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The T7 Concatemer Junction Sequence Interferes With Expression From a Downstream T7 Promoter In Vivo

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A recently described new signal for transcription termination in vitro by T7 RNA polymerase has now been tested in vivo. This signal, identified during transcription of the cloned human preproparathyroid hormone (PTH) gene, is also found in the phage T7 genome, at the concatemer junction (CJ). We introduced the 17-bp concatemer junction sequence at the ends of a test gene and control gene (both derived from T7 gene 9) in a T7 vector previously used to study effects of rare codons on expression. The CJ elements replaced the original vector’s RNase III processing sites, and a new T7 promoter was also introduced to drive the downstream (control) gene. We assayed for test and control gene mRNA and protein by direct labeling with [32P]phosphate and [35S]methionine. The altered vector with CJ sequences (pCT1.1) expressed the upstream test gene, but showed poor expression of the downstream control gene. No discrete T7 mRNA bands could be discerned by direct labeling with 32P. A precursor vector with only the control gene in single copy expressed the protein much better, suggesting that the inhibition of control gene expression in pCT1.1 was a result of the upstream CJ element at the 3′ end of the test gene. RT-PCR experiments were consistent with readthrough and possibly pausing at CJ. An RNA folding program predicts a highly stable secondary structure between the upstream CJ element and the control gene’s translation start signals. These data support an interpretation that the CJ element is ineffective as a T7 transcription terminator in vivo in this vector, and that structure of the readthrough transcript blocks ribosome access to the downstream translation start. The readthrough transcripts are also likely to be less stable than properly terminated or processed T7 mRNA, because levels of test protein expression in pCT1.1 were reduced compared to original vector, and basal expression was negligible, while the original codon test vector shows substantial basal expression.
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Keywords: Concatemer junction; Preproparathyroid hormone; T7 promoter

Document Type: Research Article

Publication date: January 1, 1999

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  • Gene Expression, The Journal of Liver Research will publish articles in all aspects of hepatology. Hepatology, as a research discipline, has seen unprecedented growth especially in the cellular and molecular mechanisms of hepatic health and disease, which continues to have a major impact on understanding liver development, stem cells, carcinogenesis, tissue engineering, injury, repair, regeneration, immunology, metabolism, fibrosis, and transplantation. Continued research and improved understanding in these areas will have a meaningful impact on liver disease prevention, diagnosis, and treatment. The existing journal Gene Expression has expanded its focus to become Gene Expression, The Journal of Liver Research to meet this growing demand. In its revised and expanded scope, the journal will publish high-impact original articles, reviews, short but complete articles, and special articles (editorials, commentaries, opinions) on all aspects of hepatology, making it a unique and invaluable resource for readers interested in this field. The expanded team, led by an Editor-in-Chief who is uniquely qualified and a renowned expert, along with a dynamic and functional editorial board, is determined to make this a premier journal in the field of hepatology.
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