We are studying the specification of E cell fate during embryogenesis. To identify possible mechanisms of specification, maternal effect lethal mutations in ten gut genes were isolated whose inviable progeny fail to make differentiated products of the intestine. In these mutants, the E cells fail to gastrulate, and embryos arrest as balls of cells following an approximately wild-type number of embryonic cell divisions. A cell-cycle defect is observable in the intestinal lineage at the two- to four-E cell division when the E cells in gut mutant embryos fail to elongate their cell cycles as in wild type, and, instead, divide immediately following the division of the two MS cells. In addition to E lineage defects, mutant embryos fail to differentiate pharyngeal muscle and therefore appear to have defects in the MS lineage. We have focused our efforts on the molecular cloning and characterization of
gut-2. We chose to study
gut-2 because we had multiple alleles (two, both EMS-generated) and the maternal effect lethal phenotype of
gut-2 homozygous mutant embryos did not appear any different from that of mutant opposite a deficiency for the
gut-2 region. In a non-complementation screen for new
gut-2 alleles we have, however, isolated a new class of allele with a more severe mutant phenotype. These new alleles are sterile and slow-growing, and most likely represent small deficiencies which remove additional loci in the
gut-2 region. We have previously rescued both allele classes using the same genomic fragment; these rescue results suggest that the severe alleles may represent the null phenotype. Using PCR we have demonstrated that the
gut-2 locus is removed in the more severe alleles, while sequencing of the two EMS-generated alleles demonstrated that they are different missense mutations affecting the same codon. The
gut-2 sequence has revealed that the gene encodes a small polypeptide with strong sequence similarity to a S. cerevisae gene product predicted by the yeast sequencing project. The worm and yeast gene products appear to belong to a family of proteins related to core spliceosomal snRNP proteins (Sm proteins). These core proteins have been previously identified biochemically, but the newly-identified members of the family had not been observed in previous biochemical characterization of spliceosomal snRNPs, and are of unknown function. Amino acid comparisons among family members has defined an SM domain of high sequence homology, and GUT-2 shares most of these conserved residues, and shares all of the absolutely conserved residues. The missense mutations in the maternal effect lethal alleles change a highly conserved glycine residue. We are currently investigating whether or not
gut-2 affects the expression or distribution of other gene products during embryogenesis. We are also analyzing the expression and distribution of GUT-2 protein using an epitope-tagged
gut-2 transgene.