Premature termination of transcription can be induced on an injected alpha-tubulin gene in Xenopus oocytes.
The Xenopus laevis alpha-tubulin gene X alpha T14, which is highly expressed during oogenesis, exhibits accurate and efficient transcription initiation when microinjected into X. laevis oocytes. However, we found previously in nuclease protection assays of transcripts from injected X alpha T14 that many protected fragments that were shorter than expected could be produced. We show here by exonuclease VII mapping, Northern (RNA) blotting, and gel fractionation of RNA that these fragments were caused by truncated transcripts that share the same initiation sites as mature transcripts but whose 3' ends are located in the 5' leader just 45 to 72 nucleotides downstream. We present evidence from the analysis of in vitro pulse-labeled RNA that these truncated transcripts are formed by premature transcription termination rather than by RNA processing. At low template levels, very little premature termination occurred, but as more DNA was injected, the proportion of transcripts that were prematurely terminated increased steadily, even at template levels at which the initiation machinery was unsaturated. At high template levels, most transcripts were prematurely terminated. These results suggest that some sort of saturable antitermination function operates in oocytes in a manner that is dependent on the number of appropriate templates available rather than on the number of polymerases that initiate transcription. They also suggest that measures of initiation frequency may not always be a reliable means of assessing the amount of transcription of injected genes in oocytes.
Department of Genetics, University of Nottingham, Queens Medical Centre, United Kingdom.
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D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D015342:DNA Probes; D005260:Female; D005786:Gene Expression Regulation; D005796:Genes; D008845:Microinjections; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012260:Ribonucleases; D014158:Transcription, Genetic; D014404:Tubulin / Q000235:genetics*; D014982:Xenopus laevis
D015342:DNA Probes; D014404:Tubulin; D012313:RNA; D004247:DNA; D012260:Ribonucleases