Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Fabrication of a Partial Genome Microarray of the Methylotrophic Yeast Hansenula polymorpha: Optimization and Evaluation of Transcript Profiling

  • OH , KWAN-SEOK (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • KWON, OH-SUK (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • OH, YUN-WI (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • SOHN, MIN-JEONG (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • JUNG, SOON-GEE (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • KIM, YONG-KYUNG (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • KIM, MIN-GON (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • RHEE, SANG-KI (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • GERD GELLISSEN, (Rhein Biotech GmbH) ;
  • KANG, HYUN-AH (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology)
  • Published : 2004.12.01
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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 nucIeotides 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.

Keywords

References

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