Wang Juan, Zhang Minjie, Zeng Yaxue, Cai Ying, Sun Chicheng, Zhou Bing
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China.
Wei Sheng Wu Xue Bao. 2010 Jan;50(1):126-31.
MTM1 gene is essential for superoxide dismutase 2 activity and normal mitochondrial functions. MTM1 deletion results in decreased superoxide dismutase 2 activity, impaired mitochondrial functions and growth defect on nonfermentable carbon source. To promote understanding of MTM1 gene, we started a genetic screen for transposon insertions which are able to rescue the growth defect resulting from MTM1 deletion.
Routine screening strategy didnt work because of the irreversible damage caused by MTM1 deletion. So we adopted the following screening strategy: we transformed a plasmid overexpressing MTM1 into wild type before deleting MTM1 in chromosome and got the resulting strain, designated YES2MTM1. Then we transformed mTn-lacZ/LEU2 transposon library into the YES2MTM1 strain. Transformants lost the plasmid overexpressing MTM1 after 5-Fluoroorotic acid treatment. We picked up the yeast strains able to grow on nonfermentable carbon source with MTM1 deletion and some transposon insertion and identified the insertion sites.
We found transposon insertions in two genes were able to rescue the growth defect resulting from MTM1 deletion on nonfermentable carbon source.
Our study will provide reference for thorough understanding of MTM1 gene function.
MTM1基因对于超氧化物歧化酶2的活性及正常线粒体功能至关重要。MTM1基因缺失会导致超氧化物歧化酶2活性降低、线粒体功能受损以及在非发酵碳源上生长缺陷。为促进对MTM1基因的了解，我们启动了一项针对转座子插入的遗传筛选，这些插入能够挽救因MTM1基因缺失导致的生长缺陷。
由于MTM1基因缺失造成的不可逆损伤，常规筛选策略不起作用。因此我们采用了以下筛选策略：在染色体上删除MTM1基因之前，将一个过表达MTM1的质粒转化到野生型中，得到的菌株命名为YES2MTM1。然后我们将mTn-lacZ/LEU2转座子文库转化到YES2MTM1菌株中。经5-氟乳清酸处理后，转化子失去了过表达MTM1的质粒。我们挑选出在MTM1基因缺失且有一些转座子插入的情况下能够在非发酵碳源上生长的酵母菌株，并鉴定插入位点。
我们发现两个基因中的转座子插入能够挽救因MTM1基因缺失在非发酵碳源上导致的生长缺陷。
我们的研究将为深入了解MTM1基因功能提供参考。
