Worm Breeder's Gazette 13(1): 58 (October 1, 1993)

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.

Feminization of XO animals by TRA-1

David Zarkower, Jonathan Hodgkin

MRC-LMB, Hills Road, Cambridge CB2 2QH, England

The tra-1 gene is necessary and sufficient for female somatic development, while absence of tra-1 results in male somatic development. Two mRNAs are transcribed from the tra-1 locus, both encoding proteins with zinc fingers. The longer protein, TRA-1 A(formerly Tra-1 L),has five zinc fingers that are remarkably similar to those of the mammalian proteins GLI and GLI3 and the Drosophila protein ciD. The shorter protein, TRA-1 B(formerly Tra-1 S),is a truncated form of TRA-1 Awith only the first two zinc fingers.

Surprisingly, TRA-1 Bappears not to bind DNA in vitro (Zarkower and Hodgkin, NAR 21:3691, 1993). Not only does TRA-1 Bfail to select binding sites from a pool of random oligonucleotides, but removing fingers 1 and 2 from TRA-1 Adoes not abolish binding. Furthermore the site selected by TRA-1 Amissing fingers 1 and 2 differs by only one C residue from the site preferred by the intact protein. These results make us very suspicious that TRA-1 Bdoes not bind DNA in vivo even though its mRNA is more abundant than that of TRA-1 A.What is TRA-1 Bdoing? It may do nothing. However both proteins contain a domain (the "gf" domain; WBG 12:3, p29 )in which missense mutations can render tra-1 insensitive to regulation. This suggests that TRA-1 Bmight act to enhance the activity of TRA-1 Aby titrating negative regulators (eg the FEMs).

If TRA-1 Bacts to enhance TRA-1 A,several predictions follow. First TRA-1 A,if expressed at high enough levels, should be able to perform all tra-l functions in the absence of TRA-1 B.Second, TRA-1 Bshould have no effect on animals that lack tra-1 even if expressed at high levels. Third, coexpression of TRA-1 Aat a given level with TRA-1 should provide more tra-1 function than the same level of TRA-1 Aalone. Fourth, this enhancement of Tra-1 Aactivity by Tra-1 Bshould require an intact gf domain in Tra-1 B.We want to test these predictions, so we have made heatshock constructs containing each cDNA and begun to assay them in transmitting transgenic lines.

We find that a Tra-1 Aheatshock construct can feminize XO males. The resulting rather gender-confused animals are feminized in all tissues examined: they have a two-armed gonad with sperm and a few oocytes, have vulvae, make yolk, have a variably feminized tail, and do not display mating behavior. XX animals are unaffected. Only Tra-1 Awith a mutant gf domain causes the feminization; presumably XO animals contain sufficient fem activity to regulate the wild type protein. In these experiments endogenous Tra-1 also is present, so we do not know if any of the effects are mediated by the endogenous protein. We are now testing the predictions further in animals that lack tra-1 .

How is tra-1 regulated? The existence of the gf domain and presence of similar mRNA levels in both sexes suggest that regulation is post-translational. One possible model is that in XX animals the Tra-1 proteins are nuclear, but in XO animals they are sequestered in the cytoplasm by the FEMs. As a test of this model we have tagged heatshock Tra-1 A(gf domain wild-type) with a c-myc epitope and stained both sexes with the 9E10 monoclonal antibody. There is no staining in animals without the transgene or in the absence of heatshock. In both sexes the 9E10 staining is predominantly nuclear. Thus it appears that Tra-1 Ais nuclear even when it is inactive (in XO animals). Again, we will need to stain animals that lack tra-1 . If this result holds up, the model will need revision. Perhaps in XX animals one or more of the FEM proteins either is not in the nucleus or is in an inactive state and thus unable to regulate Tra-1 A.One possibility is that TRA-2 sequesters the FEMs in the cytoplasm or at the cell membrane of XX animals.