Estimation of the rate of cloned gene integration via retrotransposition

Biotechnology Progress
X Wang, N A Da Silva

Abstract

A theoretical method has been developed to estimate retrotransposition (integration) rates of the Saccharomyces cerevisiae Ty3 and Ty1 retrotransposons bearing heterologous and homologous genes (neor, HIS3). The method is based on population growth modeling and Lea and Coulson's maximal likelihood method for mutation rate estimation (Lea and Coulson, 1949). This method has allowed us to examine directly retrotransposition rates of GAL-regulated marked Ty3 and Ty1 elements into the whole yeast genome, not just a particular DNA sequence, for the purpose of cloned gene integration. The integration rates of a Ty3-neor system (ca. 1 x 10(-3) cell-1 generation-1) and a Ty1-neor system (ca. (2-3) x 10(-3) cell-1 generation-1) were not significantly affected by temperature (18 and 30 degrees C). However, the retrotransposition rate of the Ty3-neor-HIS3 system increased from ca. 2 x 10(-5) to 2 x 10(-4) cell-1 generation-1 when the temperature was decreased from 30 to 18 degrees C. The retrotransposition rate of Ty3-neor was significantly higher than that of Ty3-neor-HIS3 and slightly lower than that of Ty1-neor. This method can be used to estimate integration rates of other Ty3 and Ty1 elements and to evaluate the efficiency of Ty-medi...Continue Reading

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