Functional analysis of the gene bli-4 in caenorhabditis elegans

• Main Author: Burgstrome Jones, Alana Kristine
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/5904

Many biologically active compounds are first formed as inactive precursors. Their activation results from proteolytic cleavage, largely accomplished by a group of enzymes known as KEX2/subtilisin-like proprotein convertases, or kexins. This family of enzymes has been described in a number of species, from prokaryotes (B. subtilis), to unicellular eukaryotes (S. cerevisiae), as well as multicellular organisms (Drosophila, mouse, human). Recently, an additional member of this convertase family was identified in the nematode Caenorhabditis elegans (Peters et al, 1991). The gene encoding the proprotein protease (bli-4) produces four distinct protein isoforms, all of which show sequence similarity to members of the kexin family. The purpose of the series of experiments reported here was twofold. First, to determine whether there was a conservation of function between the four isoenzymes produced by the gene bli-4 and kexin members from S. cerevisiae and humans. In order to achieve this goal, I have tested two putative homologues of bli-4, KEX2 and hfur, for functional rescue of the viable blistering mutation in bli- 4. The second goal of this research was to analyze the control of bli-4 expression. To address this question, deletion analysis of the 5' flanking DNA was performed, searching for critical regions that may function in the control of timing and tissue specificity of bli-4. The approach taken to address the question of functional conservation of the four isozymes of bli-4 and other members of the kexin family took advantage of the ability to genetically transform C. elegans through germline injection. A genomic fragment containing the yeast gene KEX2 (provided by R. Fuller, Stanford, CA) and a cDNA clone of the human gene hfur (provided by G. Thomas, Portland, OR) were cloned into an ectopic expression vector carrying a heat-shock promoter (provided by A. Fire, Carnegie). The heat shock promoter (P. Candido, University of British Columbia) expresses in many of the same tissues as bli-4. A cDNA clone of one of the BLI-4 isoforms was used as a positive control. These constructs were injected into the gonad of bli-4(e937) homozygous hermaphrodites. Progeny of injected worms were heat-shocked to induce expression of the transgenes, and then screened for phenotypic rescue. In addition to this approach, functional rescue of S. cerevisiae KEX2 deletion mutants using one bli-4 isoform was tested. The isoform chosen has a predicted structure similar to the native yeast kex2p. However, this construct, when expressed in yeast, did not rescue KEX2 mutants, even though the control experiment using a KEX2 clone did rescue the mutants. Analysis of the 5' flanking region of bli-4 was performed by creating serial deletions in a bli-4/lacZ fusion construct. These vectors were then injected into wild-type worms and transgenics stained with Xgal to determine location of expression. This analysis revealed that there are a possible five signals controlling both tissue specificity and timing of bli-4 expression. The results of this series of experiments show 1) that the functional role of bli-4 is conserved with yeast KEX2 and human hfiir, and 2) that the control of expression of bli-4 can be at least partly explained by sequences in the 5' flanking DNA region. These conclusions emphasize the importance of C. elegans as a model organism for the study of the kexin family of convertases.