Biomedical subjects

C Bernelot-Moens

Publications and source records attributed to C Bernelot-Moens.

5 recordsLinked to original sources

PubMed1993

Genome manipulation in embryonic stem cells.

Embryonic stem (ES) cells derived from pluripotent cells of the early mouse embryo provide a powerful tool for genome manipulation in mammals. Dominantly acting effects can be achieved by introducing constructs to misexpress or ectopically express a gene product, express an altered product or express antisense constructs. Use of ES cell chimeras to analyse the effects of such alterations may provide information not readily available from transgenic mice. However, the most important use of ES cells, to date, is in the generation of recessive mutations, either in known genes by targeted mutagenesis or randomly by insertional mutagenesis. Examples of these approaches and possible future strategies are discussed.

Animals↗

PubMed1993

Spontaneous mutagenesis in Escherichia coli harbouring plasmid pKM101: DNA sequence analysis of forward lacI- mutations.

To investigate the influence of plasmid pKM101 on spontaneous mutagenesis, 198 lacI- mutations generated in Escherichia coli harbouring pKM101 were characterized at the DNA sequence level. pKM101 by itself did not enhance the lacI- forward mutation frequency. In general, the resultant distribution of mutation highlights particular sequences at which a variety of mutational events repeatedly occur, including 5'-(G/C)TGG-3', 5'-CCAGG-3', 5'-GATC-3' and 5'-TCGCG-3' sequences. Specifically, the distribution of mutation within base substitution and deletion classes distinguishes the pKM101 spectrum from the wild-type distribution (i.e. absence of pKM101). An even distribution amongst base substitutions was observed which corresponds to a 2.9- to 6.3-fold increase in occurrence of low frequency events (A-->G,T,C; G-->C); high frequency events in the wild-type distribution (G-->A,T) were not influenced by the presence of pKM101. One complex event was recovered which was comprised of two base substitutions separated by 4 bp. An 11-fold increase in small deletion events (3-6 bp) was also observed. The observed pKM101 spectrum does not closely resemble the mutational consequences of SOS induction in the absence of mutagenic treatment (recA441 spectrum) but does return a distribution like that obtained in E. coli deficient in polymerase I activity (polA1 spectrum).

Amino Acid Sequence↗

PubMed1990

Induction of specific frameshift and base substitution events by benzo[a]pyrene diol epoxide in excision-repair-deficient Escherichia coli.

We have determined the DNA alterations recovered after treatment with (+-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+-)-anti-BPDE] in the lacI gene of excision-repair-deficient (Uvr-) Escherichia coli. The high induction of -(G:C) frameshifts, G:C----T:A and A:T----T:A transversions, and the presence of complex mutations of a particular motif, distinguish the mutational distribution recovered in the Uvr- strain, although other mutational classes, including -(A:T) frameshifts and duplications, were also moderately induced. The great majority of -(G:C) frameshifts, the predominant mutation recovered, occurred in runs of G residues. The G:C----T:A transversion was found to occur more frequently (10/12 occurrences) at 5'-Y-G-3' sites, sequences at which the labile BPDE:N7G adduct has been predicted to occur. Moreover, the relative proportions of G:C----T:A, A:T----T:A and G:C----A:T mutations correlate well with the expected proportions of alkali-labile lesions at G, A and C residues. These results support the model that (+-)-anti-BPDE-induced base substitution mutagenesis in E.coli proceeds through abasic intermediates across from which adenine is preferentially incorporated during replication.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1989

Multiple DNA repair activities for 3'-deoxyribose fragments in Escherichia coli.

Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolaldehyde, PGA) from the 3' termini of a synthetic DNA substrate. The major such activities are the main bacterial apurinic endonucleases, exonuclease III and endonuclease IV. In a double mutant deficient in both of these oxidation repair enzymes, Mg++-dependent 3'-PGA diesterase was detected at 3% the level found in wild-type bacteria. Gel filtration fractionated this residual diesterase activity into two peaks of Mr 40,000-52,000 (Pool A) and Mr 22,000-30,000 (Pool B) with differing abilities to remove 3'-phosphates from DNA. These multiple repair activities were resolved in 3'-PGA diesterase activity gels. The exonuclease III and endonuclease IV bands were identified using the purified proteins and by their specific absence from strains defective for the respective structural genes. Gel filtration Pool B yielded two activity bands of apparent Mr 25,000 and 28,000, but Pool A did not form a new band in the activity gels. Incubation of activity gels in different transition metals or boiling of the samples before electrophoresis also served to distinguish the various activities. The possible identities of the novel E. coli 3'-PGA diesterases and the importance of multiple repair enzymes for 3' damages are discussed.

Chromatography, Gel↗

PubMed1988

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein.

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Amino Acid Sequence↗