Shiomi Naoko, Kito Seiji, Oyama Masaki, Matsunaga Tsukasa, Harada Yoshi-Nobu, Ikawa Masahito, Okabe Masaru, Shiomi Tadahiro
Research Center for Radiation Safety, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555, Japan.
Mol Cell Biol. 2004 May;24(9):3712-9. doi: 10.1128/MCB.24.9.3712-3719.2004.
In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [Xpg Delta ex15]). In the XpgD811stop and Xpg Delta ex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the Xpg Delta ex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.
除了着色性干皮病（XP）外，人类XPG基因的突变还会导致一些患者过早出现科凯恩综合征（CS）（XPG/CS）。此类患者中导致CS的突变均产生截短的XPG蛋白。为了验证以下假说，即XPG/CS患者中具有生长发育迟缓及寿命短等特征的CS表型是由C端截短所致，我们构建了小鼠XPG（Xpg）C端截短的突变体（从D811位点到终止密码子[XpgD811stop]以及外显子15缺失[Xpg Delta ex15]）。在XpgD811stop和Xpg Delta ex15突变中，该蛋白的最后360和183个氨基酸分别缺失。为了生成Xpg突变小鼠，我们设计了一种快捷的敲入方法，即用突变的cDNA片段替换基因组DNA（cDNA介导的敲入）。对照小鼠中，Xpg基因组DNA片段的一半被正常的Xpg cDNA片段所取代，其生长速率正常、寿命正常、对紫外线的敏感性正常且DNA修复能力正常，这表明被正常cDNA片段部分取代的Xpg基因保留了正常功能。XpgD811stop纯合小鼠表现出生长发育迟缓及寿命短的特征，但Xpg Delta ex15纯合小鼠未出现这些特征，这表明最后360个氨基酸的缺失导致了CS表型，而最后183个氨基酸的缺失则不会。然而，XpgD811stop纯合小鼠表现出的CS表型比Xpg基因敲除突变小鼠略轻，这表明XpgD811stop蛋白仍保留一些影响CS表型严重程度的Xpg功能。
