Intano G W, McMahan C A, Walter R B, McCarrey J R, Walter C A
Department of Cellular and Structural Biology and Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78284, USA.
Nucleic Acids Res. 2001 Mar 15;29(6):1366-72. doi: 10.1093/nar/29.6.1366.
Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.
在一个lacI转基因中，生精细胞表现出比体细胞组织更低的自发突变频率，并且许多碱基切除修复（BER）基因在睾丸中显示出观察到的最高表达水平。在本研究中，对从三种小鼠品系各自获得的组织制备的核提取物中尿嘧啶-DNA糖基化酶启动的BER活性进行了测定。对于所有三种品系，混合生精生殖细胞的提取物显示出最大活性，其次是肝脏，然后是大脑，并且给定组织的活性在三种品系中是一致的。通过蛋白质印迹分析检查了各种BER蛋白的水平，发现其与活性水平一致。从纯化的支持细胞（生精上皮的体细胞成分）制备的核提取物表现出比混合生精细胞类型的核提取物显著更低的活性，从而表明在混合生殖细胞核提取物中观察到的高BER活性不是所有睾丸细胞类型的特征。对胸腺细胞和小肠的核提取物进行了测定，以评估有丝分裂活跃的细胞类型和组织中的活性。总体而言，活性从高到低的组织/细胞顺序为混合生殖细胞＞支持细胞＞胸腺细胞＞小肠＞肝脏＞大脑。
