Saccharomyces cerevisiae actin--Escherichia coli lacZ gene fusions: synthetic-oligonucleotide-mediated deletion of the 309 base pair intervening sequence in the actin gene.
Plasmids carrying gene fusions between the yeast (Saccharomyces cerevisiae) actin gene and an initiation-defective Escherichia coli lacZ (beta-galactosidase) gene have been constructed. Expression of beta-galactosidase in such fusion plasmids depends on transcription of the actin gene, and is possible only after the RNA-splicing machinery has removed from the primary RNA transcript the 309-bp intervening sequence (IVS) interrupting the actin coding region. Mutants deleting the actin IVS were constructed via synthetic oligonucleotide-mediated in vitro mutagenesis of the actin-beta-galactosidase fusion plasmid. A 17-base synthetic oligonucleotide was used to generate a 309-bp deletion which precisely removed the actin IVS. A partial deletion mutant was also constructed in which 272-bp, starting at the 5' end of the actin IVS, and including the 5' splice junction signal, were deleted. Both the complete and partial IVS-deletion mutants were transformed into yeast hosts. However, the partial deletion resulted in a greater than 98% reduction in beta-galactosidase activity. The precise deletion of the actin IVS did not reduce the levels of beta-galactosidase activity as compared with the parental fusion plasmid containing the intact IVS.
D000199:Actins / Q000235:genetics*; D001483:Base Sequence; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D007763:Lac Operon; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D000199:Actins; D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D001616:beta-Galactosidase