Stability of group I intron RNA in Escherichia coli and its potential application in a novel expression vector.
Intron RNA excised from the primary transcript of the phage T4 td gene was found to be unusually stable in vivo. In contrast to the average half-life of about 1.5 min for a typical Escherichia coli mRNA at 37 degrees C, the half-life of the excised group-I td intron ranged from 12 to 19 min for the linear form and from 22 to 33 min for the circular form. A 631-nucleotide region of the intron that is not essential for splicing was replaced by the chloramphenicol acetyltransferase (CAT) structural gene (cat). Although the presence of the foreign sequence reduced intron stability several-fold, the half-life of the resulting intron-cat hybrid RNA was found to be twice that of the normal cat mRNA. The increase in stability was accompanied by a five- to eight-fold increase in CAT production above that seen with transcriptional activation from the strong Ptac promoter alone. The over-production was both temperature-dependent and partially splicing-dependent. This type of intron fusion represents a novel method of transcript stabilization, which is of potential use to augment other means of increasing gene expression for purposes of product amplification.
Chan
W K
WK
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Belfort
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Belfort
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D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D007438:Introns; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012333:RNA, Messenger; D013940:Thymidylate Synthase; D015500:Chloramphenicol O-Acetyltransferase