In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC) in two closely related species of yeast – Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR) activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ), the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%), while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).