteins for the three operator sites is different: whereas lambda repressor binds with an affinity OR1 greater than OR2 greater than OR3, the order for cro protein is OR3 greater than (OR1, OR2). As predicted by these results, cro protein, like the lambda repressor, blocks in vitro transcription of cI and cro from the two divergent promoters that overlap OR. Also as predicted, transcription of cI is turned off at lower cro protein concentrations than is transcription of cro, whereas the opposite order of repression is obtained with lambda repressor. These results describe the molecular mechanism of cro protein action and show that two regulatory proteins can bind to the same three adjacent sites in DNA with markedly different consequences.

Base Sequence↗
PubMed1977

Sites of contact between lambda operators and lambda repressor.

DNA bearing lambda operator sequences was methylated by dimethyl sulfate (DMS) in the presence or absence of lambda repressor. Under the experimental conditions, DMS methylates only the purine residues. The presence of lambda repressor affects only the methylation of certain G residues in the operators. Repressor blocks the methylation of certain G's and enhances the methylation of other G's. Since the reactive ring-nitrogen of G lies in the major groove of double-stranded DNA, and the reactive ring-nitrogen of A lies in the minor groove, the above results imply that the repressor makes contacts in the major groove of the helix. The repressor effect on G-methylation is sharply confined to the three 17 base pair units within each lambda operator previously proposed as the repressor-binding sites.

Base Sequence↗
PubMed1976

Construction of plasmids carrying the cI gene of bacteriophage lambda.

By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.

Coliphages↗
PubMed1976

Novel properties of a restriction endonuclease isolated from Haemophilus parahaemolyticus.

The sequences in lambda DNA in and around six sites cut by Hph, a restriction enzyme isolated from Haemophilus parahaemolyticus, are compared. The enzyme produces a staggered cut around an AT or TA base pair, but the sequences immediately surroinding the cleavage sites bear no obvious relation to one another. Eight (in some cases nine) base pairs to one side of each cleavage site is the common sequence TCACC AGTGG. Two lines of evidence indicate that these bases constitute part or all of the Hph recognition site. First, mutations in this sequence prevent Hph cutting. Second, dimethylsulfate-mediated methylation of Gs and As in this site prevent cutting, whereas methylation of purines in the region between this sequence and the cleavage sites has no such effect. There is discernible 2-fold rotational symmetry neither in the common sequence nor around the cleavage sites.

Base Sequence↗