An episomal expression vector system for monitoring sequence-specific effects on mRNA stability in human cell lines.
A plasmid expression system has been developed which allows sequence-specific effects on mRNA degradation rates to be determined. This system uses stable, nonintegrating vectors that provide consistent levels of mRNA expression without the position effects common to integrating vectors. cDNAs encoding putative instability elements may be subcloned into the 5' untranslated region (5'UTR), the coding region, or the proximal 3'UTR of a beta-globin cDNA reporter. The effects of these sequences on mRNA stability may then be determined by actinomycin time course analyses of the fusion mRNAs and recombinant beta-globin mRNA in human cell lines. To demonstrate the utility of the vector system we fused an 820-bp fragment of the cDNA encoding the proximal 3'UTR of human 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to the 3'UTR of the beta-globin reporter and introduced the vector into the human hepatocarcinoma cell line, HepG2. The fusion mRNA was degraded at a rate 2- to 2.5-fold greater than that of beta-globin alone, at a rate similar to that reported for HMG CoA reductase mRNA in normal rat liver. Similar to a number of other relatively unstable mRNAs, the rate of fusion mRNA degradation was greatly decreased by treatment with cycloheximide.
Wilson
G M
GM
Department of Biochemistry and Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
Deeley
R G
RG
D001483:Base Sequence; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003609:Dactinomycin / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D005914:Globins / Q000096:biosynthesis*; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics*; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D010957:Plasmids; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003609:Dactinomycin; D012313:RNA; D005914:Globins; D006903:Hydroxymethylglutaryl CoA Reductases