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M P Calos

Publications and source records attributed to M P Calos.

18 recordsLinked to original sources
PubMed1992

Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication.

Chinese hamster genomic DNA sequences from the region downstream of the dihydrofolate reductase (DHFR) gene reported to contain a chromosomal origin of bidirectional DNA replication (OBR-1) were tested for their ability to support autonomous DNA replication in human cells. A 13.3 kilobase fragment containing OBR-1 and surrounding sequences supported replication in short-term and long-term replication assays, while a 4.5 kb fragment containing OBR-1 did not support substantial replication in either assay. These results are consistent with our previous observations that large fragments of human DNA support replication, while smaller fragments are less efficient. The replication activities of plasmids containing OBR-1 were no greater than those of randomly chosen human fragments of similar size. Furthermore, two-dimensional gel analysis of plasmids containing OBR-1 indicated that initiation does not preferentially occur within the OBR-1 region. These results suggest that in the context of autonomous replication, the DHFR sequences tested do not contain genetic information specifying site-specific replication initiation. Possible implications of these results for chromosomal replication are discussed.

Animals↗
PubMed1991

Replication control of autonomously replicating human sequences.

Three autonomously replicating plasmids carrying human genomic DNA and a vector derived from Epstein-Barr virus were studied by density labelling to determine the number of times per cell cycle these plasmids replicate in human cells. Each of the plasmids replicated semi-conservatively once per cell cycle. The results suggest that these human autonomously replicating sequences undergo replication following the same controls as chromosomal DNA and represent a good model system for studying chromosomal replication. We also determined the time within the S phase of the cell cycle that three of the plasmids replicate. Centromeric alpha sequences, which normally replicate late in S phase when in their chromosomal context, were found to replicate earlier when they mediate replication on an extrachromosomal vector. Reproducible patterns of replication within S phase were found for the plasmids, suggesting that the mechanism specifying time of replication may be subject to experimental analysis with this system.

Bromodeoxyuridine↗
PubMed1991

Replication initiates at multiple locations on an autonomously replicating plasmid in human cells.

We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise sequence requirements which target initiation to specific locations.

Cell Cycle↗
PubMed1991

Autonomous DNA replication in human cells is affected by the size and the source of the DNA.

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.

Animals↗
PubMed1989

Comparison of ultraviolet irradiation-induced mutagenesis of the lacI gene in Escherichia coli and in human 293 cells.

We report the sequence changes in the Escherichia coli lacI gene in 133 mutants detected after passage of an ultraviolet-irradiated shuttle vector human 293 cells. The results are compared with our previous studies of the lacI gene after ultraviolet light treatment in E. coli. In human cells, base substitutions predominate, and frameshifts are found much less frequently than in bacteria. The most frequent base change is the G.C to A.T transition. Overall, 110 to 112 transitions were G.C to A.T. Some of the hotspots seen in lacI in bacteria are prominent also in human 293 cells, suggesting that the same lesions are targeting mutations in both systems. Transitions are found almost exclusively at sequences at which pyrimidine-pyrimidine photoproducts can form. The data are consistent with the notion that a significant fraction of ultraviolet irradiation-induced mutagenesis in mammalian systems occurs by adding an A across from a photolesion. Double mutations are significantly more frequent in human cells than in bacteria. Reasons for this difference are discussed.

Base Sequence↗
PubMed1989

Isolation of human sequences that replicate autonomously in human cells.

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.

Base Sequence↗
PubMed1988

Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells.

We have increased the copy number of Epstein-Barr virus vectors that also carry the origin of replication of simian virus 40 (SV40) by providing a transient dose of SV40 T antigen. T antigen was supplied in trans by transfection of a nonreplicating plasmid which expresses T antigen into cells carrying Epstein-Barr virus-SV40 vectors. A significant increase in vector copy number occurred over the next few days. We also observed a high frequency of intramolecular recombination when the vector carried a repeat segment in direct orientation, but not when the repeat was in inverted orientation or absent. Furthermore, by following the mutation frequency for a marker on the vector after induction of SV40 replication, it was determined that SV40 replication generates a detectable increase in the deletion frequency but no measurable increase in the frequency of point mutations.

Antigens, Polyomavirus Transforming↗
PubMed1987

Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.

Cell Line↗
PubMed1985

The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.

A system has been devised that allows the effect of mutagens acting in human cells to be readily analyzed at the DNA sequence level. The bacterial gene lacI, carried on a shuttle vector, is introduced into human tissue culture cells by transfection and allowed to replicate in the cell nucleus. Twenty-four to 48 hr after transfection, the cells are exposed to a mutagen. After 1-2 days of further replication, vector DNA is purified and transfected back into Escherichia coli for scoring and analysis of mutations in lacI. The nucleotide sequence changes for 53 UV light-induced mutations have been deduced in this way. Most of the mutations are transitions and occur at pyrimidine-pyrimidine sequences. The mutagenic specificity observed closely resembles that of UV light in E. coli, suggesting that human and bacterial cells respond similarly to damage from UV light. Use of the lacI shuttle in this way should permit determination of the mutagenic specificity of a wide range of mutagens and carcinogens in human cells.

Base Sequence↗
PubMed1985

Amplification of a bovine papillomavirus-simian virus 40 chimera.

A chimeric plasmid, pBOP, containing bovine papillomavirus (BPV) and the origin of replication from simian virus 40 (SV40) was constructed. The plasmid was established in mouse cells, where it was maintained stably as an autonomous BPV replicon. Lines carrying pBOP were fused to cells of COS-7, a simian line producing SV40 T antigen. Replication dependent on the SV40 origin and having the kinetics and approximate amplitude of an SV40 infection ensued. SV40 replication is therefore dominant over BPV replication, and the SV40 origin can conveniently be used to amplify lower-copy-number plasmids in mammalian cells.

Animals↗
PubMed1978

The transposon Tn9 generates a 9 bp repeated sequence during integration.

We performed a genetic and sequencing analysis of insertions of the transposon Tn9 into the lac operon of E. coli. Genetic mapping of 70 insertions into lacl and Z shows that starting from the same point on the chromosome, Tn9 goes to at least 50 different points in these two genes. Although there are preferred regions for insertion, these consist of multiple integration points within a small area, as demonstrated by pairwise crosses and restriction mapping. Sequence analysis of three Tn9 insertions reveals that Tn9 integration is associated with a direct repeat of 9 base pairs (bp) of host sequence. We show that these extra 9 nucleotide pairs are generated upon insertion and not brought in with the element.

Base Sequence↗
PubMed1978

DNA sequence at the integration sites of the insertion element IS1.

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.

Base Sequence↗
PubMedDate not supplied

Improved EBV shuttle vectors.

Shuttle vectors based on Epstein-Barr virus (EBV) replicate autonomously in the nuclei of human cells. These vectors represent reasonable models for chromosomes, have low background mutation frequencies, and have been useful for studying induced mutation in human cells. Two improvements in the EBV vector system are discussed. Attempts are described to increase vector copy number per cell by using a limited period of replication driven by the simian virus 40 (SV40) origin of replication. Isolation of human sequences that can replace the viral origin of replication in providing for autonomous replication of the vectors is also described. These improvements are leading toward shuttle vectors that are more efficient and more closely resemble authentic chromosomes.

Cells, Cultured↗
PubMedDate not supplied

Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression.

Simian virus 40 (SV40) replicates efficiently in monkey kidney cells. However, we have now found that SV40-based vectors transfected into most human cells replicate poorly, if at all. In contrast, strong SV40 replication is observed in human embryonic kidney (HEK) cells transformed with the adenovirus early region, but not in untransformed HEK cells. Vector replication in adenovirus-transformed cells is dependent on the presence of the SV40 origin of replication and large-T antigen. However, vigorous replication occurs at levels of large-T antigen that are undetectable by immunofluorescence. These data suggest that the adenovirus oncogenes create a replication-permissive environment to which the SV40 replicon responds. Furthermore, replication and gene expression seem to be antagonistic on our vectors. High levels of large-T antigen are observed only when vector replication is blocked by mutations in the gene for large-T antigen or the origin of replication, or by direct inhibition of DNA polymerase with aphidicolin.

Adenoviridae↗