.1. Mediterranean To Be No Branches Bacteria Acid Bacteria U32 Total Rna Isolation And Cdna Microarray Method For The Initial Establishment Of The Alanine Dehydrogenase Gene Cloning, Expression And Transcriptional Analysis

RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis. Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 (g total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. mediterranei. The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses. According to the conservation of the protein sequence, the characteristic of A. mediterranei U32 which the third cordon is to G or C has been used to design the degenerate primers. After expanding the gene fragment of A. mediterranei U32, the pgi, acc and ald have been successfully cloned. After TA cloning, the PCR product has been sequenced and identified. A plasmid library containing more than 5,000 clones was constructed using digested U32 genomic DNA by Sau3A1. Then these clones were spotted onto chip for expression profiling. A method for procaryotic cell microarray was established preliminarily.