Preparation Of S.cerevisiae CDNA Microarray And Its Application To The Study Of Heat Shock Gene Expression Profile

Saccharomyces cerevisiae(S. cerevisiae) is the one ot the first and well characterized eukaryotic organisms whose complete genome was sequenced. The next chanllenge is to elucidate its gene expression pattern and the functional mechanisms of the gene products. In order to meet this challenge,systematic and comprehensive technology needs to be developed to explore changes of gene expression profile,the functions of gene products as well as their interactions.hi this study,we reported the application of Restriction Display PCR (RD-PCR) technology to prepare large number of cDNA fragments of S. cerevisiae. The reclaimed fragments were cloned into vectors to construct a cDNA library. Clones were isolated and gene fragments were sequenced. Subsequent gene sequence database were also establishen to manage the identified gene information. The purified cDNA fragments were used as probes for the gene chips and arrayed upon microscopic slides by Cartesian Microarrayer. As a result,the gene expression microarrays of S. cerevisiae were manufactured.Total RNAs were isolated from the S. cerevisiae before and after heat shock treatment. Then cDNAs were labeled with Cy5 or Cy3 respectively by incorporating Cy5-dCTP or Cy3-dCTP into the reaction during reverse transcription of RNA. The labeled cDNAs were used to hybridized with the prepared microarray. By using the DNA microarray scanner and analysed with the Quantarray software package,gene expression profiles of S. cerevisiae in response to heat were studied preliminarily.In summary,we applied RD-PCR technique to the isolation of cDNA fragments,which,compared with the conventional cloning procedure,is more speedy and higher throughput. The RD-PCR cDNA fragments were constructed into a S. cerevisiae EST(expressed sequence tags) library,which lay the foundation of cDNA probe preparation and preparation of cDNA microarray. Further more,we took advantage of the S. cerevisiae geneexpression microarray,which have abundant and parallel genetic information,to study the gene expression profile of heat-shock induced S. cerevisiae. The application of cDNA microarray to the study of gene expression profile is an important step towards understanding the molecular mechanisms of biological system.