Espression studies and progress towards identifying substrates for
bli-4. Colin Thacker, Martin Srayko, Readman Chiu, and 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 studies. 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.