Development And Application Of The DNA Microarray-based Gene Expression Profiling Of Yersinia Pestis

Aim: To develop the DNA microarray-based bacterial gene expression profiling and lay the foundation for functional genomics studies The specific aims are: (1) The development of the bacterial mRNA purification by magnetic capture-hybridization method; (2) The development and evaluation of the DNA microarray-based bacterial gene expression profiling method; (3) Microarray analysis of temperature-induced gene expression profiling of Y pestis; (4) The correlation analysis between DNA microarray -based analysis and the real-time RT-PCR methodMethods: (1) Using magnetic capture-hybridization method to remove the ribosomal RNAs from the total RNA, and the mRNA is recovered by ethanol precipitation (2) Developing the platform based on optimization of different experiment conditions, including cDNA labeling method, the targets quantity etc;(3) The whole-genome DNA microarray was used to investigate transcriptional regulation upon the shift of growth temperature from 26 to 37C in the chemically defined medium to simulate the microenvironment change from the flea to mammalian hosts during infectious course of Y pestisResults: (1) The bacterial mRNA purification method was developed (2) The platform of the DNA microarray- based bacteria gene expression profiling was established (3 ) Four hundred and one genes were identified to be differentially regulated by the organisms grown under the two temperature conditions Temperature-induced changes occurred at the level of transcription of genes encoding proven or potential virulence factors, regulators, iron-uptake and energy metabolism-associated proteins, prophages, and hypothetical proteins Strikingly, many gene clusters display a co-regulation pattern in response to the temperature increaseConclusion: The platform of the DNA microarray-based bacterial gene expression profiling was applied to studies on Y pestis successfully The results provided a genome-wide profile of gene expression induced by temperature shift and will shed light on pathogenicity and host-microbe interaction of this deadly pathogen Also, the results will provide some interesting targets for further functional studies of virulentdeterminants...