aphidicolin and Chromosome-Inversion

Gene amplification is frequently associated with tumor progression, hence, understanding the underlying mechanisms is important. The study of in vitro model systems indicated that different initial mechanisms accumulate amplified copies within the chromosomes (hsr) or on extra-chromosomal elements (dmin). It has long been suggested that formation of dmin could also occur following hsr breakdown. In order to check this hypothesis, we developed an approach based on the properties of the I-SceI meganuclease, which induces targeted DNA double-strand breaks. A clone containing an I-SceI site, integrated by chance close to an endogenous dhfr gene locus, was used to select for methotrexate resistant mutants. We recovered clones in which the I-SceI site was passively co-amplified with the dhfr gene within the same hsr. We show that I-SceI-induced hsr breakdown leads to the formation of dmin and creates different types of chromosomal rearrangements, including inversions. This demonstrates, for the first time, a direct relationship between double-strand breaks and inversions. Finally, we show that activation of fragile sites by aphidicolin or hypoxia in hsr-containing cells also generates dmin and a variety of chromosomal rearrangements. This may constitute a valuable model to study the consequences of breaks induced in hsr of cancer cells in vivo.

Topics: Animals; Aphidicolin; Cell Hypoxia; Cells, Cultured; Chromosome Breakage; Chromosome Fragile Sites; Chromosome Fragility; Chromosome Inversion; Cricetinae; Deoxyribonucleases, Type II Site-Specific; DNA; Extrachromosomal Inheritance; Gene Rearrangement; Genes, MDR; Genetic Engineering; Molecular Biology; Saccharomyces cerevisiae Proteins; Tetrahydrofolate Dehydrogenase

Aphidicolin (APC)-induced chromosomal gaps and breaks were analyzed for ten deer mice (Peromyscus maniculatus) from a natural population. The FSM statistical methodology was used to identify fragile sites as chromosomal loci exhibiting significantly non-random numbers of gaps/breaks in each individual and enabled an assessment of variation in fragile sites among the individuals. The individual deer mice exhibited as few as 7 to as many as 19 of the populational total of 34 sites. Two sites were fragile in all individuals and 13 sites were fragile in single individuals only. Defined by populational frequencies of greater than 50%, high-frequency fragile sites constituted 26% of the populational total. Approximately 35% of the total fragile sites were fragile in 20-40% of the population (low-frequency fragile sites) and about 38% were fragile in single individuals only. Analysis of the data pooled over all individuals identified significantly non-random breakage at 80 sites, 47 of which were not identified as fragile in any single individual. It appears, therefore, that fragile site identifications from pooled data have fostered an inflated estimate of the numbers and frequencies of common fragile sites. Comparison of the fragile site and spontaneous breakage (control) data suggest that APC-induced fragile sites represent regions of chromosomes that experience elevated levels of somatic mutation. Additionally, the occurrence of APC-induced fragile sites at or near the interstitial breakpoints of two pericentric-inversion polymorphisms in this population supports the hypothesis that fragile sites experience an increased rate of meiotic chromosomal mutation and are predisposed to undergo phylogenetic rearrangement.

Topics: Animals; Aphidicolin; Chromosome Aberrations; Chromosome Banding; Chromosome Fragile Sites; Chromosome Fragility; Chromosome Inversion; Genetic Variation; Peromyscus

To determine the replicative mechanism for human cytomegalovirus (HCMV) DNA, field inversion gel electrophoresis was used to separate HCMV replicative DNAs during lytic infection. Unit-length circular HCMV genomes lacking terminal restriction fragments were detected starting 4 h after infection even when cells were treated with aphidicolin, phosphonoacetic acid, or cycloheximide. Viral DNA synthesis began 24 h after infection and produced large amounts of high-molecular-weight replicative DNA that was a precursor of progeny genomes. Replicative DNA contained rare terminal restriction fragments, and long-arm termini were much less frequent than short-arm termini. Replicative DNA was not composed of unit-length circles because low-dose gamma irradiation of replicative DNA generated numerous random high-molecular-weight fragments rather than unit-length molecules. PacI digestion of replicative DNA from a recombinant HCMV with two closely spaced PacI sites revealed that replicative DNA is concatemeric and genome segment inversion occurs after concatemer synthesis. These results show that after circularization of the parental genome, DNA synthesis produces concatemers and genomic inversion occurs within concatemeric DNA. The results further suggest that concatemers acquire genomic termini during the cleavage/packaging process which preferentially inserts short-arm termini into empty capsids, causing a predominance of short-arm termini on the concatemer.

Topics: Aphidicolin; Cells, Cultured; Chromosome Inversion; Cycloheximide; Cytomegalovirus; Deoxyribonucleases, Type II Site-Specific; DNA Replication; DNA, Circular; DNA, Recombinant; DNA, Viral; Electrophoresis, Agar Gel; Fibroblasts; Gamma Rays; Genome, Viral; Humans; Isotope Labeling; Micrococcal Nuclease; Nucleic Acid Conformation; Phosphonoacetic Acid