Inhibition of repressor formation in the lactose system of Escherichia coli by inhibitors of protein synthesis
TL;DR: The re-establishment of repression after heat treatment was almost completely inhibited by chloramphenicol, puromycin or methionine starvation and to some extent by 5-methyltryptophan, leading to the conclusion that protein synthesis is required for the establishment of repression.
About: This article is published in Journal of Molecular Biology.The article was published on 1966-10-01. It has received 18 citations till now. The article focuses on the topics: Repressor & Puromycin.
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TL;DR: This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea, which results in the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6).
Abstract: A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.
214 citations
TL;DR: Thirteen Escherichia coli clones containing the whole or a part of the fibroin gene (16 kilobases long) have been isolated and the cleavage sites for endodeoxyribonucleases EcoRI, HindIII, and BamHI have been established for the cloned sequences.
Abstract: Thirteen Escherichia coli clones containing the whole or a part of the fibroin gene (16 kilobases long) have been isolated. The starting material was DNA extracted from the posterior silk glands of Bombyx mori. Most clones were obtained from sheared DNA fragments linked by poly(dA)-poly(dT) joints to the plasmid pMB9. One of them includes the 5' end of the fibroin gene with a flanking sequence of 12 kilobases, and another includes the 3' end of the gene with a flanking sequence of about 1 kilobase. One clone was obtained by ligation, to pMB9, of a fragment generated by endodeoxy-ribonuclease EcoRI. This clone has a 21-kilobase insertion that probably includes the entire fibroin gene with flanking sequences at both ends. The cleavage sites for endodeoxyribonucleases EcoRI, HindIII, and BamHI have been established for the cloned sequences.
74 citations
49 citations
TL;DR: This chapter reviews the variety of possible mechanisms for the relaxed control phenomenon, without expressing the prejudices toward any of them, and discusses the concept of relaxed control to the synthesis of DNA as well.
Abstract: Publisher Summary This chapter reviews the variety of possible mechanisms for the relaxed control phenomenon, without expressing the prejudices toward any of them It reviews that a single genetic locus in control of RNA synthesis as the “RC” locus and considered its effectiveness to be “relaxed” in all derivatives of the original mutant regardless of the amino acid deficiency imposed While all species of RNA accumulated in RC mutants, excess DNA synthesis seemed to be relatively minor during an absolute amino acid starvation It discusses the concept of relaxed control to the synthesis of DNA as well Since a significant synthesis of this macromolecule is observed only when a gradual depletion of the amino acid supply occurs, it is our view that its magnitude reflects the adequacy of the amino acid starvation rather than a genetically controlled relaxation Synthesis of DNA to the completion of its replication cycle in amino acid-starved strains that exhibit stringent control over RNA synthesis is also discussed New insights and new skills are needed in this field and the solicitation of these is the goal of this chapter It is hoped that amino acid auxotrophs of mammalian cells have become a reality, the question of the possible existence of a similar control phenomenon for RNA synthesis in mammalian cells may also be open to exploration
44 citations
TL;DR: The results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon.
Abstract: Mutants of Escherichia coli exhibiting temperature-sensitive repression of the tryptophan operon have been isolated among the revertants of a tryptophan auxotroph, trpS5, that produces an altered tryptophanyl transfer ribonucleic acid (tRNA) synthetase. Unlike the parental strain, these mutants grew in the absence of tryptophan at high but not at low temperature. When grown at 43.5 C with excess tryptophan (repression conditions), they produced 10 times more anthranilate synthetase than when grown at 36 C or lower temperatures. Similar, though less striking, temperature-sensitivity was observed with respect to the formation of tryptophan synthetase. Transduction mapping by phage P1 revealed that these mutants carry a mutation cotransducible with thr at 60 to 80%, in addition to trpS5, and that the former mutation is primarily responsible for the temperature-sensitive repression. These results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon. In agreement with this conclusion, tRNA of several trpR mutants was found to be normal with respect to its tryptophan acceptability. It was also shown that the trpS5 allele, whether present in trpR or trpR(+) strains, produced appreciably higher amounts of anthranilate synthetase than the corresponding trpS(+) strains under repression conditions. This was particularly true at higher temperatures. These results provide further evidence for our previous conclusion that tryptophanyl-tRNA synthetase is somehow involved in repression of this operon.
28 citations
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Journal Article•
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: The synthesis of enzymes in bacteria follows a double genetic control, which appears to operate directly at the level of the synthesis by the gene of a shortlived intermediate, or messenger, which becomes associated with the ribosomes where protein synthesis takes place.
5,588 citations
TL;DR: Results of cis-trans tests show that the constitutive alleles of the two genes are recessive in both cis and trans positions relative to the structural gene, which suggests that each regulator gene plays an essential role in the formation of a repressor for alkaline phosphatase.
306 citations
TL;DR: During the induction phase a messenger RNA specific for β-galactosidase is produced which directs the subsequent synthesis of the enzyme in the inducer-free medium, which largely prevents the subsequent production of normal enzyme and causes the formation of an altered protein serologically related to β-Galactosids.
260 citations
TL;DR: By experiments with mutants having temperature-sensitive repression systems for the Lac operon, it has been possible to provide evidence indicating that the i -gene product is the repressor, or physically included in it.
237 citations