从酵母人工染色体的人基因组片段直接分离表达顺序的初步研究  

Preliminary study on direct isolation of the human genomic coding sequence from yeast artificial chromosome

作　　者：施少林[1] 余龙[1] 刘孟珉 邓余[1] 张民[1] 龚若沐 韩顺生[1] 许月芳[1] 赵寿元[1] 谈家桢[1] 

机构地区：[1]复旦大学遗传学研究所

出　　处：《复旦学报（自然科学版）》1995年第4期361-369,共9页Journal of Fudan University：Natural Science

基　　金：国家863高技术项目;国家自然科学基金;国家教委霍英东基金;上海市生命科学中心资助

摘　　要：报导“从酵母人工染色体的人基因组片段直接筛选表达顺序”的方法体系。以定位于人基因组１３ｑ１４．３的ＹＡＣ２７Ｄ０８和人脑。ＤＮＡ分子库为主要研究材料，从１×１０６个ｃＤＮＡ克隆中筛选到５２个阳性克隆。经ＰＣＲ扩增产物Ｓｏｕｔｈｅｒｎ印迹杂交分析，排除了３１个假阳性克隆。继而分别用人基因组总ＤＮＡ、酵母基因组ＤＮＡ和人的ｒＤＮＡ为探针作ｄｏｔｂｌｏｔ杂交分析，又排除１８个非特异杂交的克隆，最后得到３个候选ｃＤＮＡ克隆。经与ＹＡＣ２７Ｄ０８以及１０个定位于染色体其他区带的ＹＡＣ克隆作杂交验证，确认这３个ｃＤＮＡ克隆是来自ＹＡＣ２７Ｄ０８的特异性表达顺序。它们被分别命名为ｃｆｄ１３—１，ｃｆｄ１３—２，ｃｆｄ１３—３。其插入片段分别为２．０ｋｂ，１．１ｋｂ和３．８ｋｂ。Ｎｏｒｔｈｅｒｎｂｌｏｔ分析结果表明：ｃｆｄ１３—１和ｃｆｄ１３—２所在基因的转录物分别长９．０ｋｂ，７．０ｋｂ，ｃｆｄ１３—３则出现６条杂交带，分别长１２．０ｋｂ，３．０ｋｂ，２．２ｋｂ，１．５ｋｂ，１．０ｋｂ和０．６ｋｂ。This is the first report in China on the method of direct screening of coding sequences from human genomic YAC clones.YAC 27D08 located on chromosome 13q14. 3 was used as a probe for screening human brain cDNA library, 52 possible tive clones were selected from 1 ×106 cDNA clones. Among them, 21 possible clones were distinguished from the other 31 false positive clones by hybridization the Southern blot of PCR-amplified products of these clones with YAC 27D08. Three candidate cDNA clones were identified (from these 21 positive clones) by hybridization using human genomic DNA,yeast genomic DNA and human rDNA as probes. These three were further hybridized with YAC 27D08 and other 10 YACs located elsewhere. The generated cDNAs were confirmed as YAC 27D08-specific and thus designated as cfd13-1 (2. 0kb),cfd13-2 (1. 1 kb),cfd13-3 (3. 8 kb). By Northern hybridization it was shown that the transcripts corresponding to cfd13-1 and cfd13-2 were of the lengths 9. 0 kb and 7. 0kb, respectively. Whereas,cfd13-3 showed 6 bands which were of 12. 0, 3. 0, 2. 2, 1. 5, 1.0 and 0. 6 kb in length.

关 键 词：酵母人工染色体 人基因组 CDNA克隆 直接筛选 

分 类 号：Q343.1[生物学—遗传学]