Worm Breeder's Gazette 15(3): 28 (June 1, 1998)
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.
|1||Department of MCD Biology, University of Colorado, Boulder 80309-0347|
|2||Current address: Zoologisches Institut, Abt. Steinmann-Zwicky, 8057 Zuerich, Switzerland|
The male-determining gene her-1 has two sex-specific transcripts: the larger one, which is necessary and sufficient for masculinization, driven by an upstream promoter P1 and the smaller one, which has no known function, by an internal promoter P2 in the large second intron of the gene. We have reported previously that transgenic arrays of a 3.4-kb P2-region fragment including all of intron 2 appear to mimic phenotypes resulting from sdc loss-of-function [sdc(lf)] mutants, including suppression of XO lethality and masculinization of XX and XO animals in a xol-1(lf) background. We have obtained evidence that these transgene effects are copy-number dependent, supporting our view that they result from in vivo titration of negative regulators encoded by the sdc genes. We analyzed two lines that carried integrated arrays of P2 sequences, driving a lacZ reporter in one case (ctIs1) and a GFP reporter in the other (ctIs32). One copy of the ctIs1 array did not suppress xol-1(lf) XO lethality whereas one copy of ctIs32 did so. Moreover, the ctIs1 reporter was expressed male-specifically whereas the ctIs32 reporter was expressed in both sexes, suggesting that the P2 promoter might be present in too many copies to be appropriately regulated. Southern blot analysis showed that ctIs32 includes 6.4 times more P2 sequences than ctIs1, consistent with titration as the cause of the observed sdc-like phenotypes. We have taken advantage of the masculinizing effects of P2 promoter sequences in xol-1(lf) XX animals to perform a deletion analysis, in order to define the sites involved in causing these effects. We prepared a series of deletion constructs missing various segments of the 3.4-kb P2 region, injected each into xol-1(lf) animals, and scored between 5 and 25 transmitting lines carrying each construct for masculinized animals in the F2 progeny. We find that multiple regions in P2 contribute to the putative titration effects. A 0.4-kb segment near the middle of the intron masculinizes weakly alone, but its effect is enhanced by the presence of sequences in other more P2-proximal and P2-distal segments of the intron. P1 sequences do not masculinize alone. They can enhance masculinization by P2 sequences, but only if the putative negative regulatory site defined by her-1(gf) mutations just upstream of the transcription start site is intact. These results suggest a cooperative mechanism of her-1 regulation, specifically, that the repression of transcription from P1 in XX animals is likely to be assisted by sequences in the P2 region.