Examination of coding regions in the dpy-14 regions of Caenorhabditis elegans

The purpose of this study was to identify and clone the genes rescued by the cosmid T21G5 located in the dpy-14 region of chromosome I. Mutants in four genes, let- 394, let-534, let-545 and dpy-14 were rescued by transgenic C. elegans carrying T21G5 cosmic DNA created by McKay et al, 1993. Using the transgenic of hEx24 created by S. McKay, phenotypic rescue was confirmed. In order to isolate the individual genes corresponding with each of the mutant phenotypes, a set of plasmid subclones was constructed from T21G5 cosmid DNA. Each subclone was injected into wild-type C. elegans along with a plasmid carrying the dominant marker, Rol-6. Heritable transmission of the Rol-6 plasmid was detected by the roller phenotype. DNA molecules that share sequence identity could recombine to form an extrachromosomal arrays after injection into the gonad. In order to confirm the presence of the T21G5 plasmid subclone in the transgenic strain, PCR analysis with a set of primers from the Bluescript vector was used. The generation of the plasmid containing transgenics and PCR detection of the cosmid subclones distinct from the Rol-6 plasmid was successful. However, none of the plasmid transgenics rescued any of the mutants. In fact, three new T21G5 transgenics were generated, and none of these rescued the mutants. A number of controls were performed leading to the conclusion that hEx24 was exceptional in its ability to express and rescue the mutants in the germline. It has been shown that transgenic arrays often do not express well in the germline. An examination of the DNA sequence of T21G5 reveals two genes proposed to exhibit germline specific expression (gld-1-like, Ross etal, 1995) and (glh-1, Bennett et al, personal communication). Thus, this cosmid may not normally express well explaining the difficulty in repeating the rescuing ability of hEx24.Medicine, Faculty ofMedical Genetics, Department ofGraduat