PhD

dissertationChromosome translocations occur when DNA is transferred from one chromosome to another or is exchanged between chromosomes. The process is known to start with two nonhomologous, intact chromosomes and to end with two nonhomologous derivative chromosomes carrying DNA from the translocation partner. However, what happens in between is very poorly understood. There are a number of reasons for this. One is that chromosome translocations probably occur by a number of different mechanisms. Based on breakpoint sequences and epidemiological studies of diseases characterized by chromosome translocations, many hypotheses have been made as to how translocations actually arise. These include misrepair of double-strand breaks by homologous and nonhomologous recombinational repair pathways and errors of site-directed recombination pathways. The process of translocation is poorly understood also because there is no selection system in a model organism to detect the untargeted types of translocations present in human diseases. It has become apparent over the last 10 years that although various eukaryotes preferentially use different double-strand break repair systems, all eukaryotes are capable of repairing double-strand breaks in essentially the same ways. Therefore the development of a chromosome translocation selection system in a model organism would be of great benefit in investigating this process. This dissertation describes the development and characterization of a novel genetic selection system for chromosome translocations in the yeast, Saccharomyces cerevisiae. The system specifically selects translocations between a translocation YAC and any chromosome in the yeast genome. The translocation products are analyzed by pulsed-field gel electrophoresis, Southern blot hybridization, PCR, and breakpoint sequencing. Such analyses provide insights into how translocations are happening within yeast and provide directions to pursue in future assays. Production of a specific double-strand break in the YAC increases the translocation rate as would be expected if formation of a double-strand break is rate-limiting in translocation. The assay works in yeast strains that have been made deficient in homologous recombination by deletion of RAD52 and limitation of homology between the YAC and the yeast genome. Product analysis and rates also suggest differences between haploid and diploid strains. The current results suggest that it will be possible to use this assay in yeast to study chromosome translocation in ways relevant to the processes in mammalian systems

A Novel Selection System for Chromosome Translocations in <i>Saccharomyces cerevisiae</i>

Abstract Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers. A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection. When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations. The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct. A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold. This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations.</jats:p