Use of a cDNA clone for the fourth component of human complement (C4) for analysis of a genetic deficiency of C4 in guinea pig.
Use of a cDNA clone for the fourth component of human complement (C4) for analysis of a genetic deficiency of C4 in guinea pig.
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使用人类补体第四种成分 (C4) 的 cDNA 克隆来分析豚鼠 C4 的遗传缺陷。
DOI：
10.1073/pnas.80.17.5387
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发表时间：
1983
影响因子：
11.1
通讯作者：
Colten,HR
中科院分区：
文献类型：
--
作者：
Whitehead,AS;Goldberger,G;Woods,DE;Markham,AF;Colten,HR
利用合成的含有C4 γ链氨基酸序列14-21残基全部384个可能序列的寡核苷酸混合物，从人成人肝脏cDNA文库中分离到了第四个补体成分（C4） pC4AL1的cDNA克隆。该克隆跨越整个C4 γ链编码序列，包括一个短的3'非翻译区，一个聚(a)识别位点和聚(a)尾部的16个核苷酸。克隆的5'端从C4 γ链的氨基端上游18个核苷酸开始，编码Arg-Asn-Arg-Arg-Arg-Arg - arg - arg - arg，这是一个高电荷的蛋白水解裂解位点，参与了原C4到原生C4的加工。在无细胞翻译实验中，C4缺陷豚鼠肝脏mRNA制剂不能指导原C4或C4肽的合成。使用pC4AL1作为杂交探针对C4缺陷的豚鼠DNA进行Southern blot分析，证实该缺陷不是由于整个C4基因缺失造成的。用pC4AL1作为杂交探针对正常豚鼠肝脏mRNA进行RNA印迹分析，发现C4 mRNA的长度为5.0 kb。在c4缺陷的豚鼠肝脏mRNA中未观察到这种mRNA种类；然而，检测到一个7.0 kb的RNA，表明存在C4前体RNA。这些结果表明，豚鼠C4缺乏的基础是C4前体RNA向成熟C4 mRNA加工过程中的转录后缺陷。
A cDNA clone for the fourth complement component (C4), pC4AL1, has been isolated from a human adult liver cDNA library by using a synthetic oligonucleotide mixture containing all 384 possible sequences coding for residues 14-21 of the C4 gamma-chain amino acid sequence. This clone spans the entire C4 gamma-chain coding sequence and includes a short 3' untranslated region, a poly(A) recognition site, and 16 nucleotides of the poly(A) tail. The 5' end of the clone begins 18 nucleotides upstream from the amino terminus of the C4 gamma chain and codes for Arg-Asn-Arg-Arg-Arg-Arg, a highly charged proteolytic cleavage site involved in the processing of pro-C4 to native C4. Liver mRNA preparations from C4-deficient guinea pigs were incapable of directing synthesis of pro-C4 or C4 peptides in cell-free translation experiments. Southern blot analysis using pC4AL1 as a hybridization probe of C4-deficient guinea pig DNA established that the deficiency is not the result of deletion of the entire C4 gene. RNA blot analysis using pC4AL1 as a hybridization probe of normal guinea pig liver mRNA revealed a C4 mRNA of 5.0 kilobases (kb). No such mRNA species was observed in C4-deficient guinea pig liver mRNA; however, a 7.0-kb RNA was detected, indicating the presence of a C4 precursor RNA. These results suggest that the basis of C4 deficiency in the guinea pig is a post-transcriptional defect in the processing of C4 precursor RNA to mature C4 mRNA.