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P Chartrand

Publications and source records attributed to P Chartrand.

At least 55 records · Page 3Linked to original sources
PubMed1991

Reciprocal homologous junctions generated in mouse cells.

We analysed pairs of reciprocal homologous junctions resulting from intermolecular conservative homologous recombination in mouse cells. The assay used did not rely on the reconstitution of a selectable gene. This permitted the introduction of multiple markers in the parental homologous sequences which in turn enabled us to compare the contribution of each parent to the reciprocal products of a given recombination event. In all recombinants analysed we found, when comparing the reciprocal junctions, a middle segment originating from only one parent. This segment of uniparental origin occurred randomly throughout the region of homology and could extend over a thousand base pairs. These results are consistent with a gap repair process like the one proposed for homologous recombination in yeast. However, introducing a double-strand break in the region of homology did not enhance but rather decreased the proportion of recombinants with reciprocal homologous junctions relative to other types of recombinants.

Animals↗
PubMed1991

The immune response to the HPA-1a antigen: association with HLA-DRw52a.

Antibodies to the HPA-1a antigen can elicit a condition in the new-born known as neonatal alloimmune thrombocytopenia (NAITP). Retrospective and prospective studies have shown that there is a strong correlation between the presence of HLA-DR3, HLA-DRw52 in the mother and the antibody response to HPA-1a. HLA Class II molecules play an important role in the initiation of the immune response and it has been postulated that HPA-1a antibody production could be determined by the presence of a specific HLA Class II molecule at the surface of the antigen-presenting cell. Thirty-one HPA-1a negative women with HPA-1a antibodies (responders) and nine HPA-1a negative women without HPA-1a antibodies (non-responders) were recruited. They were studied using serological HLA Class I and Class II typing and RFLP analysis with a DR beta probe. We found that all responders had the HLA-DRw52a sub-specificity confirming recently published data. Moreover, two of the nine non-responders were also found to be HLA-DRw52a. These results suggest that the HLA-DRw52a molecule is necessary for HPA-1a antibody responsiveness but not sufficient. The results also indicate that in HPA-1a negative women the absence of HLA-DRw52a is associated with a very low risk of being antibody producers and hence, is associated with a very low risk for NAITP in their new-borns.

Antigens, Human Platelet↗
PubMed1990

Genetic exchange between endogenous and exogenous LINE-1 repetitive elements in mouse cells.

The repetitive LINE (L1) elements of the mouse, which are present at about 10(5) copies per genome and share over 80% of sequence homology, were examined for their ability to undergo genetic exchange with exogenous L1 sequences. The exogenous L1 sequences, carried by a shuttle vector, consisted of an internal fragment from L1Md-A2, a previously described member of the L1 family of the mouse. Using an assay that does not require the reconstitution of a selectable marker we found that this vector, in either circular or linear form, acquired DNA sequences from endogenous L1 elements at a frequency of 10(-3) to 10(-4) per rescued vector. Physical analysis of the acquired L1 sequences revealed that distinct endogenous L1 elements acted as donors and that different subfamilies participated. These results demonstrate that L1 elements are readily capable of genetic exchange. Apart from gene conversion events, the acquisition of L1 sequences outside the region of homology suggested that a second mechanism was also involved in the genetic exchange. A model which accounts for this mechanism is presented and its potential implication on the rearrangement of L1 elements is discussed.

Animals↗
PubMed1990

Characterization of nonconservative homologous junctions in mammalian cells.

Homologous recombination in mammalian cells between extrachromosomal molecules, as well as between episomes and chromosomes, can be mediated by a nonconservative mechanism. It has been proposed that the key steps in this process are the generation (by double-strand cleavage) of overlapping homologous ends, the creation of complementary single-strand ends (either by strand-specific exonuclease degradation or by unwinding of the DNA helix), and finally the creation of heteroduplex DNA by the annealing of the single-strand ends. We have analyzed in detail the structure of nonconservative homologous junctions and determined the contribution of each end to the formation of the junction. We have also analyzed multiple descendants from single recombination events. Two types of junctions were found. The majority (90%) of the junctions were characterized by a single crossover site. These crossover sites were distributed randomly throughout the junction. The remaining 10% of the junctions had mosaic patterns of parental markers. Furthermore, in 9 of 10 cases, multiple descendants from a single recombination event were identical. Thus, it appears that in most cases few parental markers were involved in junction formation. This finding suggests that nonconservative homologous junctions are mediated mainly by short heteroduplexes of a few hundred base pairs or less. These results are discussed in terms of the current models of nonconservative homologous recombination.

Animals↗
PubMed1988

Polyoma integrates readily in mouse cellular DNA.

Although the natural host of polyoma virus is the mouse, its integration in cellular DNA has been investigated almost exclusively in rat cells. We report here studies on the integration of polyoma in mouse cells. We introduced the polyoma virus genome in two different mouse cell lines as an unselected genetic marker, by cotransfection with the tk gene of herpes simplex virus or the neo gene of E. coli. The number of TK+ or G418R clones obtained was reduced up to 50 fold by the presence of the polyoma genome. The gene coding for the early protein large T of polyoma was necessary and sufficient to produce this reduction. However, this effect appeared to be independent of polyoma replication. Surprisingly, all of the 33 clones analysed that had survived cotransfection with polyoma contained polyoma DNA integrated in their genome. Furthermore, in over 50% of these clones, the entire polyoma genome had been integrated. We conclude that polyoma integrates readily in mouse cellular DNA.

Animals↗
PubMed1988

Cloning of Trametes versicolor Genes Induced by Nitrogen Starvation.

We have screened a genomic library of Trametes versicolor for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.

Journal Article↗
PubMed1987

Integration of a vector containing rodent repetitive elements in the rat genome.

We have previously shown that integration of a polyoma vector containing rodent repetitive elements into rat cellular DNA is non-random (Wallenburg et al. J. Virol. 50: 678-683). Junctions between the polyoma vector and the host DNA occur in the repetitive sequences of the vector about ten times more frequently than would be expected if sequences from the vector were used randomly for integration. In this paper we looked at the host sequences involved in these junctions. Our analysis did not reveal any repetitive or specific sequences and we presume therefore that the repetitive sequences of the vector acted as hot spots for illegitimate recombination. We also analysed the integration mechanism and found that: First, even though the polyoma vector was transfected in the presence of carrier DNA, integration did not involve the formation of a transgenome. Second, in at least one of the clones analysed, integration resulted in deletion of host DNA sequences. Third, the host DNA displaced at the integration site was considerably longer than the integrated segment.

Animals↗
PubMed1987

Linear DNA must have free ends to transform rat cells efficiently.

We have observed that failure to remove certain restriction enzymes after digestion reduced the transforming ability of DNA from 10- to 50-fold. The DNA found integrated in the transformed cells isolated under these conditions had lost little or no sequences. We interpret these results as indicating that certain restriction enzymes remain bound to the DNA ends after digestion, thus generating a substrate unfavorable both for integration and exonucleolytic degradation. As expected from this interpretation, removal of the restriction enzymes before transfection restored the full transforming ability of linear DNA, but also resulted in the integrated sequences being significantly shorter than the transfected DNA. These findings strongly argue for the hypothesis that integration of linear DNA by illegitimate recombination requires free ends and further suggest that exonucleolytic degradation of such ends may generate a preferred substrate for integration. Finally, a comparison of the sequences found integrated after transfection with circular or linear molecules, led us to conclude that circular molecules need not be linearized to become integrated.

Animals↗
PubMed1987

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker. It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector. The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes. Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination. Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one. The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se. Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one. We propose a model to account for the generation of these recombinants.

Animals↗
PubMed1986

An excision event that may depend on patchy homology for site specificity.

In mouse cells transformed by a mutant polyomavirus genome, recombination between integrated viral DNA and flanking cellular DNA resulted in the excision of two readily amplifiable chimeras, designated RmI and RmII. The crossing-over that generated RmII was unique in that it involved a simple cellular sequence in which the triplet 5'-CTG-3' was repeated many times. We show that the sequence across the junction resulting from excision was identical in several molecules of RmII, as if the cross-over generating this junction always involved exactly the same two sites on the viral and cellular DNA. We also show that the cellular site mapped where the replacement of a G by an A in one of many successive 5'-CTG-3' triplets generated a homology of five nucleotides (5'-CTACT-3') with the viral site. Oligonucleotides on both sides of these sites are probably involved in matching the two DNAs prior to recombination.

Animals↗
PubMed1984

Random and nonrandom integration of a polyomavirus DNA molecule containing highly repetitive cellular sequences.

RmI is a circular DNA molecule that consists of a complete polyomavirus genome with an insertion (Ins) of mouse cellular DNA. This polyomavirus genome carries a mutation which renders its replication, but not its transforming ability, temperature sensitive. Ins contains both unique and repetitive cellular DNA sequences. We transfected RmI into rat cells at the permissive and nonpermissive temperatures for replication and isolated clones that had integrated RmI in their genomes. In this paper, we describe detailed mapping of the integrated RmI sequences present in 37 different cell clones. Our results indicated that transfection at the permissive temperature resulted in a random integration pattern, whereas transfection at the nonpermissive temperature resulted in a nonrandom integration pattern. The nonrandom insertions had a preferential length and preferential endpoints. We argue from these results that the nonrandom integration pattern is related to the presence of Ins and that the switch between nonrandom integration and random integration reflects a modification of the integrating substrate. When both are active, the random mechanism dominates the nonrandom mechanism.

Animals↗
PubMed1982

Polyoma virus mutant with normal transforming ability but impaired tumorigenic potential.

Cloned DNA from the P155 mutant of polyoma virus transforms cells in culture as efficiently as wild-type DNA, but has a much lower tumorigenic potential when injected into newborn rats. Like cells transformed by wild-type DNA, cells transformed by the mutant DNA grow in low serum concentrations, form colonies in agar suspension, and grow to high saturation densities compared with untransformed cells. They are, however, much less tumorigenic since they transplant 100- to 2,000-fold less efficiently than cells transformed by wild-type DNA. Substitution of the region between 89.7 and 1.8 map units by the corresponding region of P155 DNA decreased the tumorigenicity of wild-type DNA. When this region was isolated from wild-type DNA and substituted in P155 DNA, the tumorigenicity of the latter increased to values comparable to those of wild-type DNA. This showed that the lesion affecting tumorigenicity occurred between 89.7 and 1.8 map units on the polyoma virus genome. Sequence analysis in this region revealed a 12-base-pair deletion between nucleotides 1,347 and 1,360. This identified P155 as an mlt mutant, i.e., a mutant with a deletion from a region which encodes parts of the large and middle T antigens.

Animals↗
PubMed1981

Physical mapping of temperature-sensitive mutations of herpes simplex virus type 2 by marker rescue.

The physical mapping of six ts mutations of herpes simplex virus type 2 (HSV-2) is presented. The results were obtained from 14 separate intratypic marker rescue experiments and the analysis of 20 HSV-1/HSV-2 intertypic recombinants. The order of these mutations on the physical map of HSV-2 is unambiguous and correlates almost exactly with the previously published genetic map of Timbury & Calder (1976). One of the mutants studied (HSV-2 ts12) has apparently two distinct conditionally lethal ts mutations, one in the long and the other in the short region of the HSV genome.

DNA Restriction Enzymes↗
PubMed1981

Integrated polyoma genomes in inducible permissive transformed cells.

Using the approach described by Botchan, Topp, and Sambrook (Cell 9:269-287, 1976), we analyzed the organization of the integrated viral sequences in five clonal isolates from the same permissive, inducible cell line (Cyp line) transformed by the tsP155 mutant of polyoma virus. In all five clones, viral sequences were found that could be assigned to a common integration site, as they were joined to the cellular DNA in the same fashion in every instance. However, the sequences comprised between these points differed markedly from clone to clone, as if cell propagation had been accompanied by amplification or recombination or both within the viral insertion. When the clones were compared, no correlation could be found between the abundance, or the organization, of the integrated viral sequences and the amount, or the nature, of the free viral DNA molecules produced during induction. Altogether, our findings suggest that specific events, occurring during either the excision or the subsequent replication of the integrated viral sequences, are responsible for the predominant production of nondefective viral DNA molecules by permissive transformed cells, such as Cyp cells.

Animals↗