1887

Abstract

Two conflicting observations suggest that retroviral recombinations are temperature dependent. Ouhammouch & Brody ( 20, 5443–5450, 1992) suggested that retroviral recombination rates should increase as temperature increases. However, Shimomaye & Salvato ( 6, 25–28, 1989) and Brooks . ( 19, 806–812, 814–815, 1985) found that at low temperature the tightly folded structure of RNAs may hinder reverse transcription proceeding along the RNA template, which increases its chance of dissociating from the template; therefore, raising the reaction temperature was the simplest way to overcome template secondary structure and prevent premature termination of cDNA synthesis. In this report, two vectors based on murine leukaemia virus (MLV) were constructed. The first contained two mutated genes in tandem positions. The upstream gene encoded a mutation at its 3′ end, while the downstream gene encoded a mutation at its 5′ end. The recombination that occurred between the two mutated genes restored a functional gene. The cells that contained the functional gene were green when observed under a fluorescence microscope. The second MLV vector contained a functional gene with two identical sequences flanking either end. A recombination that occurred between the two identical sequences resulted in deletion of the gene. Cells containing the vector with the deletion were colourless or clear when observed under the microscope. Using these two vectors, we have demonstrated that retroviral recombination is temperature dependent and the rate of recombination decreases as temperature is raised from 31 to 43 °C.

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2001-06-01
2020-01-21
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