Worm Breeder's Gazette 10(1): 33

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.

Molecular Cloning of unc-5 While Walking to fem-1

A.M. Spence

I am attempting to clone the fem-1 gene, which is essential for male 
development and hermaphrodite spermatogenesis in C.  elegans (Doniach 
and Hodgkin.  Dev.  Biol.  106:223.  1984).  In the absence of mutator-
induced alleles of fem-1, I initiated a walk from a Bergerac (N62)
/Bristol Tc1 polymorphism, eP14, which had been cloned by J.  Hodgkin, 
and which maps 0.1 mu to the left of the gene.  A region of 60 kb 
flanking the polymorphism proved to be unrepresented in the cosmid 
libraries of A.  Coulson and J.  Sulston, and this region was 
traversed with clones derived from two of their lambda libraries.  The 
eP14 contig now spans 217 kb, including two cosmid contigs of 70 kb 
each, and both ends again lie in regions which appear to be absent 
from the cosmid libraries.
The unc-5 gene maps between eP14 and fem-1, 0.03 mu from the latter (
Doniach and Hodgkin, op.cit.), and may provide a more favorable point 
at which to resume the walk to fem-1.  D.  Moerman and R.  Hoskins 
kindly provided strains carrying the unc-5 alleles st1000, st1002, and 
st1003, which arose in the mut-6 background of RW7096.  DNA isolated 
from each strain after 6-10 outcrosses to N2 contained 2-5 Tc1-
containing EcoRI fragments not present in N2.  Two of the novel Tc1 
elements in st1003 cosegregated with unc-5 in 24/24 recombinants in 
the dpy-13-unc-5 interval and 18/18 recombinants in the unc-5-bli-6 
interval.  Both elements were absent from a revertant of st1003.  One, 
eP52, has been cloned as a 5 kb EcoRI fragment.  Sequences flanking 
the Tc1, when used to probe Southern blots, detect a different 
polymorphism between N2 and each of the mut-6-induced  
and between each allele and a revertant of that allele.  This 
observation leads me to believe that I have cloned part of the unc-5 
locus.  The st1003/N2 polymorphism can be accounted for by Tc1 
insertion at an EcoRI site, resulting in destruction of that site.  
Simple insertion of Tc1 cannot account for the pattern of 
hybridization in st1000 or st1002, although the polymorphic fragment 
in each case includes Tc1.  None of the patterns seen in the 
revertants can be explained by simple excision of Tc1, and none is 
identical to the wild-type hybridization pattern.  More accurate 
definition of the extent of the gene and more adequate explanation of 
the rearrangements observed in the mutants and their revertants should 
be possible when I have cloned DNA spanning the locus.  To this end, 
and to continue the walk to fem-1, I have identified several clones in 
a lambda library of C.  elegans DNA which hybridize to an eP52-derived