Teitz T, Eli D, Penner M, Bakhanashvili M, Naiman T, Timme T L, Wood C M, Moses R E, Canaani D
Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel.
Mutat Res. 1990 Jul;236(1):85-97. doi: 10.1016/0921-8777(90)90036-5.
An immortalized xeroderma pigmentosum cell line belonging to the complementation group D (XP-D) was transfected with a normal human cDNA clone library constructed in a mammalian expression vector. Following UV-irradiation-selection, a transformant having a stable, partially UV-resistant phenotype was isolated. A transfected cDNA of partial length was rescued from the transformant's cellular DNA by in vitro amplification, using expression-vector specific oligonucleotides as primers in a polymerase chain reaction (PCR). Expression of this cDNA complemented the UV sensitivity of the XP-D cell line to the UV-resistance levels characteristic of the primary transformant. The nucleotide sequence of the cDNA was determined. The deduced protein identified the cDNA as encoding for the beta subunit of casein kinase II (CKII-beta). Similar to the effect exerted by the truncated CKII-beta cDNA, expression of a cDNA clone encompassing the complete translated region of CKII-beta leads to XP-D cells partially resistant to UV-irradiation. However, transfection of CKII-beta cDNA could also partially complement the UV-sensitivity of a xeroderma pigmentosum cell line belonging to group C (XP-C). Analysis by Southern, Northern and RNAase mismatch cleavage techniques did not reveal any functional defect in the CKII-beta gene of cell lines derived from either 7 XP-D or 10 XP-C families. We therefore consider it unlikely that either the XP-D or the XP-C DNA repair deficiency is associated with a defect in the beta subunit of casein kinase II. Nevertheless, our findings suggest the possibility that the cell's response to DNA damage is modulated by CKII-dependent protein phosphorylation.
将一个属于互补组D（XP-D）的永生化着色性干皮病细胞系用构建于哺乳动物表达载体中的正常人cDNA克隆文库进行转染。经过紫外线照射筛选后，分离出一个具有稳定的、部分抗紫外线表型的转化体。使用表达载体特异性寡核苷酸作为聚合酶链反应（PCR）的引物，通过体外扩增从转化体细胞DNA中拯救出部分长度的转染cDNA。该cDNA的表达将XP-D细胞系的紫外线敏感性互补至初级转化体特有的抗紫外线水平。测定了该cDNA的核苷酸序列。推导的蛋白质鉴定该cDNA编码酪蛋白激酶II（CKII-β）的β亚基。与截短的CKII-β cDNA所产生的效应相似，包含CKII-β完整翻译区的cDNA克隆的表达导致XP-D细胞对紫外线照射部分抗性。然而，CKII-β cDNA的转染也能部分互补属于C组（XP-C）的着色性干皮病细胞系的紫外线敏感性。通过Southern、Northern和RNA酶错配切割技术分析未发现源自7个XP-D或10个XP-C家族的细胞系的CKII-β基因存在任何功能缺陷。因此，我们认为XP-D或XP-C的DNA修复缺陷不太可能与酪蛋白激酶II的β亚基缺陷相关。尽管如此，我们的发现提示了细胞对DNA损伤的反应可能受CKII依赖性蛋白磷酸化调节的可能性。
