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Modelling of genomic aberrations responsible for<br />
neoplastic transformation and tumor progression in yeast<br />
Carlo V. Bruschi, Dimitri Nikitin, Sabrina Sidari and Valentina Tosato<br />
Microbiology Group, International Centre for Genetic Engineering and Biotechnology,<br />
Area Science Park – W, Padriciano 99, I-34013 Trieste, Italy<br />
In the yeast Saccharomyces cerevisiae, aneuploidy is physiologically well tolerated<br />
and the karyotype has a remarkable degree of plasticity, making this microorganism<br />
an excellent cellular model system to investigate genome dynamics. In particular, we<br />
are trying to elucidate the molecular mechanism underlying genomic homeostasis,<br />
to ultimately address human gene functions involved in the determinism of cancer.<br />
We studied the experimental feasibility of inducing chromosomal translocations<br />
between any two desired genetic loci of the genome and constructed a system<br />
for the production of site-specific non-reciprocal translocations in wild-type yeast<br />
strains. Cells were transformed with a linear DNA cassette having the KAN R selectable<br />
marker flanked by two DNA sequences homologous to loci on two different<br />
chromosomes. Using this BIT (Bridge Induced Translocation) system, induction<br />
of targeted non-reciprocal translocations in mitosis was achieved (Tosato et al.,<br />
2005). In these strains we characterized the DNA rearrangement of the chromosome<br />
fragments deriving from the translocation. We obtained evidence that centromeredistal<br />
chromosome fragments may be processed by a break-induced replication<br />
(BIR) mechanism ensuing in partial trisomy, while many integrants may correctly<br />
integrate at only one DNA end. Several translocation mutant strains were obtained<br />
by our BIT technology, in which the induced chromosomal aberrations correlated<br />
with an abnormal cell and nuclear division. Some of the mutant strains revealed<br />
strange cell morphology by light and fluorescent microscopy. Especially odd, multibranched<br />
and multi-nucleated cells were observed in old cultures (more than 24<br />
hrs. of growth). Differences in the karyotype of these mutants was observed both,<br />
by quantitative PCR and CHEF electrophoresis. The expression of genes located<br />
close to the breakpoint regions on chromosomes VIII and XV was analyzed by<br />
quantitative RT-PCR. As could be concluded from these data, almost all genes near<br />
translocation breakpoints were 1.5 - 4.5 times more expressed then in parental<br />
strain. Experiments are in progress for chromatin immunoprecipitation (ChIP)<br />
analysis of RNA-polymerase II presence on the promoters of genes located close to<br />
translocation breakpoints and for whole-cells transcriptome profiling of wild type<br />
and mutant strains using systemic analysis of gene expression (SAGE).<br />
56l38<br />
The straightforward technology employed could be instrumental in elucidating the<br />
molecular mechanisms underlying Gross Chromosomal Rearrangements generated<br />
by genome DNA integration and characteristic of many types of cancer.
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