Worm Breeder's Gazette 13(3): 36 (June 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.
Genetic analysis suggests that the fem genes of C. elegans promote male development in the somatic tissues of X0 animals by negatively regulating tra-1 In XX animals, the activity of one or more of the fem genes is negatively regulated by tra-2 and tra-3 .(1)We are interested in the molecular mechanisms involved in the regulation and action of the FEM proteins We have tested for direct interactions involving either FEM-1 or FEM-3 using the yeast two-hybrid system and by immunoprecipitation of proteins synthesized in rabbit reticulocyte Iysates
In both assays, a C-terminal, 344 amino acid fragment of TRA-2 Ainteracts specifically with FEM-3 (We are grateful to J. Kimble for kindly providing a cDNA clone encoding full-length FEM-3 ) The sequence of tra-2 predicts that TRA-2 Ais an integral membrane protein with a C-terminal intracellular domain(2), whereas FEM-3 is likely to be an intracellular protein.(3) Yeast which coexpress the C-terminal region of TRA-2 Afused to the activation domain of GAL4 with FEM-3 fused to the DNA-binding domain of GAL4 also express reporter genes under the control of a GAL UAS. Neither fusion protein alone activated reporter gene expression. Immunoprecipitations using the monoclonal antibody 9E10, which recognizes a c-myc epitope, provided further evidence for a direct interaction between FEM-3 and TRA-2 A.Following cotranslation in reticulocyte Iysates of mRNAs encoding myc epitope-tagged FEM-3 and the C-terminal fragment of TRA-2 A,both proteins coimmunoprecipitate with 9E10, although the antibody does not recognize the TRA-2 AC-terminal fragment alone. We are currently trying to map the domains in each protein which mediate their interaction.
One of us previously showed that the C-terminal region of TRA-2 Acauses partial feminization of X0 animals when overexpressed, suggesting that it is capable of regulating at least one of the fem gene products.(4) Direct binding of FEM-3 to the cytoplasmic, C-terminal region of TRA-2 Amay be crucial to the regulation of FEM activity in XX animals. We tested whether excess FEM-3 could saturate the regulatory capacity of tra-2 and tra-3 and cause inappropriate masculinization in transgenic nematodes carrying fem-3 fused to the heat shock promoter. Such animals exhibit extensive masculinization of somatic tissues following heat shock, suggesting that in the soma, as in the germline, fem-3 is a key target of regulation by upstream genes in the sex determination pathway. We are currently testing the requirements for the chromosomal fem genes in our heat shock fem-3 lines, and we are examining the localization of myc-tagged FEM-3 using monoclonal antibody 9E10.
(2) Kuwabara, P., P. Okkema, and J. Kimble. 1992. Mol. Biol. Cell 3: 461-473.
(3) Ahringer, J., T. Rosenquist, D. Lawson, and J. Kimble. 1992. EMBO J. 11: 2303-2310.
(4) Kuwabara, P. 1993. C. elegans Meeting Abstracts, p. 256.