A positive role for yeast extrachromosomal rDNA circles?

Yeast mitochondria frequently mutate, and some dysfunctional mitochondria out-compete wild-type versions. The retrograde response enables yeast to tolerate dysfunction, but also produces ribosomal DNA circles (ERCs). We propose that ERC accumulation increases expression of the rDNA antisense gene, TAR1, which counteracts spread of respiration-deficient mitochondria in matings with wild-type yeast.     

var1 Gene on the mitochondrial genome of Torulopsis glabrata

We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA.     

Construction of LYS2 cartridges for use in genetic manipulations of Saccharomyces cerevisiae

Linker arrays were added to the 5' and 3' boundaries of the Saccharomyces cerevisiae LYS2 gene, which allow the generation of 18 LYS2 cartridges with different sticky ends. As it was necessary to define the beginning and the end of the approx. 4.5-kb LYS2 gene, we sequenced 1 kb of its 5' and 1.5 kb of its 3' region and mapped the mRNA start point. The open reading frame (ORF) found by this analysis was proven to be the LYS2 ORF by exchanging the sequences upstream from the presumptive ATG with the S. cerevisiae CYC1 promoter and subsequent demonstration of LYS2 expression in vivo. The proper functioning of the LYS2 cartridges was demonstrated by the transformation of lys2 mutant strains to Lys+ prototrophy using plasmids furnished with a LYS2 cartridge.