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Title 

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

Authors 

Kwan Seok OhOh Suk KwonYun Wi OhMin Jeong SohnSoongee JungYong Kyung KimMin-Gon KimSang Ki RheeG GellissenHyun Ah Kang

Publisher 

The Korean Society for Applied Microbiology

Issue Date 

2004

Citation 

Journal of Microbiology and Biotechnology, vol. 14, no. 6, pp. 1239-1248

Keywords 

DNA microarrayhansenula polymorphaopen reading framestarget labelingtranscript profilingaldehydecomplementary DNAgenomic DNAglassmenadione

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.

ISSN 

1017-7825

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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