Mutagenic DNA repair in escherichia coli. V. Mutation frequency decline and error-free post-replication repair in an excision-proficient strain.

Mutation frequency decline (MFD) is an irreversible loss of newly-induced suppressor mutations occurring in excision-proficient Escherichia coli during a short period of incubation in minimal medium before plating on broth- or Casamino acids-enriched selective agar. It is known that MFD of UV-induced mutations may occur before DNA containing pre-mutagenic lesions is replicated, but we conclude that MFD can also occur after the damaged DNA has been replicated on the basis of the following evidence. (1) Mutation fixation in rich medium (i.

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.

Short term screening tests for carcinogens.

There are now short term tests with a high predictive value for mammalian carcinogens. Many of them are based on the ability to detect damage to DNA in bacteria or mammalian cells after metabolic activation by microsomal enzymes. Their introduction will enable provisional safety assessments to be made for the many thousands of industrial and environmental chemicals for which long-term animal testing cannot at present be considered.

Mutagenic DNA repair in Escherichia coli. III. Requirement for a function of DNA polymerase III in ultraviolet-light mutagenesis.

The polC (= dnaE) temperature-sensitive DNA polymerase III mutation from Escherichia coli BT1026 has been transduced into E. coli WP2 (to give CM731) and WP2 uvr A (to give CM741). In excision-deficient CM741 UV-induced Trp+ mutations progressively lost their photoreversibility during post-irradiation incubation at 34 degrees.

Use of a simplified fluctuation test to detect low levels of mutagens.

As a mutagen screening procedure we have used a modification of the Luria and Delbrück fluctuation test in which the individual tubes are scored by eye for the presence or absence of a mutation. The test is simple and extremely sensitive, detecting concentrations of mutagens up to 100-fold lower than conventional tests. Measuring mutation to tryptophan independence in Escherichia coli strain WP2 we have found that methyl methanesulphonate (0.

Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity.

1. Isolated fat-cells and intact epididymal fat-pads were incubated in medium containing 45Ca2+ and the incorporation of 45Ca into mitochondrial and extramitochondrial fractions was studied. Redistribution of 45Ca between these fractions was essentially prevented by the addition of EGTA [ethanedioxybis(ethylamine)tetra-acetate] and Ruthenium Red to the sucrose-based extraction medium.

Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet.

1. Pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads was measured by using pig heart pyruvate dehydrogenase [32P]phosphate. About 80% was found to be extramitochondrial and therefore probably not directly concerned with the regulation of pyruvate dehydrogenase activity.

Mutagenic DNA repair in Escherichia coli: conditions for error-free filling of daughter strand gaps.

Two situations have been observed in which daughter strand gaps in DNA synthesized after exposure of excision-deficient Escherichia coli to ultraviolet light are filled but in which no mutations are formed as judged by loss of photoreversibility: (i) during the first 20 min of growth after u.v. irradiation, and (ii) when repair is allowed to occur in buffer.

Hypothetical dose-response curves for chronic exposures to mutagens or carcinogens subject to simple enzymatic detoxification in the mammalian body.

Dose-response curves for chemical carcinogenesis and mutagenesis in the whole mammal may be influenced (a) by processes affecting the delivery of the applied dose to the DNA of the target cell or (b) by processes affecting the response of the cell to the initial DNA damage. As an example of the former, the effect of a simple enzymatic detoxification system on the delivery to the target organ of an applied chronic dose has been considered. In certain circumstances as the applied dose is lowered, the delivered dose may become proportionately less than at high doses so that a new linear relation of lower slope is established.

Regulation of mammalian pyruvate dehydrogenase.

In mammalian tissues, two types of regulation of the pyruvate dehydrogenase complex have been described: end product inhibition by acetyl CoA and NADH: and the interconversion of an inactive phosphorylated form and an active nonphosphorylated form by an ATP requiring kinase and a specific phosphatase. This article is largely concerned with the latter type of regulation of the complex in adipose tissue by insulin (and other hormones) and in heart muscle by lipid fuels. Effectors of the two interconverting enzymes include pyruvate and ADP which inhibit the kinase, acetoin which activates the kinase and Ca2+ and Mg2+ which both activate the phosphatase and inhibit the kinase.

Mutagenic DNA repair in Escherichia coli. II. Factors affecting loss of photoreversibility of UV induced mutations.

The photoreversibility of UV-induced mutations to Trp+ in strain Escherichia coli WP2 uvrA trp (unable to excise pyrimidine dimers) was lost at different rates during incubation in different media. In Casamino acids medium after a short initial lag, photoreversibility was lost over about one generation time; in minimal medium with tryptophan, photoreversibility persisted for more than two generations; in Casamino acids medium with pantoyl lactone photoreversibility was lost extremely slowly. The rate of loss of photoreversibility was unaffected by UV dose in either Casamino acids medium or in minimal medium.

Effect of photoreactivation on the filling of gaps in deoxyribonucleic acid synthesized after exposure of Escherichia coli to ultraviolet light.

Daughter-strand gaps in deoxyribonucleic acid (DNA) synthesized after exposure of excision-deficient Escherichia coli to ultraviolet light are filled during subsequent incubation in buffer, and the rate of filling is increased when the incubation in buffer is carried out in the presence of 360-nm light. It is concluded that daughter-strand discontinuities are prevented from being rapidly sealed in the dark not because of some structural feature of the daughter-strand but because of the presence of a pyrimidine dimer on the opposite (parental) strand. "Photoreactivation-stimulated gap filling" is dependent on the polA(+) and recA(+) but not the exrA(+) genes.