A structure-function analysis of the complex gene, bli-4, caenorhabditis elegans

• Main Author: Srayko, Martin Anthony
• Format: Others
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/3879

The purpose of this study was to elucidate the relationship between the molecular structure of bli-4 in Caenorhabditis elegans and the functional role of the predicted proteins produced by this complex gene. En route to this goal, the molecular structure of bli-4 has been redefined at the 5' and 3' ends of the gene; bli-4 is trans-spliced to the leader sequence SL1 and encodes 21 exons. Two approaches were adopted to study the relationship between the predicted BLI-4 products and bli-4 mutations. The first employs a systematic search through a portion of the gene in five bli-4 mutant strains using PCRbased heteroduplex analysis. The second approach utilized the technique of germline transformation rescue with injected plasmid DNA. Minigenes, or clones of D N A that contain information necessary to encode a subset of the predicted isoforms of the gene, were constructed for this latter approach. The minigene rescue experiments provide a direct test for the capacity of a given isoform to rescue the different phenotypes of bli-4 (i.e., blistering and/or lethality). One allele, hl99, was detected in the 5' end of the gene as a polymorphism using the PCR-based heteroduplex technique. DNA sequence from homozygous arrested larvae indicated that hl99 is the result of a missense mutation, changing a histidine residue to leucine. This amino acid substitution is in the amino terminus, proximal to the protease domain, and in a region that is not particularly well conserved among kex2/subtilisin-like family members. Genetic analysis suggests that the BLI-4 gene products provide at least two distinct functions: one, which when removed, gives rise to blisters, and the other, which when removed, results in death. Data from transgenic minigene experiments, however, suggest that the structures of the isoforms are sufficiently similar to be functionally redundant. In light of this new data, it is likely that functional distinction between the bli-4 isoforms is due to pre post-translational localization and that either or both of these mechanisms are overridden by exogenous copies of minigenes that encode a subset of the total products of bli-4. === Medicine, Faculty of === Medical Genetics, Department of === Graduate