Open Collections

Mapping, characterization and cosmid rescue of essential genes in the dpy-5 unc-13 (I) region of Caenorhabditis elegans

University of British Columbia

This thesis describes the analysis of essential genes in the 1.5 map unit dpy-5 unc-13 region of chromosome I in the C. elegans genome. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described in Howell et al. (1987). The 15 map unit sDp2 region is 80% saturated for essential genes (McDowall 1990). This set of lethals comprises a substantial 'mutant library', which should play an important role in correlating the function of essential genes with the sequence currently being generated by the C. elegans genome project (Waterston and Sulston 1995). In order to fulfill this role, these lethal mutations must first be placed on both the genetic and physical maps, so as to render them accessible for cloning and functional analysis. The goal of this thesis is to place a subset of these lethal mutations on both maps, and to further define their mutant phenotypes. In this study, I report the identification, mapping, cosmid rescue and characterization of essential genes in the dpy-5 unc-13 region. Forty lethal mutations lying between the breakpoints of hDp13 and hDp16 in the dpy-5 unc-13 region have been mapped and complementation tested. Seventeen of these lethals identify six new essential genes, while the remaining 23 lethals were allelic to nine known genes. This analysis brings the number of essential genes in the dpy-5 unc-13 region to 64, as defined by the sDp2 rescue of lethal mutants. 61% of these essential genes are identified by more than one mutation. Positioning of mutations lying between hDpl3 and hDp!6 was done using the breakpoints of six duplications. These genes appear to be nonrandomly distributed throughout the region, based on their arrest phenotypes. This completes the genetic mapping of the entire sDp2 set of lethal mutations between dpy-5 and unc-13. The mutant phenotypes of the loci essential for fertility were further characterized by Nomarski microscopy and DAPI-staining of their nuclei. In total, 14 genes in the dpy-5 unc-13 region, plus one gene to the right of unc-13, were characterized. None of the mutants were rescued by male sperm, indicating that none of the mutations caused defects in spermatogenesis alone. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Seven genes, let-355, let-367, let-384, let-513, let-544, let-545, and let-606, produced mutants that affected germ cell proliferation or gametogenesis. Mutants of the remaining four genes produced eggs that failed to develop or hatch, let-370, let-538, let-599 and let-604, thereby acting as maternal effect lethals. Finally, a subset of the essential loci in the dpy-5 unc-13 region were positioned on the physical map by phenotypically rescuing lethal mutations with transgenic strains. Germline transformation with cosmid DNA was used to create 30 heritable extrachromosomal array-bearing transgenic strains. These arrays were used as small duplications for fine scale mapping. Mutations in 13 essential genes have been phenotypically rescued by 10 different transgenic strains. These rescues provide an alignment of the genetic and physical maps in this region. The frequency of transmission of extrachromosomal arrays to hermaphrodite gametes was calculated. In 12/16 transgenic strains, the arrays were found to be transmitted 2- to 7-fold less frequently in oocytes compared to hermaphrodite sperm. Three strains showed a subsequent increase in array stability in oocytes. This phenomenon may be influenced by cosmid sequences. Early mitotic loss of arrays was observed in all 17 strains examined, suggesting that loss of the array can occur at any time during development when cell divisions are occurring. This research has helped to refine the genetic map of the dpy-5 unc-13 region of chromosome I, as well as integrate the genetic and physical maps over 0.5 map units. There are now 64 essential genes in the dpy-5 unc-13 region identified from the sDp2 set of lethal mutations, of which 22% have been characterized with respect to their mutant phenotypes. As a result of this work, 13 of these loci are anchored to the physical map, providing links between the genetic and physical maps on average every 85 kb. These links will facilitate the future cloning and functional analysis of these loci.

Thesis/Dissertation

application/pdf

2009-02-19

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/4803

Genetics

University of British Columbia

UBCV

DSpace