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Degree

PhD, University of Cincinnati, Medicine : Molecular Genetics, Biochemistry & Microbiology, 2002.

Abstract

Bloom’s syndrome (BS) is a rare, recessive chromosome breakage disorder characterized by small stature, sun sensitivity, facial erythema, immunodeficiency, female subfertility, male infertility, and a predisposition to a variety of cancers. When this body of work was started, the gene for Bloom’s syndrome (BLM) had yet to be identified. This work presents characterization of the genomic region at BLM and the identification of BLM. With the cloning of the gene, the answers to a number of questions could be investigated. Additional chapters present data that demonstrate that increased genomic instability and recombination are the result of loss of function of the Bloom’s syndrome gene product. Somatic cells from BS individuals are characterized by a high frequency of chromatid exchanges both between and within chromosomes, as well as by a high mutation rate at specific loci. DNAs from BS and normal clonal cell lines were first examined for alterations at microsatellite repeat loci. Alterations in size microsatellite repeats were observed at a 10-fold increase in frequency in BS clones compared to normal clones. A contiguous representation of 2-Mb region that contains the BLM gene was generated. YAC and P1 clones from the region were identified and ordered using genetic markers in the region along with newly developed sequence tagged sites from radiation-reduced hybrids, polymorphic dinucleotide repeat loci, and end-sequences of YACs and P1s. The physical map, and DNA markers derived from it, was instrumental in identifying BLM. With the gene identified, the genomic structure was determined and a rapid DNA screening test was developed for the identification of Blm ^Ash, the most common mutation in BS. To determine whether BLM can suppress recombination we over-expressed BLM in separate cell lines capable of identifying recombination or frameshift events. However, no significant difference was noted between cells transfected with BLM and those transfected with vector alone. Finally, we established a mouse model of BS using homologous recombination to disrupt mouse Blm. Genotyping offspring from heterozygous parents did not identify any offspring homozygous for the knockout allele, suggesting embryonic lethal phenotype. In long-term studies, heterozygosity for Blm increases tumor formation compared to wild-type littermates.

Keywords

Bloom's Syndrome; recombination; cancer

Advisor

Dr. Joanna Groden

Pages

140p.

Document number: ucin1022269717
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=ucin1022269717

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