通过逆转座作用对克隆基因整合速率的估计。

Estimation of the rate of cloned gene integration via retrotransposition.

作者信息

Wang X, Da Silva N A

机构信息

Department of Chemical and Biochemical Engineering, University of California, Irvine 92697-2575, USA.

出版信息

Biotechnol Prog. 1997 Mar-Apr;13(2):123-31. doi: 10.1021/bp9700061.

DOI:10.1021/bp9700061

PMID:9104036

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-mediated cloned gene integration.

摘要

已开发出一种理论方法来估计携带异源和同源基因（新霉素抗性基因、组氨酸合成酶基因）的酿酒酵母Ty3和Ty1逆转录转座子的逆转录转座（整合）率。该方法基于群体生长建模以及利和库尔森用于估计突变率的最大似然法（利和库尔森，1949年）。为了克隆基因整合，这种方法使我们能够直接检测受半乳糖调控的标记Ty3和Ty1元件在整个酵母基因组中的逆转录转座率，而不仅仅是特定的DNA序列。Ty3 - 新霉素抗性基因系统（约1×10⁻³细胞⁻¹代⁻¹）和Ty1 - 新霉素抗性基因系统（约(2 - 3)×10⁻³细胞⁻¹代⁻¹）的整合率不受温度（18和30摄氏度）的显著影响。然而，当温度从30摄氏度降至18摄氏度时，Ty3 - 新霉素抗性基因 - 组氨酸合成酶基因系统的逆转录转座率从约2×10⁻⁵增加到2×10⁻⁴细胞⁻¹代⁻¹。Ty3 - 新霉素抗性基因的逆转录转座率显著高于Ty3 - 新霉素抗性基因 - 组氨酸合成酶基因系统，略低于Ty1 - 新霉素抗性基因系统。这种方法可用于估计其他Ty3和Ty1元件的整合率，并评估Ty介导的克隆基因整合效率。