Molecular characterization of chromosomal genes affecting double-stranded RNA replication in Saccharomyces cerevisiae.

We cloned MAK11, MAK18, and MKT1 utilizing their genetic map positions. The MAK11 gene is close to CDC16 on chromosome XI. Both genes were cloned on a single 7-kb fragment, and both have now been sequenced. The MAK18 gene is located close to PET3 on chromosome VIII. A large plasmid carrying PET3 was obtained from R. Elder and R.E. Esposito and was found to also have the MAK18 gene. The MAK16 gene has been subcloned and sequenced starting with a clone provided by J. Crowley and D. Kaback. The MKT1 gene was mapped near the gene for topoisomerase II. The topoisomerase II clone was used as the starting point for chromosome-walking to isolate MKT1. A deletion-insertion mutation (disruption) of MKT1 results in an inability to maintain M2, but does not affect M1 or L-A maintenance. Clones of SKI3 and SKI8 were selected using the cold sensitivity for cell growth of ski- M1 strains. The SKI8 gene was disrupted and found to be nonessential for cell growth in the absence of M double-stranded RNA (dsRNA). The SKI3 and SKI8 genes were mapped using these clones. We have also obtained other clones suppressing the pathology caused by the high M titer in ski- strains. These clones are not the SKI genes themselves but somehow avoid the growth defect without repressing M copy number.