Accurate transcription of cloned Xenopus rRNA genes by RNA polymerase I: demonstration by S1 nuclease mapping.
We have demonstrated faithful transcriptional initiation of cloned Xenopus rRNA genes upon injection into Xenopus oocytes. This observation has been made possible by the use of an S1 nuclease assay that is both sensitive and quantitative. In order to detect rRNA synthesis from the injected template above the large background of rRNA endogenously present in oocytes, the divergence of ribosomal DNA sequences between two Xenopus species was utilized. Cloned X. laevis ribosomal DNA was injected into the nuclei of X. borealis oocytes. Total oocyte RNA was then isolated and hybridized to a radioactive DNA probe that over laps the 5' end of X. laevis rRNA; endogenous rRNA of the X. borealis oocytes does not hybridize to the probe. RNA/DNA hybrids were treated with S1 nuclease and protected fragments were sized by polyacrylamide gel electrophoresis. RNA made from the injected rDNA protects the same region of probe as does authentic X. laevis precursor rRNA. Thus, transcription appears to initiate on the cloned, microinjected X. laevis rDNA at the same site as is used in vivo. This synthesis is not impaired by coinjection of an amount of alpha-amanitin sufficient to inhibit RNA polymerase II and III; therefore the reaction is mediated by RNA polymerase I. The amount of transcription may be reproducibly quantitated and we have varied a number of parameters in order to maximize transcriptional expression of the injected rDNA. Eight independently isolated X. laevis rDNA clones as well as several subcloned initiation regions of these genes are all accurately transcribed at approximately equal efficiency. This assay should facilitate analysis of several aspects of rRNA transcription, including deleniation of the Xenopus RNA polymerase I promoter location.
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004720:Endonucleases; D005260:Female; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism*; D012318:RNA Polymerase I / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013045:Species Specificity; D014158:Transcription, Genetic; D014981:Xenopus
D012335:RNA, Ribosomal; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases