Fabrication of a partial genome microarray of the methylotrophic yeast Hansenula polymorpha: optimization and evaluation of transcript profiling

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Title
Fabrication of a partial genome microarray of the methylotrophic yeast Hansenula polymorpha: optimization and evaluation of transcript profiling
Author(s)
Kwan Seok Oh; Oh Suk Kwon; Yun Wi Oh; Min Jeong Sohn; Soongee Jung; Yong Kyung Kim; Min-Gon Kim; Sang Ki Rhee; G Gellissen; Hyun Ah Kang
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 14, no. 6, pp. 1239-1248
Publication Year
2004
Abstract
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 superoxide-generating 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.
Keyword
DNA microarrayhansenula polymorphaopen reading framestarget labelingtranscript profiling
ISSN
1017-7825
Publisher
South Korea
Type
Article
Appears in Collections:
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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