Worm Breeder's Gazette 11(4): 56
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.
The one-cell C. elegans embryo must support two distinct patterns of cell division. Shortly after fertilization, the embryo undergoes the two meiotic divisions; and about twenty minutes later, the first mitotic cleavage occurs. Essentially the same cytoplasm must support both the meiotic and mitotic spindles, which differ in many aspects, including location, morphology and chromosomal alignment. Clearly, the activities of the gene products that govern the features that distinguish meiosis from mitosis are carefully regulated during this time. We have identified maternal effect mutations in four loci that disrupt either the meiotic or the mitotic divisions. The dominant ts gain-of-function (gf) mutations mei-1(ct46) I and mel-26(ct61) I and recessive loss-of-function (lf) mutations of zyg-9 II show similar defects in the first mitotic division, and these mutations are strong enhancers of one another's defects. If mutations of mei-1 and mei-2 result in recessive meiotic abnormalities but act as dominant suppressors of the mitotic defects of mei-1(ct46gf and mel-26(ct61gf). We are focusing our attention on mei-1, which can be mutated to affect either meiosis or mitosis. The molecular cloning of this gene has been straightforward, since it maps between lin-10 and lin-28, both of which have been located on the physical map. A total of 14 overlapping cosmids (provided by Alan Coulson and John Sulston) span the region, and we have used these to rescue the recessive maternal- effect lethality of mei-1(lf) by germline transformation. The individual cosmids were co-injected with a plasmid containing the dominant rol-6 mutation (described by Craig Mello) into mei-1(lf) /+ hermaphrodites (with appropriate markers in cis), and transgenic lines were established. Segregants homozygous for mei-1 that rolled were then tested for fertility. Since in previous mapping experiments recombinants between mei-1 and either lin-10 or lin-28 were easily obtained, we started testing cosmids in the center of the region and worked our way out. Naturally the rescuing activity was very near one end. Two overlapping cosmids, T01G9 (which also includes lin-10) and F54A4 provide mei-1+ activity. Injecting F54A4 directly into homozygous mei-1 hermaphrodites does not result in even transient rescue. We tested F54A4 cut with various restriction enzymes for rescuing activity in order to identify enzymes that did not cut within the gene, as described by Kim and Horvitz [Genes Devel. 4: 357 (90)]. The SalI digest was positive. Since only one of the four fragments produced by this enzyme was also cut by every enzyme that abolished rescuing activity, we could predict which SalI fragment should rescue. Being dubious about taking the (negative) results of failure to rescue too seriously we injected the other three gel-purified fragments anyway (since we can inject faster than we can analyze). However, only the predicted 10 kb SalI fragment contained mei-1+ activity. We are repeating this strategy with additional enzymes to whittle the clone down to a more convenient size.