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M R Chevallier

Publications and source records attributed to M R Chevallier.

At least 37 records · Page 2Linked to original sources
PubMed1980

Transcriptional and translational expression of a chimeric bacterial-yeast plasmid in yeasts.

Chimeric plasmids composed of the bacterial plasmid pBR322, 2 micron yeast plasmid fragments and the 1.1 kb ura3+ fragment of yeast chromosomal DNA which codes for orotidine-5'-phosphate (OMP) decarboxylase were constructed and used to transform Escherichia coli and Saccharomyces cerevisiae recipient cells. The expression in yeast of one such plasmid was studied and compared to the expression of a chromosomally integrated bacterial plasmid. In the strain carrying the chimeric plasmid the level of OMP decarboxylase activity is about 25 times that found in either the wild-type strain or in the strain carrying the chromosomally integrated plasmid. The ampicillin gene of pBR322 is expressed in yeast. Labeling kinetics of RNA and measurements of the polyadenylated fractions showed that RNA hybridizing to the pBR322 plasmid was polyadenylated to the same extent as RNA hybridizing to the ura3+ gene. Half-lives of 10 and 20 min were estimated for the ura3+ and pBR322 transcripts respectively.

Chimera↗
PubMed1979

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis.

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts. Two-dimensional patterns of ghost proteins were not heavily contaminated (less than or equal to 25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8--10 fold over whole cells. Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.

Cell Membrane↗
PubMed1978

Properties of three distinct pyrimide transport systems in yeast. Evidence for distinct energy coupling.

In Saccharomyces cerevisiae the uptake of cytosine, uracil and uridine is mediated by three permeases. Using mutants blocked in the metabolic utilization of these three compounds we were able to study their specific uptake. Cytosine and uridine show simple saturation kinetics, whereas uracil uptake is a biphasic process. A comparison of the effects of several inhibitors of energy metabolism on these uptake systems was made. Striking differences were found. 2,4-Dinitrophenol (10(-3) M) and NaN3 (10(-2) M) inhibit the entry of the three compounds to similar extent, but chlorhexidine (10(-5) M) and Dio 9 (50 microgram/ml) which are ATPase inhibitors in vitro strongly impaired cytosine and uridine entry and remained without effect on uracil uptake. We provisionally conclude that these systems may be energized by different mechanisms. In the case of cytosine and uridine permease, a membrane ATPase is possibly involved in the process of energetic coupling whereas this does not seem to be so for uracil.

Adenosine Triphosphatases↗
PubMed1977

Ureidosuccinic acid permeation in Saccharomyces cerevisiae.

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20-25 mumol/ml cell water per min. The apparent Km is 3-10(-5) M. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one. In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyldrazone (CCCP) or NaN3 inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs. The specificity of the permease is very strict, only amino acids carrying an alpha-N-carbamyl group are strongly competitive inhibitors. The high concentration capacity of the cells and lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.

Amino Acids↗
PubMed1975

Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease.

In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.

Adenosine Triphosphate↗
PubMed1975

Characterization of cytosine permeation in Saccharomyces cerevisiae.

Cytosine permeation in Saccharomyces cerevisiae has been studied. Cytosine uptake is mediated by a permease which is also responsible for purines transport. The Km for the transport of various substrates of this permease have been determined. By means of appropriate selective techniques, mutants with altered Km and mutants lacking the permease have been selected. Cytosine transport is active and is inhibited by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, and by N-ethylmaleimide, a reagent of--SH group. Internal labeled cytosine is chased by addition of unlabeled cytosine in the medium. These results support the hypothesis of a carrier-mediated transport, with reduced internal affinity, allowing the release and accumulation of cytosine in the inner compartment. The efflux of cytosine from cytosine permease-less cells has also been studied and shows first order kinetics. A diffusion coefficient of 5.7 per 10- minus 8 cm per S- minus 1 has been evaluated for this efflux.

Adenine↗
PubMed1973

UV sensitivity of several genetic markers of Haemophilus influenzae DNA.

Studies on inactivation by UV of several genetic markers of the streptomycin and novobiocin resistance regions of H. influenzae DNA have shown that two kinds of markers may be found with respect to frequency of integration, UV sensitivity and reactivability. One class of markers has high integration frequency and low UV sensitivity, which can be partly explained by the reactivability of the irradiated markers. The second class of markers has low integration frequency and high UV sensitivity. Markers of each class have been found in the novobiocin region.-In one recipient strain with decreased transformability the difference in integration frequency between the two classes is enhanced.

Acridines↗