Amber Hohl and Heather Brockway will present their work on 9th June 2011 Thursday 2-501 BSB at noon in Student Seminar
Background for Amber Hohls’ talk :
IDENTIFICATION OF MUTATIONS IN TOPOISOMERASE 2, A PROTEIN REQUIRED FOR SOMATIC PAIRING IN DROSOPHILA
Amber M. Hohl1,2, Morgan Thompson2, Jianhong Wu3, James Morris4, Tao Shih-Hsieh3, Ting Wu2, and Pamela K. Geyer1,5
1Graduate Program in Genetics, University of Iowa, Iowa City IA 2 Department of Genetics, Harvard Medical School, Boston MA; 3Department of Biochemistry, Duke University, Durham NC; 4Department of Biology, Brandeis University, Waltham MA; 5Department of Biochemistry, University of Iowa, Iowa City IA
Homologous chromosomes display associations in many organisms. Many of these interactions lead to changes in gene expression, as exemplified by processes of transvection, mammalian X-inactivation, and imprinting. Drosophila serves as an excellent model to study pairing interactions because homologous chromosomes in all cells are fully aligned. Using a cell culture based assay, Topoisomerase 2 (Top2) was found to be required for chromosome pairing. This gene encodes an ATP dependent homodimeric enzyme that generates double stranded breaks to change DNA topology. Top2 has many known functions, including DNA replication, chromosome segregation and transcription. To expand our understanding of the role of Top2 in chromosome pairing, we conducted a genetic screen to isolate germ line mutations in Top2. Fourteen recessive lethal alleles were identified from a screen of three thousand chromosomes. Molecular analyses of these alleles uncovered single or multiple base pair substitutions within each mutant line. These included changes that generated premature stop codons or missense mutations within previously identified functional domains. Complete loss of Top2 is lethal, with evidence of decreased cell division. Even though flies carrying each missense allele in trans to a deficiency produce inviability, hetero-allelic combinations of several missense alleles produce live flies. Current studies are directed at using the viable hetero-allelic combinations to examine chromosome structure and function during development. Further, these studies indicate that female germline development is particularly sensitive to decreased Top2 activity, implying roles for this enzyme in meiotic pairing. These data suggest that the collection of Top2 mutations will provide a resource for understanding chromosome functions that require homolog interactions.
Background for Heather Brockways’ Talk:
Breaking It Down: Investigating Synaptonemal Complex Disassembly In C. elegans Using RNAi
Heather M Brockway1, Sarit Smolikove1,2
1University of Iowa Interdisciplinary Program in Genetics, 2University of Iowa Department of Biological Sciences
The events in meiotic prophase I are essential for proper chromosome segregation. In prophase I, homologous chromosomes pair, synapse, and form crossovers to recombine the genetic material. Once these events are completed, the homologous chromosomes segregate in preparation for meiosis II, at which time the sister chromatids separate. This process is highly regulated by a series of molecular checkpoints, ensuring that each step proceeds correctly. The synaptonemal complex (SC) is involved in most prophase I events and is a highly conserved protein structure formed between homologous chromosomes during meiosis. The absence of a functional SC leads to missegregation of chromosomes and generates aneuploid gametes. We utilize the model organism C. elegans to elucidate the underlying molecular mechanisms of the SC disassembly pathway. Our research employs RNAi methodology as a means of discovering the proteins associated with SC disassembly. Prior screens conducted in our lab have uncovered a novel mutant possessing meiotic defects specific to SC disassembly. Using this new mutant, we have initiated an RNAi screen to search for enhancers and suppressors of the embryonic lethal phenotype associated with this mutation. The goal of this screen is to identify SC disassembly proteins via their interaction with this new mutant. To date, 4% of the RNAi library has been screened. In this pilot, we identified three suppressors and seven enhancers that were mutant-specific. Eight of these clones have not shown an embryonic lethal phenotype in other screens, including a screen on the wild-type background, thus indicating we are capable of identifying novel clones with this method. Of these eight clones, two are known genes while the rest are uncharacterized ORFs. Cytological analyses are conducted to determine if the changes in embryonic lethality are associated with defects in SC disassembly. Identifying meiotic genes involved in SC disassembly will lead to a better understanding of the molecular mechanisms involved with the disassembly process and chromosome segregation in general.