Title

Evaluation of genetic instability in eutherian hybrids

Date of Completion

January 2007

Keywords

Biology, Genetics

Degree

Ph.D.

Abstract

Interspecific hybridization of marsupial species leads to genomic instability as a result of methylation aberrations and retroelement activation. Eutherian mammals last shared a common ancestor with marsupial mammals approximately 147mya. However, no study has successfully addressed whether retroelement demethylation contributes to eutherian hybrid genomic instability, yet phenotypes and preliminary evidence suggest eutherian interspecific hybrids may be subject to the same patterns of genomic instability as that observed in marsupials. This thesis examines the hypothesis that interspecific hybridization of eutherian species results in genomic instability as a result of aberrant methylation and retroelement activation. Genomic instability manifesting as double minute chromosomes in cell lines from a 16d Mus musculus X Mus caroli hybrid was characterized and determined to be amplification of an oncogene, mdm2. Hybrid-specific methylation abnormalities were isolated using a novel application of a methylation-sensitive substractive hybridization technique. These data showed that the double minutes were a result of demethylation targeted to retroelements surrounding the mdm2 locus that was amplified on the double minute chromosomes. This is the first demonstration of retroelement demethylation in a eutherian hybrid. Additional M. musculus X M. caroli hybrids were produced using artificial insemination to allow for an examination of developmental timing of genetic instability as well as to further quantify the underlying cause of these destabilizing events. These hybrids were growth retarded and morphologically abnormal, with hypoplastic placentas. Isolation of hybrid-specific methylation changes produced a library of sequences enriched in retroelements. A functional genomics approach revealed the pathology observed in two 13d M. musculus X M. caroli hybrids was the result of overexpression of the retroelement mvL30-1. The activation of mvL30-1 was a direct result of undermethylation in the hybrid genomes and was most pronounced in the placenta. The results presented herein provide evidence that the placenta is a specific site of genomic instability via demethylation and retroelement activation in interspecitic hybridization. This research will lead to a further understanding of hybrid dysgenesis and genomic instability and contributes to our understanding of the mechanisms and genes that, in concert, play a role in abnormal placental growth. ^