Worm Breeder's Gazette 14(2): 54 (February 1, 1996)
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
FEM-2 falls into a class of PP2C enzymes that contain a long amino terminus in addition to the six conserved domains that are common to all PP2C classes. We have shown that the phosphatase activity of FEM-2 is not dependent upon the amino terminus. A GST-FEM-2 fusion protein missing the first 87 amino acids of FEM-2 was produced in bacteria. When assayed in vitro for its ability to dephosphorylate 32P-casein, truncated FEM-2 was indistinguishable from the full-length version. In the same assay, GST-FEM-2, with the full amino terminus, but engineered to carry the glycine to argenine change found in the temperature sensitive b245 allele (Pilgrim et al., 1995), showed greatly reduced phosphatase activity at 20°C. This finding suggests that the temperature sensitive phenotype may be due to general reduction in enzyme activity superimposed upon a temperature dependent process, rather than due to the production of an unstable protein. Truncated FEM-2 was also assayed for its ability to rescue a S. cerevisiae PP2C mutant (ptc1). FEM-2 missing 127 amino acids and expressed in yeast from the ADH1 promoter, was able to rescue the growth defect of ptc1 mutant in a pattern indistinguishable from full length FEM-2. This result suggests that the amino terminus is not directly involved in the dephosphorylating reaction but possibly in the regulation of this activity. It is unlikely that the amino terminus serves as a domain for the binding of a positive regulator because full length FEM-2 is able to dephosphorylate casein in vitro where no other proteins are present. Furthermore, no difference in activity is seen in full length and truncated forms. A polyclonal antibody was produced against a bacterially expressed GST-FEM-2 fusion protein. Using this antibody we investigated the expression of FEM-2 protein in wildtype worms using Western blotting and immunofluorescence on whole mount animals. Preliminary results on Western blots indicate that FEM-2 protein is present throughout the stages of the hermaphrodite life cycle, most strongly in the embryo. The antibody could also detect a cross reacting band of higher molecular weight in worm extracts of C. briggsae, but not in extracts of C. remanei. When used for immunofluorescence, expression was predominantly seen in the hermaphrodite germline, and in early embryos. Staining appeared to be strongest in the cytoplasm of the oocytes and early embryos. No staining above background was seen in the null mutants of fem-2. Characterization of FEM-2 expression in other stages and in males is under way. Pilgrim et al (1995). Molecular Biology of the Cell 6:1159