Worm Breeder's Gazette 13(2): 74 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
As part of our efforts to understand posterior patterning in C. elegans embryos, we are cloning and characterizing one of several loci defined by mutations that result in the Nob (no back end) phenotype. nob-1 ,the first locus thus identified, is defined by two psoralen-induced mutations: ct223 ,which results in 100% L1 lethality and the Nob phenotype and ct230 ,a weaker allele which results in only 15% lethality and production of viable, fertile animals with variable tail abnormalities.
Thus far, our cloning endeavor has been directed towards correlating the genetic and physical maps on the right arm of LGIII, where nob-1 resides approximately 2 map units right of spe-6 and 1 map unit left of pie-1 .In this region, no genes have been cloned so far, and ambiguities have been found in the cosmid map. Therefore, we constructed a congenic strain to identify and map Bergerac-specific Tc1 elements that might be useful in correlating the genetic and physical maps. We identified two Tc1 elements that map approximately 2.5 map units left of nob-1 .
When attempts to clone the flanking genomic sequences by inverse PCR met with difficulties, we tried a different strategy that in our hands has proved to be more efficient. Using size-selected, EcoRI-digested genomic DNA, we first performed a primer extension using a Tc1 -specificprimer that was directed outward. This resulted in double-stranded, blunt-ended molecules to which we ligated a linker-adaptor molecule that contained a unique NotI restriction site. We then performed two rounds of PCR with nested primers specific to sequences in Tc1 and the linker adaptor. After the second round of PCR, we observed strong signals from each of the expected Tc1 elements in our size-selected genomic fractions. These PCR products were digested with NotI and EcoRV (which cuts at a site in the 54 bp terminal repeat of Tc1 )and cloned efficiently into a plasmid vector. Subsequent probing of Southern blots with these cloned genomic fragments have confirmed the identity of the PCR products, which fortunately appear to contain unique sequence. We can now use these fragments as probes to assay for presence of the nob-1 region in members of a set of ten YAC clones identified by genomic DNA flanking a mutator allele of pie-1 (Craig Mello and Alan Coulson, personal communications and thank you's) and thus proceed towards transformation rescue of nob-1 .