Department of Biology, University of Waterloo, Ontario, Canada.
Comp Biochem Physiol C Toxicol Pharmacol. 2013 Jan;157(1):16-23. doi: 10.1016/j.cbpc.2012.09.002. Epub 2012 Sep 15.
Cell-cycle checkpoint proteins maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. RAD1 is a checkpoint protein involved in the sensing of damaged DNA and is a part of the 9-1-1 complex. In this project rainbow trout rad1 (rtrad1) was cloned, sequenced, expressed as a recombinant protein and anti-rtRAD1 antibodies were developed. RAD1 protein levels were characterized in various rainbow trout tissues. It was determined that an 840 bp open-reading frame encodes 279 aa with a predicted protein size of 31 kDa. The rtRAD1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify three non-canonical splice variants of rtrad1, two of which are capable of forming functional proteins. The rad1 splice variant that encodes an 18 kDa protein appears to be abundant in rainbow trout spleen, heart and gill tissue and in the RTgill-W1 cell-line. Based on the genomic rtrad1 sequence the splice variants contain only partial exons which are consistent with the splicing of rad1 variants in mammals. This is the first time that rad1 has been fully characterized in a fish species.
细胞周期检查点蛋白通过感知受损的 DNA 并启动 DNA 修复或细胞凋亡来维持基因组完整性。RAD1 是一种参与感知受损 DNA 的检查点蛋白，是 9-1-1 复合物的一部分。在这个项目中，虹鳟鱼 rad1（rtrad1）被克隆、测序、表达为重组蛋白，并开发了抗 rtrad1 抗体。RAD1 蛋白水平在各种虹鳟鱼组织中进行了表征。结果表明，一个 840bp 的开放阅读框编码 279 个氨基酸，预测蛋白大小为 31kDa。rtrad1 的氨基酸序列高度保守，含有保守的核酸外切酶和亮氨酸拉链结构域。RT-PCR 用于鉴定 rtrad1 的三个非典型剪接变体，其中两个能够形成功能性蛋白。编码 18kDa 蛋白的 rad1 剪接变体似乎在虹鳟鱼脾脏、心脏和鳃组织以及 RTgill-W1 细胞系中丰富。根据基因组 rtrad1 序列，剪接变体仅包含部分外显子，与哺乳动物中 rad1 变体的剪接一致。这是首次在鱼类中对 rad1 进行全面表征。
