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Biomedical subjects

M P Calos

Publications and source records attributed to M P Calos.

At least 19 recordsLinked to original sources
PubMed1995

Autonomous replication in Drosophila melanogaster tissue culture cells.

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and test ed them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA were able to replicate autonomously, as were random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Xenopus eggs and may be general properties of autonomous replication in animal cells.

Animals↗
PubMed1995

Replication of centromere II of Schizosaccharomyces pombe.

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.

Base Sequence↗
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↗
PubMed1984

Specificity of mutations induced in transfected DNA by mammalian cells.

DNA transfected into mammalian cells is subject to the high mutation frequency of approximately 1% per gene. We present data bearing on the derivation of the two main classes of mutations detected, base substitutions and deletions. The DNA sequence change is reported for nearly 100 independent base substitution mutations that occurred in shuttle vectors as a result of passage in simian cells. All of the mutations occur at G:C base pairs and involve either transition to A:T or transversion to T:A. To identify possible mutational intermediates, various topological forms of the vector DNA were introduced separately. Supercoiled and relaxed DNA are mutated at equal frequencies. However, linearized DNA leads to a greatly elevated frequency of deletions. Nicked and gapped templates stimulate both deletions and base substitutions. We discuss a model involving intracellular degradation of the transfected DNA which explains these observations.

Animals↗
PubMed1984

Deletion of plasmid sequences during Saccharomyces cerevisiae transformation.

Saccharomyces cerevisiae was transformed with DNA by the lithium acetate method. Mutation of nonselected markers on the transforming vector was observed at a frequency several orders of magnitude higher than spontaneous mutation frequencies. These mutations were shown to be deletions. Linearization of the vector before transformation stimulated deletion formation.

Base Sequence↗
PubMed1984

Transfected DNA is mutated in monkey, mouse, and human cells.

Papovavirus-based shuttle vectors containing the bacterial lacI gene were used to show that a mutation frequency in the range of 1% occurs in lacI when such vectors are transfected into COS7 and CV-1 simian cells, NIH 3T3, 3T6, L, and C127 mouse cells, and human 293 and HeLa cells. This frequency is approximately four orders of magnitude higher than the spontaneous mutation frequency in either mammalian or bacterial cells. The mutations are predominantly base substitutions and deletions and also include insertions from the mammalian genome. Time course experiments argue that mutagenesis occurs soon after arrival of the DNA into the nucleus. However, replication of the vector is not required since mutations occur even when the vector lacks all viral sequences. The high mutation frequency appears to be the characteristic outcome of transfection of DNA into mammalian cells.

Animals↗
PubMed1983

High mutation frequency in DNA transfected into mammalian cells.

The lacI gene of Escherichia coli was used to score mutation in mammalian cells of simian virus 40-based recombinant DNA vectors that provide for replication and selection in both bacterial and mammalian cells. Plasmid DNA was introduced into COS7 simian cells by DEAE-dextran transfection, allowed to replicate in the mammalian cells, and then returned to E. coli for analysis. Mutants in lacI were observed at frequencies of one to several percent, compared with a spontaneous mutation rate in E. coli of less than 10(-5). The lesions include a large number of base substitutions, in addition to deletions, duplications, and more complex rearrangements, including insertion into the plasmid of sequences originating in the host genome. We discuss possible sources of the high mutation frequency and its implications for experiments involving DNA transfer.

Animals↗