Worm Breeder's Gazette 14(4): 25 (October 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.


Feizhou Liu1, Chris C. Bauer1, Irving Ortiz1, Richard G. Cook2, Henry F. Epstein3

1 Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
2 Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
3 Departments of Neurology, Biochemistry, and Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030

 C. elegans thick filaments are complex structures consisting of
multiple protein molecules. Biochemical and electron microscope studies
show that the outer layer of the filaments contain myosins A and B,
which are differentially localized in central and polar regions
respectively. The underlying layers contain paramyosin, a homolog of
myosin rod. The cores contain a second population of paramyosin
(distinguishable by 2D IEF/PAGE) associated stoichiometrically with
three proteins, designated as P20, P28, and P30 (Deitiker and Epstein,
J. Cell Biol., 123, 303-311, 1993). Image processing and structural
modeling of electron micrographs show that the cores consist of seven
subfilaments (two strands of paramyosin for each) cross-linked by
internal protein rings (Epstein et al., J. Struct. Biol., 115, 163-174,
1995). In order to study potential roles of P20, P28, and P30 in the
assembly of thick filaments, efforts were made to purify these three
proteins. Thick filaments were purified by sedimentation on 19-38%
sucrose gradients and the proteins were precipitated from the
filament-containing gradient fractions. The proteins were then separated
on SDS-PAGE and transferred to PVDF membranes. Protein bands of interest
were subjected to endoproteinase Lys-C digestion, and the resulting
peptides were separated by HPLC and then microsequenced. Both P28 and
P30 are novel proteins, and cosmids (T14G12 and C46G7) containing the
genes encoding them have been completely sequenced by the C. Elegans
Genome Project. P28 is a 201 amino acid protein that is composed of only
beta sheets interspersed with turns as predicted by secondary structure
programs. P30 is a 250 amino acid protein that is predicted to have both
beta sheets and alpha helices.  Each of the three proteins have been
mapped to different chromosomes. P20, which has not been completely
sequenced, is located on chromosome II near the unc-52 gene,, whereas
P28 maps to the X chromosome and P30 to chromosome IV near unc-82.
Anti-peptide antibodies have been raised against both P28 and P30. In
preliminary experiments the antiserums have been shown to label
body-wall muscle A-bands and isolated thick filaments and cores by
immunofluorescence and immunoelectron microscopy. Anti-P28 has been
affinity purified against its peptide antigen. The resulting antibody
labels isolated cores with a paramyosin-like periodicity and appears to
react along the entire length of both cores and thick filaments. These
results are consistent with the predictions of the structural model of
Epstein et al. (1995) for the inner core proteins. Present approaches to
characterize the function of P28 include isolation of a strain with a
transposon insertion in the P28 gene by R. H. A Plasterk and K. van der
Linden and injection of constructs driven by the unc-54 promoter (Okkema
et al., Genetics, 135, 385-404, 1993) which produce anti-sense RNA to
P28 mRNA. The possible identity of P30 and unc-82 is being tested by
cosmid rescue experiments. Both P28 and P30 have been expressed in E.
coli as GST fusion proteins for future biochemical experiments.