Worm Breeder's Gazette 13(4): 43 (October 1, 1994)

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.

Some fem-2 Constructs Show Dominant/Negative Feminization of the Germline in Transgenic Worms.

Troy Johnson, Petra Jackle, David Pilgrim

Department of Biological Sciences, University of Alberta, Edmonton, Alberta.

  We have previously reported the preliminary molecular characterization of fem-2
(WBG 13(1) p59 ).We recently observed that transgenic lines carrying two altered fem-2
constructs resulted in a feminization of the germline (FOG) phenotype in transgenic
XO and XX animals.
  DP# DBP023 306Bamwas derived from a rescuing clone (023) by deleting the four
control bases of the Bam HI site in fem-2 .The deletion lies at the extreme 5' end of exon
4 and alters the consensus splice site. Transgenic XX b245ts animals containing the
306Bam construct are feminized, even at the normally permissive temperature of 20260C.
b245 /+XX animals appear to be normal hermaphrodites. The feminization is most
severe in b245 / b245 transgenic daughters of b245 / b245 transgenic mothers; these
animals appear to make no sperm at all, as assayed by DAPI staining and
self-sterility. They are fertile, however, if crossed to males. XO b245 transgenic
animals are also feminized at 20260C, but only in the germline, where apparent oocytes
are produced. The soma appears perfectly normal with a good fan and rays.
  The second plasmid (TJ004) was constructed by fusing 2.0 kb of fem-2 upstream
region and the first 400 bp of fem-2 coding region into Andy Fire's pPD22
.04lacZ-vector. Transgenic lines carrying TJ004 show the same FOG phenotype seen
above even in a N2 background at 20260C. We are not sure if this is due to differences in
the constructs or copy number in the arrays. The presence but not the relative level of
the transgene has been confirmed by PCR. We have not observed this effect in
transgenic worms carrying TJ011 which is identical to TJ004 except that lacZ is out of
frame. X-gal staining of worms containing TJ004 showed LacZ expression at the very
distal end of the gonad in X0 adults in only 1 or 2 cells. XX egg to L2 staining reveals
three blue cells whose identity is not yet confirmed (but two appear to be at the distal
ends of the gonad arms).
  The observation of a dominant-negative phenotype in transgenic animals
carrying the above constructs has suggested that overexpression of N-terminal parts of
fem-2 is able to interfere with normal fem-2 function in the germline but has no
apparent effect on the soma in the TJ004 animals. This suggests that the amino
terminal part of fem-2 may play a different role in the two tissues. In addition LacZ
expression appears to be tightly localized but the dominant mutant phenotype is seen at
the posterior part of the gonad, suggesting posterior transcriptional regulation of fem-2
activity. There is both a temporal and spatial difference in fem-2 activity between
males and hermaphrodites as indicated by X-gal staining. It is our hope that further
characterization of this phenotype will allow us to better understand the role of fem-2
in the germline.