Worm Breeder's Gazette 13(4): 58 (October 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.
Dept. of Genetics and CeU Biology, University of Minnesota, St. Paul, MN 55108
We have previously described the identification of a group of maternal effect lethal mutants in gut genes. These genes appear to be involved in the determination and/or differentiation of the intestinal cell lineage. The E cell is affected in two ways in these mutants. First, the dead embryos produced by mutant animals lack gut granules and gut specific antigens. Second, the E cell division rate and gastrulation are altered with respect to wild type. During normal development the E cells lengthen their cell cycles at the division from two to four E cells and the two E cells gastrulate. In gut mutants there is no elongation of the E cell cycle at this time; the two E cells divide shortly following division of two MS cells to four. Furthermore, the E cells in mutant embryos do not gastrulate, and the terminal embryos show no evidence of having undergone morphogenesis. Although the mutant embryos lack gut tissue, antigens specific to differentiated hypodermal, neuronal, and body wall muscle cells are evident. We have concentrated our efforts on the gut-2 gene. Multiple alleles of this gene had previously been recovered ( it92 and lw6 ,both EMS-generated), and these alleles over deficiency nDf42 show no obvious phenotypic differences from homozygous gut-2 mutants. We have performed a non-complementation screen to isolate new gut-2 alleles, and have recovered three different phenotypic classes of new alleles. We recovered one new psoralen/UV allele which resembles the original maternal effect lethal alleles. We recovered three new alleles ( lw63 ,gamma-induced; lw65 and lw66 ,both psoralen/UV-induced) which have a sterile, slow-growing phenotype. At least one of these alleles, lw66 ,is a deficiency for the region; it fails to complement mes-4 ,a neighboring gene. We recovered nine new alleles which are embryonic lethal, and appear to be deficiencies since each removes at least one neighboring gene. Some trans-heterozygous combinations of these deficiencies resemble the sterile, slow-growing alleles. As a start towards cloning the gut-2 locus, we had previously determined the physical map position for gut-2 by mapping Bergerac-derived Tc1 'sin the region. We injected YAC Y53F11 which is contained within the determined physical boundaries, and found it to show rescuing ability. We have recently performed Southern blots on many of the new alleles and deficiencies we generated for the region, probing with cosmids underlying YAC Y53F11 .Genetic and physical map data placed gut-2 between the right breakpoint of deficiency lwDf2 and the left end of Y53F11 ,and identification of the right lwDf2 breakpoint by Southern blot revealed that this region is entirely contained on cosmid K10B8 .This cosmid and an overlapping cosmid T10G8 were individually tested for rescue, and both were found to restore a wild-type phenotype to gut-2 mutants. We have partially mapped these cosmids to determine their overlap with each other and with YAC Y53F11 .We have recently injected a 7.2 kb plasmid subclone contained within this overlap which appears to have rescuing ability. We are now in the process of defining the smallest genomic rescuing fragment and isolating cDNA's within the 7.2 kb region.