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Molecular analysis of the DPY-14 region of chromosome I in Caenorhabditis elegans

University of British Columbia

This thesis describes the alignment of cloned DNA with the genetic map, and the identification of coding elements within the aligned DNA. The region of study was the dpy-5 unc-29 interval from chromosome I of the nematode Caenorhabditis elegans, with an emphasis on the region surrounding the gene dpy-14. The objectives of this thesis were: 1) to align the physical and genetic maps .of the region; 2) to identify and characterize the coding elements in the vicinity of dpy-14; and 3) to cross-hybridize the identified C. elegans coding elements to mammalian DNA in an attempt to identify evolutionarily conserved genes. Six polymorphisms from the dpy-5 unc-29 interval were mapped with respect to the free duplication sDp2. The polymorphisms hP5, sPl, and hP9 were found to.be inside the region spanned by sDp2 while the polymorphisms hP4, hP6, and hP7 were found to be outside this interval. In addition, these six polymorphisms were mapped with respect to visible markers from the dpy-5 unc-29 interval. These analyses demonstrated the genetic order to be dpy-5, hP5, unc-37, (dpy-14, sPl), hP9, unc-13, hP7, (hP4, hP6), unc-29. Lambda phage containing the hP5, sPl, and hP6 sites identified and anchored cosmid contigs to the genetic map. The interval from the left of hP5 to the right of unc-13 is contained in a single contig of approximately 1400 Kb. The amount of DNA in Kb across the hP5 and unc-13 interval was compared to the genetic distance in map units. The DNA per map unit value was found to vary in this interval with the greatest value found between hP9 and unc-13. Seven cosmids representing 173 Kb of N2 genomic DNA near the gene dpy-14 were isolated. Using cross-species hybridization to C. briggsae DNA ten conserved regions were identified within these seven cosmids. The ten conserved fragments were used to identify seven cDNAs, six of which also identified RNAs on Northern blots. The relative abundance of the isolated cDNAs varied 250 fold with the most abundant having a level similar to that found for actin. The first comprehensive survey of mammalian homologies in a contiguous set of ten coding regions found three coding elements to be. conserved. One was demonstrated to be the small nuclear RNA gene U1. Another shared sequence similarities with the gene S-adenosyl-L-homocysteine hydrolase. No detectable homologies were identified with the third. A formaldehyde-induced mutation that failed to complement the genes unc-37, unc-87, dpy-14, let-83 and let-86 was isolated. This mutation appeared to be the result of a DNA rearrangement which had one breakpoint within the cosmid C14A12. Using the conserved elements identified in this thesis together with the rearrangements and mapped genes from the region, a detailed physical and genetic map in the vicinity of dpy-14 was constructed.

Text

2011-01-26

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http://hdl.handle.net/2429/30852

Genetics

University of British Columbia

Graduate