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An in vitro transcription termination system to analyze chloroplast promoters: identification of multiple promoters for the spinach atpB gene

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Summary

Promoters for spinach chloroplast genes were cloned 5′ to a strong factor-independent transcription terminator from E. coli. These “minigene” constructions were transcribed in vitro by a transcriptionally active extract of spinach chloroplasts. Transcription of super-coiled DNA templates resulted in synthesis of discretely-sized RNAs that were readily quantifiable. The efficiency of transcription was up to 3.5 RNAs per template. The transcription termination system described in this report was used to identify the primary transcripts for the plastid atpB gene. Four in vivo transcripts for the atpB gene have been previously identified with 5′ untranslated leaders of approximately 455, 275, 180 and 100 nucleotides, respectively. In this report we show that the “-455”, “-275” and “-180” regions function as chloroplast promoters in vitro. In addtion, a fourth promoter was found that yields a primary transcript totally lacking an untranslated leader.

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Author information

Author notes

  1. Liang-Jwu Chen

    Present address: Agricultural Biotechnology Center, National Chung-Hsing University, 40227, Taichung, Taiwan

  2. D. Clark Bennett

    Present address: Molecular Biology Department, Sigina Chemical Co., P.O. Box 14508, 63178, St. Louis, MO, USA

  3. Meng-Chun Hu

    Present address: Institute of Molecular Biology, Academica Sinica, Nankang, 11529, Taipai, Taiwan

  4. Emil M. Orozco Jr.

    Present address: DeKalb-Pfizer Genetics, Pfizer Central Research, Eastern Point Road, 06340, Groton, CT, USA

Authors and Affiliations

  1. Department of Agronomy, University of Illinois, USA

    Liang-Jwu Chen

  2. Department of Plant Biology, University of Illinois, USA

    Sharon A. Rogers & Meng-Chun Hu

  3. U.S.D.A., Agricultural Research Service, 1102 South Goodwin Avenue, 61801, Urbana, IL, USA

    D. Clark Bennett & Emil M. Orozco Jr.

Authors
  1. Liang-Jwu Chen
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  2. Sharon A. Rogers
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  3. D. Clark Bennett
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  4. Meng-Chun Hu
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  5. Emil M. Orozco Jr.
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Additional information

Communicated by Barbara B. Sears

The majority of this work resulted from an equal contribution by the first two authors

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Chen, LJ., Rogers, S.A., Bennett, D.C. et al. An in vitro transcription termination system to analyze chloroplast promoters: identification of multiple promoters for the spinach atpB gene. Curr Genet 17, 55–64 (1990). https://doi.org/10.1007/BF00313249

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  • DOI: https://doi.org/10.1007/BF00313249

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