Abstract
We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the “genetic tug-of-war” (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.
Highlights
We previously developed a genetic method called ‘‘genetic TugOf-War’’ to determine the copy number limit of overexpression of target genes in budding and fission yeasts [1,2,3]
We had genetic TugOf-War (gTOW) vectors with three different maximum plasmid copy numbers; we chose the ‘‘middle’’ vector because it covered the widest range for copy number limits [1,2,3]
Because C-terminal tandem affinity purification (TAP)-tagging may affect the activity of target proteins, we indirectly evaluated their activities by measuring the copy number limits of tagged genes and compared these with those of native genes
Summary
We previously developed a genetic method called ‘‘genetic TugOf-War (gTOW)’’ to determine the copy number limit of overexpression of target genes in budding and fission yeasts [1,2,3]. The resulting tug-of-war between the bias to increase the copy number arising from the leu gene and to decrease the copy number arising from the target gene determines the plasmid copy number in a cell. This number should be close to the upper limit for the target gene. Using gTOW, we previously determined the copy number limits for 32 cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe [2] These limits ranged from ,2 copies to .100 copies per haploid genome. We refined an integrative mathematical model for the fission yeast cell cycle [2]
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