Biomedical subjects
Publications and source records attributed to C Hamelin.
A simplified restriction endonuclease analysis procedure is described which allows the characterization of baculovirus DNA obtained directly from a single larvae without purification of virus. This rapid method was used to demonstrate the genomic stability of nuclear polyhedrosis viruses (NPVs) from Agrotis segetum, Euxoa messoria and Mamestra brassicae after several passages in Euxoa scandens.
Alkaline sucrose gradient sedimentation indicates that ozone can produce DNA single- and double-strand breaks in wild-type E. coli and ozone-sensitive mutant MQ1844(ozrB). Another type of DNA damage repaired only by the ozrB gene product may also be responsible for the killing effect of ozone on E. coli cells.
Restriction endonuclease analysis was used to compare the genome of mouse adenovirus (MAd) strains FL and K87. Large differences were found between the Kpn I, PaeR7, Pvu I, Sal I and Sma I restriction profiles of the prototype strains. MAd-FL and MAd-K87 thus represent two distinct species of mouse adenovirus.
Restriction endonuclease cleavage site analysis was used to differentiate between mouse adenovirus (MAV) types 1 and 2 strains. Viral DNA of suitable purity and quantity for multiple enzymatic digestions was obtained from cloned CMT-93 mouse tumor cells infected with each type of MAV. Clear differences between the MAV-1 (FL) and MAV-2 (K87) genomes were observed after cleavage with restriction enzymes such as BglII, EcoRI, and PaeR7. Fast electrophoresis of DNA fragments in miniature agarose slab gels allowed rapid and unequivocal identification of the MAV strains. This relatively simple and accurate method should be quite useful to determine the different modes of transmission of mouse adenoviruses and their presence in various animal populations.
The survival of u.v.-irradiated human cytomegalovirus (HCMV) on u.v.-irradiated human IAFP-1 cells was increased over that on unirradiated cells. Irradiated virus had a higher forward mutation frequency towards temperature sensitivity in irradiated than in unirradiated cells. Enhanced reactivation of u.v.-irradiated HCMV is thus mutagenic in normal human cells. This observation supports the possible induction of an error-prone mode of DNA repair in u.v.-irradiated mammalian cells.
Enhanced survival of UV-irradiated human cytomegalovirus (HCMV) is demonstrated in normal human cells exposed to UV light prior to infection. The UV fluence that gave rise to maximum UV reactivation falls in the range of 15 J/m2. A large number of temperature-sensitive HCMV mutants were found under the peak of reactivation. These results confirm the existence of inducible SOS functions in human cells.
Thirteen temperature-sensitive (ts) mutants of human cytomegalovirus (HCMV) have been isolated after mutagenesis with nitrosoguanidine or ultraviolet light. Four mutants defective in viral DNA synthesis at nonpermissive temperature (DNA-) were classified into two separate complementation groups. The other nine HCMV ts mutants, capable of synthesizing viral DNA at the elevated temperature (DNA+), belonged to seven independent complementation groups. The number of essential genes encoded by HCMV is thus raised here by nine.
Cloned HindIII fragments of the type 1 and type 2 canine adenovirus (canAd) genomes were mapped with respect to the BamHI, EcoRI, HincII, PaeR7 and PstI restriction endonuclease cleavage sites. Considering the large differences found between the canAd-1 and canAd-2 DNA molecules, in terms of number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the canAd-1 and canAd-2 physical maps. Both viruses are genetically related to the extent of 57%. Our results confirm the existence of two distinct Ad species in the dog.
Insertion of foreign DNA into the unique HindIII site of the high copy number plasmid pAT153 reduces but does not completely abolish the resistance of Escherichia coli HB101 cells to tetracycline. Recombinant DNA-containing colonies could then be phenotypically differentiated from non-recombinant ones by their smaller size on nutrient agar plates with ampicillin and tetracycline at a final concentration of 50 and 4 micrograms/ml, respectively. A wide variety of human cytomegalovirus DNA fragments have been found in pAT153 molecules propagated by the ampicillin-resistant tetracycline-sensitive bacteria selected.
Dog embryo kidney cells were efficiently transformed by human cytomegalovirus (HCMV) particles or intact viral DNA. Negative results were obtained after transfection of the canine cells with recombinant plasmids carrying the HCMV Hind III-E subgenomic fragment or with limit Bgl II and Hind III digests of the viral genome. Immortalized dog cells with typical transformation properties appeared, however, after transfection with EcoR I fragments of the HCMV DNA. Distinct regions of the viral genome are probably responsible for the immortalization and the tumorigenicity of mammalian cells.
The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (+/- 0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV-2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies.
The molecular weights of canine adenovirus type 1 (canAV-1, strain Utrecht) and type 2 (canAV-2, strain Toronto A26/61) DNAs were determined by contour length measurements and restriction endonuclease analysis. In each case, an average molecular weight value of 20 (+/- 0.5) X 10(6) daltons was obtained with both methods. Similar in size, the canAV-1 and canAV-2 DNA molecules were, however, cleaved at very distinct positions by a variety of restriction endonucleases. The observed lack of DNA sequence homology confirms the existence of at least two different canine adenovirus species [Intervirology 23: 23-28 (1985)].
Two canine adenovirus (CAV) isolates, apparently distinct from type-1 (Utrecht) and type-2 (Toronto A26/61) reference strains in their biochemical and/or immunologic properties, were submitted to DNA-restriction endonuclease analysis. Both isolates, designated IAF-81-2116 and IAF-75-95, appeared as genotypic variants of CAV-2. Isolate IAF-81-2116 was recovered from the intestine of a young pup with diarrheal disease. Seemingly, relatively small changes in the original CAV-2 DNA sequence allowed the virus to replicate at an unusual site in the dog.
Agarose gel electrophoresis and electron microscopy were used to determine the type of lesions produced in DNA by ozone. This strong oxidizing agent was found to relax, linearize, then degrade native plasmid (pAT153) DNA molecules in solution. Ozone, like ionizing radiation, thus produced DNA breakage. To ascertain this point, wild-type and radiosensitive strains of Escherichia coli were transfected with control or ozonated plasmid DNA, and the host cells were selected for antibiotic resistance. A significant reduction in the transforming ability of pAT153 was observed following ozonation. Mutants deficient in the repair of DNA single-strand breaks yielded less ampicillin- or tetracycline-resistant clones than repair-proficient strains. In E. coli, the same gene products are probably involved in the repair of both radiation- and ozone-induced DNA breaks.
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Identification of canine adenovirus-1 ( CAV -1) and canine adenovirus-2 ( CAV -2) strains was done by electrophoresis of restriction endonuclease-fragmented viral DNA. Results obtained with this sensitive and reproducible technique clearly show that CAV -2 is not a variant of CAV -1 but a distinct virus.
Chromatin-associated DNA topoisomerase I activity was measured in human diploid fibroblasts during in vitro aging. No difference was detected as a function of cell age in the nicking and the closing activities of the DNA-unwinding enzyme. The capacity of type-I topoisomerase to relax superhelical DNA molecules was, however, increased in aged cells. An age-related increase in nucleoprotein content was also observed.
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