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.

• PMID: 6407898
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Comparative Study | Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Larson GP, Itakura K, Ito H, Rossi JJ

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