Worm Breeder's Gazette 10(2): 25

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.

X-linked Sequences That Cause Sex Transformation of Males

William McCoubrey, Kim Nordstrom and Philip Meneely

Figure 1

In the last WBG (10(1): 62-63), we described our initial experiments 
to define the molecular basis of the X-linked signal for sex 
determination in C.  elegans.  To summarize, cloned X-linked sequences 
were injected into the distal arm of the gonad of tetraploid 
hermaphrodites.  The injected animals were mated to diploid males 
heterozygous for the X-chromosome duplication mnDp8 to produce 3A;2X 
cross progeny.  In the absence of injected DNA, the 3A;2X + mnDp8 
progeny are males (Madl and Herman, Genetics 93: 393-402, 1979).  
Injection of the act-4 plasmid pCeA4 (provided by Mike Krause) led to 
intersexual development of 3A;2X + Dp progeny.  The intersexes are of 
a mosaic type in which nearly normal structures of both sexes are 
observed in the same animal.  Injection of vector sequences, lambda 
DNA, or the autosomal gene act-3 had no effect on sex determination.  
We concluded that pCeA4 contained a sequence or sequences that are 
involved in assessment of the X/A ratio.
By testing subclones of pCeA4 we find that the transforming activity 
of pCeA4 resides in the 5' 783 bp of the plasmid (plasmid pCeA4.8, see 
figure); no detectable feminizing activity could be found in the 
remainder (plasmid pCeA4.4) by this assay.  Injection of a second 
subclone, pCeA130, which contains only 130 bp of the large intron and 
1 bp from the coding region, also led to production of intersexual 
progeny.  The transformation frequencies observed for all plasmids 
which give positive results are quite high: approximately one of every 
four fertile injected hermaphrodites produces intersexual offspring 
and about one-third of the 3A;2X + Dp offspring of such animals are 
intersexes.  We are currently using exonuclease III/S1 deletions of 
pCeA130 to determine the minimal sequence required to obtain 
transformation.  Initial results indicate that the insert retains 
feminizing activity after deletion of as much as 70 base pairs from 
the 5' end.  Thus we have identified a sequence of less than 130 base 
pairs, contained in the large intron of act-4, that appears to 
function as part of the signal for sex determination.
We have attempted to correlate the intersexual phenotype with the 
presence of the injected DNA using single worm dot blots probed with 
either vector or insert sequences.  One prediction is that all 
intersexes have the injected DNA while their normal siblings lack it.  
This proves not to be the case since some of the wild type males have 
the DNA and, in fact, some of the intersexes do not.  This procedure, 
however, only allows us to detect relatively high copy number 
sequences (>20,000 copies per worm based on hybridization with Tc1) at 
the level of the whole adult worm.  It is likely that the DNA needs to 
be present only within specific lineages and at specific stages of 
development to cause transformation.  In addition Stinchcomb et al.  (
Mol.  and Cell Biol.  5: 3484-3496, 1985) have observed that DNA 
microinjected into the distal arm of the gonad is maintained as an 
extra chromosomal array and is subject to mitotic loss.  Considering 
these complications, it is perhaps not surprising that we cannot find 
a correlation between transformation and detectable levels of the 
injected sequences.  We have tested normal males arising from the 
injection of pCeA4.4 and found the injected DNA.  Thus, this clone is 
present among the progeny at approximately the same level as the 
clones that give intersexes.
We have used the same assay to examine other X-linked genes for 
effects on sex determination.  A plasmid containing the myosin light 
chain genes, mlc-1 and mlc-2, (kindly provided by Claudia Cummins and 
Phil Anderson) has the same hermaphroditizing effect as the act-4 
plasmids.  This was also true for a phage containing part of the X-
linked myosin heavy chain gene, myo-2.  A plasmid, pGBA2.2 (provided 
by Guy Benian and Bob Waterston), containing a 2.2 kb region from the 
coding region of the same gene, however, had no effect.  This means 
that for all three regions of the X-chromosome tested, at least some 
sequence has had an effect on sex determination.  This leads us to 
believe that similar sites are very common on the X-chromosome, 
perhaps as frequent as one per gene.  We are currently subcloning both 
the mlc and myo-2 clones in hopes of identifying a consensus sequence 
for the X-linked sites involved in the sex determination signal.  So 
far we have injected only one autosomal gene; we are starting 
injections using other autosomal genes hoping to demonstrate that this 
sequence is rare or absent from the autosomes.

Figure 1