Worm Breeder's Gazette 13(5): 54 (February 1, 1995)

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.

Expression Studies and Progress Towards Identifying Substrates For bli-4.

Colin Thacker, Martin Srayko, Readman Chiu, Ann Rose

Department of Medical Genetics, University of British Columbia, Vancouver, B.C., V6T lZ3.

bli-4 encodes at least four gene products with sequence
similarity to the kex2/subtilisin-like family of proprotein
convertases. These different isoforms have structural
similarities to their mammalian counterparts which participate
in both the regulated and constitutive secretory pathways.
This suggests that the individual bli-4 gene products
may show differences in tissue expression patterns. Furthermore,
the blisterases differ only at their carboxy-termini
which we speculate provide information that directs the
various isoforms to specific intracellular locations.
Staining of transgenic lines that carry a bli4-lacZ promoter
fusion construct show b-GAL expression in a variety of
tissues including hypodermal and neural cells. Expression
of bli-4 in hypodermal cells is not unexpected considering
that the e937 mutation results in blistering of the adult
cuticle which is secreted by these tissues. Staining is
first observed in hypodermal cells of three-fold embryos.
This developmental period corresponds to the arrest stage
of the lethal alleles isolated for bli-4. Preliminary
examination of arrested embryos produced by various bli-4
lethal alleles showed an inability to complete elongation.
This may be due to the lack of production of a intact cuticle.
We are currently raising antibodies against the different
isoforms in order to more definitively characterize their
expression patterns and intracellular localization.
This will be achieved by performing immunofluorescence
to determine the temporal and spatial expression of each
blisterase product whereas the intracellular localization
can be delineated by immunoelectron microscopy.
The ultimate goal of our research efforts is to understand
the functional role of the bli-4 convertases, thus it is
important to identify the substrates which are processed
by the blisterases. Towards this end, we are attempting
to clone and characterize some of the other blister genes
which may encode substrates or accessory enzymes that
are involved in the cleavage process. We are also trying
to identify substrates by screening for extragenic suppressors.
A lethal allele that we have characterized, hl99, contains
an amino acid substitution (His-Leu) in the N-terminus
in a region proximal to the protease domain. hl99 in trans
with e937 gives less than 5percent blistered hermaphrodites,
compared to 100percent for most other bli-4 lethals, suggesting
that h199 is a hypomorph retaining some protease activity.
hl99 thus represents a good candidate to initiate suppressor
A second member of the kex2/subtilisin-like family that
we are studying was originally identified as a sequenced
cDNA (cml0b9) characterized by the genome group. We have
generated more extensive clones and sequence and found
it to be identical to a group of cDNA clones isolated by Yuji
Kohara and colleagues designated YK538. This gene maps
just to the left of unc-101 on chromosome I.
This work was supported by grants from the MRC (Canada)
and the B.C. Health Research Foundation. Many thanks to
Yuji and colleagues for providing the YK538 cDNA clones.