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.

Deciphering the Function and Expression Pattern of FEM-2

Dave Hansen, Petra Jackle-Baldwin, Dave Pilgrim

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