Worm Breeder's Gazette 10(1): 62

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.

Transformation: DNA and Sex

W. McCoubrey and P. Meneely

Figure 1

Madl and Herman (Genetics 93: 393-402, 1979) demonstrated that C.  
elegans is sensitive to slight differences in the balance between the 
number of X chromosomes and sets of autosomes (X/A ratio).  Their 
experiments showed that 4A;3X animals (X/A=0.75) were fertile 
hermaphrodites, while 3A;2X worms (X/A=0.67) were normal males.  
Further, the presence of X duplications in such males resulted in 
hermaphroditization, the severity of which was proportional to the 
size of the duplication.  These experiments led to the hypothesis that 
there are numerous sites (at least 4) on the X chromosome which are 
somehow 'counted' and compared to the number of autosomal sets during 
sex determination in the developing animal.  To determine the nature 
and distribution of these X-linked sites, we have begun a series of 
microinjection experiments in which cloned pieces of the X chromosome 
are assayed for their ability to influence sex determination.
Briefly, X-linked clones are injected into the mitotic gonad (
Stinchcomb et al., Banbury Report 20: 251-263, 1985) of 4A;4X 
hermaphrodites.  These animals are mated to diploid (2A;1X) males 
carrying a small duplication of the X (mnDp8).  In the absence of 
injected X chromosomal material, the duplication is sufficiently small 
that there is no effect on sexual development; the duplication appears 
to increase the sensitivity of the animal to the injected DNA, however.
In the first experiments, a pool of DNA representing approximately 
120 kilobases of the X was injected.  Several of the injected worms 
produced broods which included no males among cross progeny.  We 
inferred from this result that the males may have been pushed over the 
threshold from male to hermaphrodite development by the injected DNA.
One plasmid, pCeA4, obtained from Mike Krause and containing part of 
the X-linked actin gene act-4 [See Figure 1] in pBR322, was selected 
for further study.  Mosaic intersexes and abnormal males are observed 
among the 3A;2X progeny of worms injected with this plasmid.  Abnormal 
males are those in which some sexually dimorphic characteristics are 
poorly developed, e.g.  incomplete gonads and poorly developed tails.  
By contrast, mosaic intersexes possess normal or nearly normal 
characteristics of both sexes, typically an almost normal male tail 
and body morphology and a hermaphrodite gonad, often containing 
embryos and occasionally a vulva.  The two classes occur with 
approximately equal frequencies and represent one-sixth to one-tenth 
of the 3A;2X cross progeny.  In control experiments, injection of 
either pBR322 alone or a clone of the autosomal act-3 gene led to 
neither abnormal males or intersexes among over 100 2X;3A progeny.  We 
conclude that the X-linked DNA in pCeA4 contains a sequence (or 
sequences) which is recognized as being X-linked and therefore 
influences the assessment of the X/A ratio.  Since more than 90% of 
the bases in the coding regions of act-3 and act-4 are identical and 
pCeA4 contains neither 5' nor 3' non-coding regions, the most likely 
candidate for the sequence(s) is within the introns, which are not 
We are currently attempting to define further the X-linked sequence 
involved by injecting the subclones of pCeA4 indicated in the figure.  
Preliminary results rule out the plasmid containing the two small 
introns and implicate the regions containing part of the large intron. 
In addition, we are extending our studies to examine other X-linked 
genes such as myo-2 to determine the frequency and sequence 
conservation of other hermaphroditizing sites on the X-chromosome.

Figure 1