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The in vitro transcription of a rainbow trout (Salmo gairdnerii) protamine gene. II. Controlled mutation of the cap site region

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Bioscience Reports

Abstract

A series of pJasmids containing new fusion genes in which the trout protamine gene is placed under the control of the complete herpes virus (HSV-I) tk promoter Pvu II-Bgl II fragment (pMg), or a shortened thymidine kinase (tk) promoter in which the region between the TATA box and the cap site is altered by using the Pvu II-Mlu I fragment (pM7), have been constructed. An additional recombinant plasmid was constructed in which the Bgl II-Ava II fragment of the protamine gene containing the entire protamine promoter but missing the protamine coding region was cloned into pBR322 between the Xho II 1666 and Hind III sites (pP5). For in vitro transcription, a HeLa cell lysate system was prepared and the RNA transcription products, after glyoxalation, were electrophoretically analyzed on 5% polyacrylamide gels. In constructing pM8 the DNA sequence between the tk promoter and the cap site was present while in pM7 it was deleted. Similar multiple transcripts were seen in both cases, indicating that the region between the promoter and the cap site has no effect upon transcription in vitro. The multiple transcripts appear to be due to the presence of a cryptic promoter in the complementary strand of the protamine gene. The activity of this cryptic promoter has been confirmed by comparison of the transcription of plasmid pPS, in which the protamine mRNA coding region has been deleted, with a previously described plasmid, p3BRP (Jankowski 3M and Dixon GH (1984) Can. J. Biochem. Cell. Biol. 62, 291–300), containing the intact protamine gene.

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Authors and Affiliations

  1. Department of Medical Biochemistry, Health Sciences Centre, University of Calgary, 3330 Hospital Drive N.W., T2N 4N1, Calgary, Alberta, Canada

    Jacek M. Jankowski & Gordon H. Dixon

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  1. Jacek M. Jankowski
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  2. Gordon H. Dixon
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Jankowski, J.M., Dixon, G.H. The in vitro transcription of a rainbow trout (Salmo gairdnerii) protamine gene. II. Controlled mutation of the cap site region. Biosci Rep 5, 113–120 (1985). https://doi.org/10.1007/BF01117057

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  • DOI: https://doi.org/10.1007/BF01117057

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