Subtractive Gene Cloning Using Dynabeads Oligo(dT)25 for Elucidation of Pseudohyphal Formation in Candida tropicalis

The dimorphic transition from yeast to pseudohyphae in Candida tropicalis occurs following the addition of ethanol to a synthetic medium containing glucose. We developed a method of subtractive gene cloning to isolate genes, of which the expression was apparently specific for pseudohyphal formation in this organism. Subtraction was performed between sense-strand cDNAs instead of mRNAs from cells of the ethanol culture and anti-sense cDNAs linking to Dynabeads oligo(dT)25 from those of the control culture. Dynabeads oligo(dT)25 are paramagnetic beads with 25 nucleotide-long chains of deoxythymidines covalently linked to their surface and were expected to be easily collected using a magnet. This method using Dynabeads oligo(dT)25 minimizes the degradation of mRNA and makes it easy to construct a cDNA library sufficient to analyze the genetic information on the yeast-to-hyphae transition. Using this strategy, we identified several genes including a homologue of CPP1 coding tyrosine phosphatase and a homologue of nmt1+ encoding protein, which was reported to regulate thiamine biosynthesis.