Worm Breeder's Gazette 12(5): 31 (February 1, 1993)
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.
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 lineage is affected in two ways in these mutants. First, the dead embryos produced by mutant animals lacZ 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 no elongation of E cell cycle timing is evident; the E cells divide shortly following MS cell division. 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.
Our collection of mutants and three mutants identified by Diane Morton together represent ten gut genes, four of which have multiple alleles. We are concentrating our efforts on the gut-2 V gene, since multiple alleles of this gene have been recovered, and alleles of this gene over the deficiency nDf42 show no obvious phenotypic differences from the homozygous gut-2 mutants.
As a first step toward the eventual molecular characterization of gut-2 ,we wished to determine its location on the physical map. We had previously determined the genetic map position of gut-2 as between lin-25 and him-S. Using a strain kindly provided by Simon Tuck) containing many Berferac-derived Tc1 'sin the interval between rol-4 and unc-76 ,we have mapped gut-2 with respect to the Tc1 polymorphisms. The polymorphism found to be the closest on the right of gut-2 was TcParl (identified by Abbey Telfer), for which the physical map location had been previously identified as cosmid clone EEEH17 .We cloned the worm DNA flanking the closest polymorphism on the left and its physical map location assigned to cosmid clone C16A5 by Alan Coulson.
Ultimately we plan to clone gut-2 using transformation rescue of the mutant phenotype. We have injected several of the cosmids within the physical boundaries denoted above, and also one YAC, Y53F11 .Unfortunately the cosmid coverage in the area is not complete, and no cosmid injected so far shows rescuing ability, however, the YAC partially restores a wild-type phenotype. We fed confident that the gut-2 gene is contained within this YAC, and will narrow down the rescuing region by additional transformation experiments.
We are also screening by pre-non-complementation for new alleles of gut-2 generated by either psoralen or gamma irradiation. So far we have isolated two new mutations. One new mutant behaves as a deficiency; it is homozygous lethal and fails to complement at least one neighboring gene. The other mutant is slow-growing and sterile as a homozygote, but complements all nearby genes we have tested. Of particular interest is a polymorphism revealed by probing the DNA from the sterile mutants with the cosmid C11H4 .We are presently doing additional Southern blots to investigate the nature of the polymorphic band in this mutant.