Worm Breeder's Gazette 13(5): 56 (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.
Department of Medical Genetics. University of British Columbia bli-4 encodes at least four protein isoforms (termed blisterases A, B, C, and D) that arise via differential splicing of a common region onto different 3' exons. Out of fourteen bli-4 alleles identified to date, only one allele, e937, causes blistering of the adult cuticle. However, this phenotype is incompletely penetrant as only about 90 percent of homozygotes exhibit blistering. From Northern and RT-PCR analysis, e937 homozygotes do not express blisterase A, but do express blisterases B, C, and D. We have also observed that blistering is more severe (and 100 percent penetrant) when e937 is in heteroallelic combination with most class II (non-complementing) lethal alleles. At this time, we have mapped 3 class II mutations to the region common to all four transcripts - suggesting that at least these lethal alleles interfere with the production and/or function of all products from bli-4. Taken together, these data indicate that reduced penetrance of blistering in e937 homozygotes may be due to functional redundancy within the gene. One possible explanation for complete penetrance of blistering in e937/class II worms is that there is one less copy of a gene capable of producing blisterase B,C and D in e937/class II worms than in e937/e937 worms. We predicted, therefore, that at least one of the other products of bli-4 can partially substitute for blisterase A activity in e937/e937 worms. In order to test the hypothesis that reduced penetrance is due to functional redundancy within the bli-4 gene, two minigenes were constructed for transformation rescue. One minigene encodes only blisterase A, the other encodes only blisterase B. Based on predicted protein structures for the blisterases, blisterase B most closely resembles blisterase A; unlike C and D, blisterases A and B lack a cycteine-rich region and/or transmembrane domain in their carboxy tail. We predicted that blisterase B alone may rescue blistering if it is expressed from a transgene in high copy number; if the B isoform has slight overlapping function with blisterase A at endogenous expression levels in the e937 homozygote, perhaps overexpression (due to high exogenous copy number) will further reduce or even completely rescue blistering in e937 homozygotes. When DNA that encodes only blisterase A is coinjected with rol-6(su1006) into bli-4(e937) homozygotes, no Rol progeny are ever blistered, as expected. However, when a transgene that encodes only blisterase B is coinjected with rol-6, some, but much less than 90% of Rol progeny blister; blisterase B partially rescues blistering. DNA injected in Stable line % Blistering Bli,Rol/total Rol e937/e937 worm of Rollers rol-6 KR2872 79 110/139 blisterase B + rol-6 KR2868 43 79/185 blisterase B + rol-6 KR2870 16.8 17/101 blisterase B + rol-6 KR2869 7.8 41/523 blisterase B + rol-6 KR2871 3.4 7/206 blisterase A + rol-6 KR2859 0 0/>1000 We are currently examining whether there is a correlation between the number of extrachromosomal copies of blisterase B (or the expression levels of blisterase B) and the penetrance of blistering. To simplify this analysis, we decided to construct strains with integrated copies of blisterase B; 480 Rollers from KR2868 were exposed to 3000 rads of gamma radiation (thanks to Andy Fire for protocol). Four independent lines were acquired that each exhibit 100% Rolling (preliminary evidence for an integrated array). Also, each of these lines exhibit different levels of blistering, ranging from 0% to approximately 15%. These integrated lines will be used to quantify blisterase B DNA and/or RNA levels. Reduced penetrance of blistering in bli-4(e937) is most likely the result of partial functional redundancy between blisterase A and other isoforms. From this analysis, blisterase B alone demonstrates functional redundancy with blisterase A and we are currently investigating the ability of the remaining isoforms to rescue blistering.