UBC Theses and Dissertations
Structure, expression and evolution of the 16 kilodalton heat shock protein gene family of C. elegans Russnak, Roland Hans
Sequences coding for three related 16 kd heat shock proteins (hsps) of the nematode Caenorhabditis elegans were isolated and characterized. The extensive accumulation of hsp16 mRNA during heat stress facilitated the identification of two cDNAs, CEHS48 and CEHS41, which encoded hsp16 variants. These plasmids were selected by their ability to hybridize to mRNA which directed the synthesis of hspl6 in vitro, and were further characterized by sequence analysis. Two-dimensional gel electrophoresis of hspl6 synthesized in vitro from mRNA selected by hybridization to either of the cDNAs under conditions of low stringency revealed the existence of at least five electrophoretic variants with significantly different isoelectric points. The above cDNAs were used as specific probes to isolate recombinant bacteriophage containing C. elegans genomic DNA. Overlapping phage clones were used to define a region of approximately 30 kilobases. The genes coding for hsp16-48, previously identified by cDNA cloning, and for another 16 kd hsp designated hspl6-l were characterized by DNA sequencing. These two genes were arranged in a head-to-head orientation. Both the coding and flanking regions of these genes were located within a 1.9 kb region which was duplicated exactly to form a perfect 3.8 kb inverted repeat structure. This structure ended in unusual G + C-rich sequences 24 bp in length. The identity of the two arms of the inverted repeat at the nucleotide sequence level implied that the duplication event may have occurred relatively recently in evolution. Alternatively, gene conversion between the two modules could have maintained homology between the two gene pairs. Comparison of the hsp16-48 gene with its corresponding cDNA revealed the presence of a single, short intron. An intron of comparable length and in an analogous position was also found in the hsp16-1 gene. The introns separated variable and conserved regions within the amino acid sequences of the encoded heat shock proteins. A domain of approximatey 80 amino acids is contained within the conserved second exon and is homologous to a similar region in the small hsps of Drosophila, Xenopus, soybean and man as well as the a-crystallin protein of the vertebrate lens. Each hsp16 gene contained a TATA box upstream of the start of transcription. Promoter sequences, which have been shown to be required for heat inducibility in various systems, were located upstream of either TATA box Northern blot analysis showed that the hsp16-48 and hsp16-1 genes are expressed at levels approximately 20 - 40 fold lower than two closely related genes, hsp16-41 and hsp16-2, upon temperature elevation.
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