| Summary: | 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
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