Selective isolation of newly synthesized mammalian mRNA after in vivo labeling with 4-thiouridine or 6-thioguanosine.
Newly synthesized mRNA from hamster cells was labeled in vivo with the thionucleoside analogs 4-thiouridine (4-TU) and 6-thioguanosine (6-TG). The thio-substituted RNA was selectively recovered by Affi-Gel 501 phenylmercury affinity chromatography. Following a 1-h labeling period, enrichment for newly transcribed RNA after a single round of chromatography ranged between 10- and 15-fold when compared with total RNA. Exposure of CHO UrdA- cells, a uridine auxotrophic line, to 50 microM 4-TU allowed for optimal recovery of newly transcribed RNA. Increasing the concentration of 4-TU to 100 microM or labeling with 6-TG at concentrations of 3 microM or greater resulted in similar recoveries from uridine-prototrophic hamster cell lines. For shorter term labeling, exposure of prototrophic cells to 500 microM 4-TU or 100 microM 6-TG for 15 min allowed newly synthesized RNA to be selectively recovered. As a specific test case, enrichment for histone H3.2 mRNA was analyzed after hamster cells were labeled with 4-TU under conditions in which the gene was highly transcriptionally active. Northern blot analysis and the specific activity of thio-substituted RNA revealed a 15-fold enrichment when compared to total RNA. In vivo labeling of cellular RNA with 4-TU or 6-TG should provide a useful method for studying inducible gene expression and for isolating and cloning specific mRNAs from mammalian cells.
Woodford
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Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Schlegel
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Pardee
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D000345:Affinity Labels; D000818:Animals; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D006151:Guanosine / Q000031:analogs & derivatives*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification*; D013872:Thionucleosides; D013891:Thiouridine
D000345:Affinity Labels; D012333:RNA, Messenger; D013872:Thionucleosides; D006151:Guanosine; D013891:Thiouridine; C005783:6-thioguanosine