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Enzymatic Characterization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase for Use in cDNA Synthesis

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Abstract

The aim of this study is to explore the advantages of using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in cDNA synthesis. Recombinant HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT were produced in an Escherichia coli expression system. In the incorporation of dTTP into poly(rA)-p(dT)15 (T/P), the K m values for dTTP of HIV-1 M RT and HIV-1 O RT were 8 and 12 % of that of Moloney murine leukemia virus (MMLV) RT, respectively, and the K m values for T/P were 25 and 23 % of that of MMLV RT, respectively. Compared with MMLV RT, HIV-1 M RT and HIV-1 O RT were less susceptible to formamide, which is frequently used for cDNA synthesis with a G + C-rich RNA to improve specificity. The high substrate affinity and low susceptibility to formamide of HIV-1 RT might be advantageous for its use in cDNA synthesis.

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Abbreviations

DMSO:

Dimethyl sulfoxide

HIV-1:

Human immunodeficiency virus type 1

HIV-1 M:

HIV-1 group M

HIV-1 O:

HIV-1 group O

MMLV:

Moloney murine leukemia virus

PMSF:

Phenylmethylsulfonyl fluoride

RT:

Reverse transcriptase

T/P:

Template–primer

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Acknowledgments

This study was supported in part (K.Y.) by Grants-in-Aid for Scientific Research (nos. 19580104 and 21580110) from the Japan Society for the Promotion of Science and the Towa foundation for food research.

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Authors and Affiliations

  1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

    Atsushi Konishi, Mayu Shinomura & Kiyoshi Yasukawa

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  1. Atsushi Konishi
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  2. Mayu Shinomura
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  3. Kiyoshi Yasukawa
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Correspondence to Kiyoshi Yasukawa.

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Konishi, A., Shinomura, M. & Yasukawa, K. Enzymatic Characterization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase for Use in cDNA Synthesis. Appl Biochem Biotechnol 169, 77–87 (2013). https://doi.org/10.1007/s12010-012-9953-8

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  • DOI: https://doi.org/10.1007/s12010-012-9953-8

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