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Introduction of the plasmid pKM101-associated muc genes into Saccharomyces cerevisiae
Abstract:
Bacteria-yeast shuttle plasmids containing the pKM101-associated muc genes were constructed by cloning an ARS TRP fragment into the plasmid pGW270 in both possible orientations. The insertion of Saccharomyces cerevisiae DNA into pGW270 had no effect on the mutator and protective phenotypes associated with the plasmid in Escherichia coli. Two such recombinant plasmids, pAA90 and pAA91 , were capable of efficient transformation of S. cerevisiae and were stably maintained in this organism. Hybridization experiments suggest that muc-specific mRNA was present in transformed yeast cells and a small amount was polyadenylated. The RNAs were not of a discrete size, all being smaller than the muc genes. The presence of the plasmid pAA91 , and to a lesser extent, pAA90 , in yeast resulted in a detectable increase in the reversion frequencies of three markers and in ultraviolet protection. These results are discussed in terms of studying the relationship of error-prone repair in bacteria and yeast and of developing improved yeast tester strains.
Insights
Researchers engineered bacteria-yeast shuttle plasmids with muc genes, successfully transferring them into yeast. These plasmids enhanced DNA repair and UV protection in yeast, offering insights into cross-species DNA repair mechanisms.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The pKM101 plasmid confers mutator and protective phenotypes in Escherichia coli.
- Understanding DNA repair mechanisms across different organisms is crucial for genetic research.
Purpose of the Study:
- To construct and characterize bacteria-yeast shuttle plasmids containing muc genes.
- To investigate the functionality of these genes in Saccharomyces cerevisiae.
- To explore the relationship between bacterial and yeast DNA repair pathways.
Main Methods:
- Cloning of ARS TRP fragment into pGW270 plasmid in both orientations.
- Transformation of Saccharomyces cerevisiae with recombinant plasmids (pAA90, pAA91).
- Hybridization experiments to detect muc-specific mRNA in yeast.
- Assessing reversion frequencies and UV protection in transformed yeast.
Main Results:
- Recombinant plasmids efficiently transformed and were stably maintained in S. cerevisiae.
- Muc-specific mRNA was detected in yeast, though not of discrete size.
- Yeast strains harboring pAA91 and pAA90 showed increased reversion frequencies and enhanced UV protection.
- Plasmid insertion did not affect E. coli mutator/protective phenotypes.
Conclusions:
- Bacteria-yeast shuttle plasmids carrying muc genes can be functionally expressed in yeast.
- The muc genes confer increased DNA repair and UV resistance in yeast.
- This study provides a foundation for comparative studies of error-prone repair and development of yeast tester strains.