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Biomedical subjects

C Cazaux

Publications and source records attributed to C Cazaux.

29 records · Page 2Linked to original sources
PubMed1994

Purification and biochemical characterization of Escherichia coli RecA proteins mutated in the putative DNA binding site.

Escherichia coli RecA protein plays a central role both in DNA repair and in recombination. We report biochemical properties of three new RecA proteins mutated at positions 199 (RecA694), 207 (RecA659), and 211 (RecA611) in the putative DNA binding site. RecA694 had a wild-type phenotype, whereas RecA611 and RecA659 were deficient in promoting both the self-cleavage of LexA repressor and the DNA-strand exchange reaction. In order to determine the origin of this inhibition, we examined the capacity of wild-type and mutant proteins to bind to single-stranded DNA (with and without single-stranded binding protein, SSB), double-stranded DNA, and ATP. DNA strand exchange defects were correlated with the inability of mutant proteins to displace SSB from DNA. For the recA659 mutation this inhibition was reversed by equimolar wild-type protein. In contrast, mixtures of either wild-type/RecA659 or wild-type/RecA611 proteins remained deficient in LexA cleavage, suggesting that the dominant negative phenotype of the mutant proteins may be a consequence of the formation heterologous RecA complexes. Various mutations in the putative DNA binding site of RecA protein altered ATP binding, ATPase activity, displacement of SSB from single-stranded DNA, and protein-protein interactions. These results are consistent with the hypothesis that DNA binding to this site of RecA relays allosteric effects to several functional domains throughout the protein.

Adenosine Triphosphatases↗
PubMed1994

Increased proliferative activity, loss of beta-adrenergic receptor function and class I major histocompatibility complex antigen surface expression in a modified lymphoma cell line.

The molecular interaction of class I major histocompatibility complex (MHC) antigens (Ag) and of beta-adrenergic receptors was previously demonstrated on lymphocytes. By long-term culturing with high concentration of foetal calf serum, the murine S49 lymphoma cell line was modified (S49m) giving phenotypic alterations in beta-adrenergic receptors and class I Ag expression. S49m cells displayed a reduced number of beta-adrenergic sites that were uncoupled to the adenylate cyclase system. These were unable to respond to beta agonist stimulation, despite the fact that direct activation of Gs could be achieved with aluminium tetrafluoride. Although S49m cells showed normal expression of the thy 1.2 Ag, they displayed no expression of class I Ag of the d haplotype. This was assessed by the evident lack of cytotoxic activity of specific monoclonal antibodies (Mo Ab) and of their binding. When performing IFI staining on permeabilized cells, we found positive staining with anti-class d Ab inside the cell. This loss of expression and activity of beta-adrenoceptors and the internalization of class I Ag were accompanied by a higher rate of proliferation in S49m cells. The possibility that the loss of both molecules would modify the biology of the cell is also discussed.

Adrenergic beta-Antagonists↗
PubMed1993

Effects of a single intrastrand d(GpG) platinum adduct on the strand separating activity of the Escherichia coli proteins RecB and RecA.

RecB and RecA proteins play key roles in the process of DNA recombination in Escherichia coli and both possess DNA unwinding activities which can displace short regions of duplex DNA in an ATP-dependent manner in vitro. We have examined the effect of the most abundant DNA adduct caused by the chemotherapeutic agent cis-diamminedichloroplatinum(II) on those activities. For this purpose, we have constructed a partially duplex synthetic oligonucleotide containing the intrastrand d(GpG) crosslink positioned at a specific site. We report here that both the DNA strand separating and DNA-dependent ATPase activities of the RecB protein are inhibited by the d(GpG) cis-DDP adduct. In contrast, neither the unwinding nor the ATPase activities of RecA protein appear to be perturbed by this lesion.

Base Sequence↗
PubMed1993

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele.

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264-->Glu) and recA665 (Tyr264-->His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.

Alleles↗
PubMed1992

Genetical and biochemical evidence for the involvement of the coprotease domain of Escherichia coli RecA protein in recombination.

RecA amino acid residue 204 is involved in the coprotease domain of the protein responsible for the induction of mutagenic repair. Two mutations were created at this site leading to the addition of either a methyl or an isopropyl group on the original glycine. Analyses of both the in vivo and the in vitro properties of these mutated proteins demonstrated that this residue 204 is involved in many RecA activities, suggesting that this site could allosterically direct conformational changes in the protein or could be situated in a region interacting with many RecA cofactors.

Adenosine Triphosphate↗
PubMed1992

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity.

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.

Alleles↗
PubMed1991

Protein A insensitive ELISA detection of staphylococcal enterotoxin B.

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin B was developed using rat monoclonal antibodies as capture antibodies and as a biotinylated conjugate. This test was sensitive, less than 1 ng/ml of enterotoxin B was detected and interference by protein A was prevented by the use of rat monoclonal antibodies of the IgG2a isotype which were insensitive to protein A even at concentrations greater than 1000 ng/ml.

Animals↗
PubMedDate not supplied

Site-directed mutagenesis in the Escherichia coli recA gene.

Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response. We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site. We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions. Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP. In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its involvement in an ATP binding site, as previously shown in vitro.

Bacterial Proteins↗
PubMedDate not supplied

The Mars Sample Return Project.

The Mars Sample Return (MSR) Project is underway. A 2003 mission to be launched on a Delta III Class vehicle and a 2005 mission launched on an Ariane 5 will culminate in carefully selected Mars samples arriving on Earth in 2008. NASA is the lead agency and will provide the Mars landed elements, namely, landers, rovers, and Mars ascent vehicles (MAVs). The French Space Agency CNES is the largest international partner and will provide for the joint NASA/CNES 2005 Mission the Ariane 5 launch and the Earth Return Mars Orbiter that will capture the sample canisters from the Mars parking orbits the MAVs place them in. The sample canisters will be returned to Earth aboard the CNES Orbiter in the Earth Entry Vehicles provided by NASA. Other national space agencies are also expected to participate in substantial roles. Italy is planning to provide a drill that will operate from the Landers to provide subsurface samples. Other experiments in addition to the MSR payload will also be carried on the Landers. This paper will present the current status of the design of the MSR missions and flight articles.

Equipment Design↗
PubMedDate not supplied

Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes.

Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain. In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity. When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR). The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK. TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.

Animals↗