The
bli-4 gene of C.elegans encodes products belonging to the
kex-2/subtilisin family of proprotein convertases. At least 4 isoforms, named blisterases A, B, C, and D, arise by alternative splicing of a 5' common region to specific 3' exons. So far, 3 classes of mutants have been identified according to complementation testing and the phenotypes observed. Class I mutants display blistering of the adult cuticle layers; class II mutants are embryonic lethals; and class III mutants are phenotypically similar to class II mutants except 30% arrest development at an early larval stage. Because of the complex structure of the gene and the different kinds of phenotypes observed, we want to correlate the function of individual blisterase with the different phenotypes. To date, only blisterase A is known to function in the assembly or maintenance of the adult cuticle. The blistered allele,
e937, was found to result from a deletion of the blisterase A specific exon. Functions of the other isoforms are still unknown. We hypothesize that the 4 blisterases may be functionally redundant to a certain degree based on their structural similarities and on the fact that the
e937 mutation is only incompletely penetrant. Furthermore, previous results indicate that a minigene encoding blisterase B, and one that encodes blisterases B, C, and D rescue
e937 animals. We are now in the process of constructing individual minigenes specific for each isoform in order to further dissect the function of each individual blisterase. However, functional redundancy observed by mutant rescue may be due to altered expression from the extrachromosomal transgenic array. Therefore, we plan to inject the constructs at both a high (100 ng/ml) and low (1 ng/ml) concentration in order to compare the effect of copy number on rescue ability. In this way, if, for example, blisterase D is playing an essential role in embryonic development rather than in adult cuticle assembly or maintenance, then a D-specific minigene may still rescue both blistering and lethality at high concentration due to overexpression, but will only rescue lethality at low concentration. On the other hand, if blisterase D is functionally redundant with blisterase A, the D minigene is expected to rescue blistering even at low concentration. We expect an interesting rescue pattern from these experiments, and hopefully we can thus delineate the individual functions of the different isoforms. This work is supported by a grant from MRC Canada.