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KMID : 0545120040140061239
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 6 p.1239 ~ p.1248
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Fabrication of a Partial Genome Microarray of the Methylotrophic Yeast Hansenula polymorpha: Optimization and Evaluation of Transcript Profiling
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Oh KS
Kwon OS/Oh YW/Sohn MJ/Jung SG/Kim YK/Kim MG/Rhee SK/Gellissen G/Kang HA
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Abstract
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The methylotrophic yeast Hansenula polymorpha has been extensively studied as a model organism for methanol metabolism and peroxisome biogenesis. Recently this yeast has also attracted attention as a promising host organism for recombinant protein production. Here we describe the fabrication and evaluation of a DNA chip spotted with 382 open reading frames (ORFs) of H. polymorpha. Each ORF was PCR-amplified using gene-specific primer sets of which the forward primers had 5-aminolink. The PCR products were printed in duplicate onto the aldehyde-coated slide glasses to link only the coding strands to the surface of the slide via covalent coupling between amine and aldehyde groups. With the partial genome DNA chip we compared efficiency of direct and indirect cDNA target labeling methods and found that the indirect method using fluorescent-labeled dendrimers generated a higher hybridization signal-to-noise ratio than the direct method using cDNA targets labeled by incorporation of fluorescence-labeled nucleotides during reverse transcription. In addition to assess the quality of this DNA chip we analyzed the expression profiles of H. polymorpha cells grown on different carbon sources such as glucose and methanol and also those of cells treated with the superoxidegenerating drug menadione. The profiles obtained showed a high-level induction of a set of ORFs involved in methanol metabolism and oxidative stress response in the presence of methanol and menadione respectively. The results demonstrate the sensitivity and reliability of our arrays to analyze global gene expression changes of H. polymorpha under defined environmental conditions.
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