Worm Breeder's Gazette 10(2): 28

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.

The Number of the Beast: Progress in Cloning tra-1

Jonathan Hodgkin

Attempts to clone the major autosomal sex-determining gene tra-1 (
Genes & Dev.  1: 731) have been frustrated by the absence of 
transposon insertions either in this gene or anywhere near it.  
Spontaneous mutations at the locus have been obtained in transposition-
active strains (e.g.  the weak tra-1 mutation e2332, described in WBG 
10-1:113) but none of these appears to have resulted in the insertion 
of Tc1, Tc3 or Tc5.  Also, natural insertions of Tc1 in the genetic 
region around tra-1 have been mapped in six high-copy-number strains, 
but only in one of these, N62, are there any Tc1 polymorphisms in the 
vab-7 - dpy-18 interval (2.5 map units flanking tra-1).  The closest 
insertion, eP1 (WBG 8-1:45) is about 0.7 map units to the right of tra-
1, and therefore likely to be at least 150kb away.  Furthermore, 
attempts to walk in either direction from eP1 were unsuccessful.  The 
eP1 insertion was cloned as a 9.6 kb restriction fragment, and unique 
genomic fragments from the 8kb flanking the insert were used to probe 
cosmid libraries (pJB8, lorist) and phage libraries (both amplified 
and primary).  In none of these were any positives found, although 
between 10 and 50 genome equivalents were probed in each experiment.  
Thus, eP1 appears to be flanked by regions that clone very poorly or 
not at all, using standard vectors.
Fortunately, the eP1 unique probe did hybridize to 2/2000 YAC 
colonies, from the herd constructed by Bob Waterston.  These two YAC's 
were kindly purified by Alan Coulson and John Sulston, who used them 
to probe the gridded array of 1250 cosmids representing the end clones 
of 625 contigs.  The smaller YAC (150kb) detected nothing, but the 
larger (200kb) detected a small contig, #666.  Two cosmids that span 
this contig were used as probes on Southern blots of genomic DNA from 
a series of tra-1 mutants.  Initially, only mutants that were good 
candidates for carrying rearrangements were examined.  One cosmid 
revealed rearrangements in three tra-1 alleles: the acetaldehyde-
induced class B1 loss-of-function allele, e1488, and two spontaneous 
complex gain-of-function mutants, e2332 and q245 (the latter isolated 
and generously donated by M.  Kathryn Barton).  In subsequent 
recombination experiments, all three of these mutations have 
consistently cosegregated with the rearrangements, placing the cosmid 
less than 0.1 map unit from tra-1.  Also, most wild strains, including 
the ancestors of the spontaneous mutants, do not have rearrangements 
in this region.  It therefore seems likely that this cosmid includes 
part of tra-1.  Rearrangements have been tentatively detected in 
several other tra-1 mutants, using either this cosmid or the adjoining 
cosmid as in a probe.  No rearrangements have yet been detected in 
putative null mutants, or in [32P]-induced mutants.
The tra-1 gene is large, in terms of recombination (ca.  0.2 map 
units), and therefore expected to be physically large as well, 
possibly too large to be contained within contig 666, which is less 
than 60 kb across.  In the hope of expanding this contig, the 200kb 
YAC was labelled and used as a probe on a primary lambda 2001 library 
constructed by Alan Coulson.  35 phage were picked, purified and 
analyzed by fingerprinting and restriction digests.  All fall within 
the same 60-70 kb, which represents a small extension of contig 666, 
but leaves at least 100 kb of YAC still unaccounted for.