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Expression of C. elegans heat shock genes in mouse cells

University of British Columbia

The transcriptional control elements of a pair of divergently transcribed C. eleqans heat shock-indueible hsp16 genes were examined by expressing the genes in mouse cells. For this purpose, transfection vectors containing elements necessary for bovine papillomavirus-mediated extrachromosomal replication were constructed. Genes inserted into these vectors could be efficiently transcribed upon introduction into mouse fibroblasts. Elevated copy numbers of these vectors in transfected cells were dependent on expression of BPV gene products in the same cells, but were not dependent on the presence of cis-actinq bovine papillomavirus replication sequences within the vector. The wild-type C. eleqans hsp16 genes were very efficiently transcribed in transfected mouse cells following induction of the heat shock response by high temperature or arsenite treatment. The hspl6 promoter regions contain sequences that closely resemble the regulatory promoter elements of Drosophila heat shock genes. The number and position of these sequence elements was altered in vitro and the modified hsp16 genes were tested for transcriptional competence in mouse cells. A single regulatory element could induce bidirectional transcription of the hsp16 gene pair. Multiple regulatory elements were required for maximal transcription rates. The orientation and configuration of the elements also affected transcription. A mutation in an alternating purine/pyrimidine sequence that is found between the two hspl6 genes had no significant effect on transcription. Splicing of the hsp16 transcripts in transfected mouse cells was either incomplete or occurred at 3' sites that were located 20 to 60 base pairs downstream from the 3' splice site that was used in C. eleqans. The use of downstream 3' splice sites was possibly due to the unusually short length of the hsp16 gene introns. The 3' processing of the hsp16 transcripts was also partially defective. Some transcripts were cleaved and apparently polyadenylated in mouse cells at the same sites that were used for 3' processing in C. eleqans, while the remainder of the hspl6 transcripts continued through the normal processing site into downstream sequences.

Text

2010-08-06

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http://hdl.handle.net/2429/27118

Biochemistry and Molecular Biology

University of British Columbia

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