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.

Towards understanding reduced penetrance of blistering in bli-4(e937)

Martin Srayko, Colin Thacker, and Ann Rose

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.