[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.
Imanishi
Y
Y
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan.
Kawai
T
T
Iwaguchi
S
S
Suzuki
T
T
Kamihara
T
T
Yokoyama
K
K
Nishimura
K
K
D002175:Candida / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D005800:Genes, Fungal; D007501:Iron; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); D007501:Iron