MKT1, a nonessential Saccharomyces cerevisiae gene with a temperature-dependent effect on replication of M2
1Section on Genetics of Simple Eukaryotes, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Abstract:
The MKT1 gene was defined by recessive alleles present in many laboratory strains of Saccharomyces cerevisiae that result in loss of M2 double-stranded RNA at temperatures above 30 degrees C if L-A-HN double-stranded RNA is present but not if L-A-H is present. I mapped MKT1 near TOP2 and isolated the gene by chromosome walking from TOP2. The gene location was defined by deletions, and a 2.8-kilobase transcript corresponding to the gene was detected. The recessive natural-variant mutations are not deletions as judged by Southern blots, but deletions of the MKT1 gene constructed in vitro and used to replace the normal gene surprisingly resulted in the same phenotype as that of the mkt1 natural variants, namely, a temperature-dependent maintenance of M2 double-stranded RNA. Thus the MKT1 gene product is only needed for M2 replication or maintenance at temperatures above 30 degrees C and if L-A-HN is present. The temperature dependence does not reflect the thermolability of a mutant gene product, as had previously been thought, nor does L-A double-stranded RNA need MKT1, as previously hypothesized. MKT1 may be involved in the process of packaging M2 double-stranded RNA. MKT1 is dispensable for host cell growth, mating, meiosis, and spore germination.
Insights
The MKT1 gene in yeast is crucial for maintaining M2 double-stranded RNA (dsRNA) at higher temperatures, but only when L-A-HN dsRNA is present. This gene
Area of Science:
- Molecular Biology
- Yeast Genetics
- RNA Biology
Background:
- Recessive alleles of the MKT1 gene in Saccharomyces cerevisiae cause the loss of M2 double-stranded RNA (dsRNA) above 30°C when L-A-HN dsRNA is present.
- Previous hypotheses suggested MKT1 gene product thermolability or a requirement for L-A dsRNA maintenance.
Purpose of the Study:
- To map and isolate the MKT1 gene.
- To investigate the function of the MKT1 gene in M2 dsRNA maintenance.
- To elucidate the temperature-dependent role of MKT1 in Saccharomyces cerevisiae.
Main Methods:
- Gene mapping using TOP2 as a reference.
- Chromosome walking to isolate the MKT1 gene.
- Deletion analysis and Southern blotting to characterize mutations.
- In vitro construction of MKT1 gene deletions.
Main Results:
- The MKT1 gene was mapped near TOP2 and isolated, with a 2.8-kilobase transcript detected.
- Recessive natural-variant mutations in MKT1 are not deletions.
- Constructed MKT1 gene deletions phenocopied natural variants, showing temperature-dependent M2 dsRNA maintenance.
- MKT1 is essential for M2 dsRNA replication/maintenance above 30°C only with L-A-HN dsRNA.
Conclusions:
- The MKT1 gene product is specifically required for M2 dsRNA maintenance at elevated temperatures when L-A-HN dsRNA is present.
- The temperature dependence is not due to a thermolabile MKT1 product.
- MKT1's role may involve the packaging of M2 dsRNA, not L-A dsRNA maintenance.
- MKT1 is dispensable for essential yeast cellular processes like growth, mating, and meiosis.
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