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Biomedical subjects

M P Calos

Publications and source records attributed to M P Calos.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1982

On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions.

Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths. We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions. Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns. Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background. The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved. The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs). Altering a single base pair within this homology reduces deletion incidence by an order of magnitude. We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements.

Base Composition↗
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↗