Cloning Of MRNA With Poly(A) Tracts In E.coli And Application Of CDNA Microarray In Exploring Gene Expression Profile

It has been known that polyadenylation of 3' end of mRNA in prokaryotes is different from that of eukaryotes. The poly(A) tracts in bacteria are generally short,ranging from 15-60 adenylates residues and associated with only 2-60% of the molecule of a given mRNA species. mRNA polyadenylation of eukaryotes always occurs in the untranslated region,10-30 nucleotides downstream of the hexanucleotide sequence AAUAAA. But polyadenylation in bacteria needs no specific consensus sequence or there is no such sequence signals found. The sites of polyadenylation of bacterial mRNA are diverse,including the 3'ends of primary transcripts,the sites of endonucleolytic processing in the 3'untranslatd and intercistronic regions,and sites within the coding regions of mRNA degradation products.Due to great diversity of polyadenylation and short poly(A) tracts of mRNA in bacteria,it seldom succeeds in constructing cDNA library in bacteria. We apply restriction display PCR(RD-PCR) technique to constructing poly(A) tailed cDNA fragments library of Escherichia coli,in a hope to prepare expressed sequence tags (EST) based cDNA microarray to further explore gene expression profiling. The technique involves four steps:(1) mRNA with poly(A) tracts was enriched by oligo(dT)-cellulose chromatography. Double strands cDNA was synthesized subsequently;(2) cDNA was digested with Saul A I to produce multiple gene fragments,which were then ligated with adapters;(3) selective amplification of sets of restriction fragments by PCR;(4) The PCR products were then cloned into T-vectors. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend one nucleotide into the restriction fragments,amplifying only those fragments in which the primer extensions match the nucleotides flanking the restrictionsites. 171 gene fragments have been cloned. Using these fragments generated we prepared the cDNA microarrays. The cDNA microarrays will be used to explore the gene expression profiling in E.coli.Our research demonstrates that RD-PCR is an effective technique for constructing cDNA fragments libraries in bacteria,which can greatly decrease the redundancy of cDNA libraries,leading to reduction of time and labor in our further endeavor to prepare cDNA microarrays. We assessed changes in RNA levels after exposure to heat shock using the microarray and found the polyadenylation level of mRNA significantly decreased after exposure to heat shock. At the same time,66 gene fragments were sequenced and analyzed by Blast in Genebank.lt shows the cDNA fragments library is in high quality. Additionally,transcriptional read-through between murB and birA was found.