PubMed Health⌕ Search

Biomedical subjects

Dominique M Galli

Publications and source records attributed to Dominique M Galli.

2 recordsLinked to original sources
PubMed2002

Parameters associated with cloning in Actinobacillus actinomycetemcomitans.

Characterization of virulence traits in Actinobacillus actinomycetemcomitans requires the application of recombinant DNA techniques. To develop appropriate genetic tools it is necessary to identify suitable host-vector systems. The current study assessed cloning parameters in A. actinomycetemcomitans for two previously described vectors, pDMG4 and pMMB67. It was determined that the maximum size of recombinant molecules that could be transferred to A. actinomycetemcomitans strain ATCC29522 via electroporation was 33 kb. The size limit for transformation of the same strain with ligation mixtures (direct cloning), however, was limited to 23-24 kb. Additional experiments included electroporation of various A. actinomycetemcomitans strains with plasmid DNA isolated from Escherichia coli and different A. actinomycetemcomitans sources. Differences in transformation efficiencies suggested the presence of a restriction modification system for pDMG4 in some strains of A. actinomycetemcomitans. Cloning of portions of the enterococcal plasmid pJH1 into A. actinomycetemcomitans resulted in the insertion of the intact vector into the chromosome.

Aggregatibacter actinomycetemcomitans↗
PubMed2002

DNA inversion on conjugative plasmid pVT745.

Plasmid pVT745 from Actinobacillus actinomycetemcomitans strain VT745 can be transferred to other A. actinomycetemcomitans strains at a frequency of 10(-6). Screening of transconjugants revealed that the DNA of pDMG21A, a pVT745 derivative containing a kanamycin resistance gene, displayed a structural rearrangement after transfer. A 9-kb segment on the plasmid had switched orientation. The inversion was independent of RecA and required the activity of the pVT745-encoded site-specific recombinase. This recombinase, termed Inv, was highly homologous to invertases of the Din family. Two recombination sites of 22 bp, which are arranged in opposite orientation and which function as DNA crossover sequences, were identified on pVT745. One of the sites was located adjacent to the 5' end of the invertase gene, inv. Inversion of the 9-kb segment on pVT745 derivatives has been observed in all A. actinomycetemcomitans strains tested except for the original host, VT745. This would suggest that a host factor that is either inactive or absent in VT745 is required for efficient recombination. Inactivation of the invertase in the donor strain resulted in a 1,000-fold increase in the number of transconjugants upon plasmid transfer. It is proposed that an activated invertase causes the immediate loss of the plasmid in most recipient cells after mating. No biological role has been associated with the invertase as of yet.

Aggregatibacter actinomycetemcomitans↗