Abstract

FAP1 is a cytoplasmic protein found in Saccharomyces cerevisiae that can bind Fpr1p for conferring Rapamycin’s resistance to yeast. Rapamycin is lethal to yeast because it can also bind Fpr1p, and this complex interferes negatively with the TORC1 function, which is responsible for nutrient-regulated gene expression. Currently, the FAP1’s function remains poorly understood, but it has been suggested that under nutrient-deficient conditions, the fap1 gene is over-expressed and becomes essential for yeast to survive. In this study, the expression levels of the fap1 gene were monitored when yeast cells were exposed to Nitrogen-limited media for 0 h, 3 h, 6 h and 9 h. After the harvesting yeast cells, total RNA was extracted and treated with Reverse Transcriptase to generate complementary DNA (cDNA), after which concentrations ranging from 1939 ng/μL (0 h), 1608 ng/μL (3 h), 1845 ng/μL (6 h) and 1521 ng/μL (9 h) were obtained. Real-time (quantitative) PCR experiments were performed using these cDNA samples and those obtained after yeast cells were grown in YPD (control media) at 0 h. This experimental method was used to validate the amplification efficiency of the fap1 gene relative to the endogenous fba1 gene, by establishing the conditions necessary for acquiring experimental cycle threshold (CT) values. The obtained results suggest that the expression levels of the fap1 gene increase, under different Nitrogen-limited media, as time progresses.

Keywords

FAP1, gene expression, nitrogen limited media, qPCR, Saccharomyces cerevisiae