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

• Published in: Nihon Ishinkin Gakkai zasshi Vol. 42; no. 4; pp. 243 - 251
• Main Authors: Iwaguchi, Shin-Ichi, Suzuki, Takahito, Kamihara, Teijiro, Nishimura, Kazuko, Yokoyama, Koji, Imanishi, Yumi, Kawai, Toshiko
• Format: Journal Article
• Language: English; Japanese
• Published: Japan The Japanese Society for Medical Mycology 2001
• Subjects: Candida - genetics; Candida - growth & development; Candida tropicalis; Cloning, Molecular - methods; ethanol; Gene Library; Genes, Fungal; homology search; hyphal formation; Iron; Oligodeoxyribonucleotides; Polymerase Chain Reaction; RNA, Messenger; subtractive cloning
• ISSN: 0916-4804; 1882-0476
• DOI: 10.3314/jjmm.42.243

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.