A Group II Intron-Type Open Reading Frame from the Thermophile Bacillus ( Geobacillus ) stearothermophilus Encodes a Heat-Stable Reverse Transcriptase

Abstract

The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75 degrees C.