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.

A 7.2 kb Genomic Fragment Rescues gut-2.

Melinda Moseley, Jocelyn Shaw

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.