engleski

Genomic instability induced by high frequency illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

Single-stranded (ss) non-replicative plasmids with no homology to Saccharomyces cerevisiae genome efficiently transform yeast cells mostly by illegitimate integratio into the chromosomes. In haploid cells plasmid integration often causes chromosomal rearrangements that could be observed as changed karyotype after separation of intact yeast DNA by pulsed-field gel electrophoresis and additional hybridization with labeled plasmid DNA. Frequently, target chromosomes showed decreased mobility on chromosomal gelas and in most cases this was not due to the presence of tandemly integrated plasmid molecules. Some transformants contained the plasmid sequence on several chromosomes. This can be explained either by multiple independent integration events or by the chromosomal size polymorphism within a single clone. Observed genomic rearrangements could be connected with changed phenotype in several transformants, noticed as poor growth of the cells or subsequent segregation of used reporter gene. To find out if observed chromosomal rearrangements could be tolerated in yeast diploid cell, a homozygous diploid strain was transformed with ss plasmid to compare transformation efficiency. Observed yield of transformants was increased nin-fold in diploid strain, suggesting that the diploid can tolerate some genetic alterations eventually lethal in haploids. This interpretation was supported by the findings that some diploid transformants showed significantly decreased sporulation efficiency and spore viability. The karyotype from randomly selected transformants with low spore viability revealed multiplicity of plasmid integration, in some cases connected with chromosomal instability, similar to previously observed genomic changes in haploids. These results strongly suggest that illegitimate integration of ss DNA can induce instability of the yeast genome. Possible mechanisms explaining this instability might be: ectopic recombination between integrated plasmid sequences, formation of dicentric chromosomes and eventually even pairing of nonhomologous chromosomes that contain tandemly integrated plasmid DNA, causing improper chromosomal segregation during meiosis and consequently, aneuploidy.

single-stranded DNA; illegitimate recombination; genomic rearrangement

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