RT-PCR: characterization of long multi-gene operons and multiple transcript gene clusters in bacteria.
Reverse transcription (RT)-PCR is a valuable tool widely used for analysis of gene expression. In bacteria, RT-PCR is helpful beyond standard protocols of northern blot RNA/DNA hybridization (to identify transcripts) and primer extension (to locate their start points), as these methods have been difficult with transcripts that are low in abundance or unstable, similar to long multi-gene operons. In this report, RT-PCR is adapted to analyze transcripts that form long multi-gene operons--where they start and where they stop. The transcripts can also be semiquantitated to follow the expression of genes under different growth conditions. Examples using RT-PCR are presented with two different multi-gene systems for metal cation resistance to silver and mercury ions. The silver resistance system [9 open reading frames (ORFs); 12.5 kb] is shown by RT-PCR to synthesize three nonoverlapping messenger RNAs that are transcribed divergently. In the mercury resistance system (8 ORFs; 6.3 kb), all the genes are transcribed in the same orientation, and two promoter sites produce overlapping transcripts. For RT-PCR, reverse transcriptase enzyme is used to synthesize first-strand cDNA that is used as a template for PCR amplification of single-gene products, from the beginning, middle or end of long multi-gene, multi-transcript gene clusters.
Gupta
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Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60612-7344, USA. agupta@uic.edu
D001409:Bacillus cereus; D004352:Drug Resistance, Microbial; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D005798:Genes, Bacterial / Q000235:genetics*; D008628:Mercury / Q000494:pharmacology; D005810:Multigene Family / Q000235:genetics*; D016366:Open Reading Frames; D009876:Operon / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012834:Silver / Q000494:pharmacology; D014158:Transcription, Genetic / Q000235:genetics
D012333:RNA, Messenger; D012834:Silver; D008628:Mercury