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.
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.