Worm Breeder's Gazette 14(1): 59 (October 1, 1995)

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.

Cosmids Rescue of ego-3

Li Qiao and Eleanor Maine

Dept. of Biology, Syracuse University, Syracuse, NY13244

     We have reported the genetic characterization of a set of ego genes
(Madison,'95; Genetics, in press). Mutations in these genes can enhance
the weak glp-1 loss-of-function mutation bn18ts. In addition, these ego
mutations cause multiple phenotypes when separated from bn18. Our current
research involves cloning and molecular characterization of some of these
ego genes, including ego-3.

     ego-3(om40) mutants have defects in both somatic and germline
tissues.  om40 animals are severely Unc during larval stages. L3 and early
L4 stage om40 larvae have arrested germline proliferation, while older
om40 adults have various defects including delayed spermatogenesis,
abnormal oogenesis and proximal proliferation. Based on the om40/yDf8
phenotype, om40 appears to be a loss-of-function mutation. Therefore we
are trying to clone ego-3 by cosmid rescue. Previously, we genetically
mapped ego-3 ~0.7 mu to the left of unc-76 and very close to unc-61 on
LGV. After comparing the genetic and physical maps, we chose 13 cosmids
spanning the ego-3 gene region (see figure, notice there are two gaps) and
injected them into N2 germlines to generate transgenic animals. (Thanks to
Siqun Xu and Andy Fire for their help with the microinjection technique!)
The resulting transformed lines were then crossed into an ego-3(om40)/DnT1
strain. ego-3(om40);rol animals were tested for rescue. Two overlapping
cosmids (F26G12 and M05D1) can rescue the Unc phenotype of om40. However,
they only partially rescue the germline phenotypes. Most om40 animals
carrying either cosmid are sterile although they are not Unc. Under the
dissecting microscope, we can observe about 25% sterile progeny from
om40/+ mothers, which becomes 21.5% and 23% for heterozygous mothers
carrying cosmid F26G12 and M05D1 respectively. It has been observed that
germline phenotypes are often difficult to rescue completely. So we
further studied various aspects of the germline phenotype in ego-3 ;rol
animals by DAPI staining. In om40 adults, oogenesis preceeds
spermatogenesis about 82% of the time. In contrast, spermatogenesis
preceeds oogenesis in more than 60% of ego-3;rol animals carrying F26G12.
om40 animals carrying M05D1 have germline phenotypes similar to om40
alone. We are examining the early germline phenotype of these transformed
animals now to see whether it is rescued. Wild type N2 animals with either
cosmid have normal germline proliferation.  Interestingly, N2
hermaphrodites with the M05D1 cosmid are all Egl. From the genetic map, we
suspect the egl-1 gene lies on this cosmid.

     Our cosmid rescue data suggest at least two possibilities. ego-3
could be in the overlapping region of cosmids F26G12 and M05D1. If so,
then germline expression of ego-3 from M05D1 must be very low.
Alternatively, the ego-3 phenotypes may be caused by mutations in two
closely linked genes. The somatic phenotype might be caused by a mutation
in a gene in the F26G12-M05D1 overlap, while the germline phenotype might
be caused by a mutation in a gene on the left part of F26G12. Currently,
we are testing whether cosmid C54G10 can rescue ego-3  (see
figure).(Thanks to Alan Coulson for providing all the cosmid strains!)

Genetic and physical map of the ego-3 region. (see WBG)