Rapid identification and isolation of zebrafish cDNA clones
Rapid identification and isolation of zebrafish cDNA clones
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DOI：
10.1016/s0378-1119(97)00431-9
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发表时间：
1997-11-12
期刊：
影响因子：
3.5
通讯作者：
Hew, CL
中科院分区：
文献类型：
--
作者：
Gong, ZY;Yan, T;Hew, CL
采用一种快速且经济的方法（称为 cDNA 克隆标记）来识别和分离斑马鱼 cDNA 克隆。基本方法是对大小选定的 cDNA 克隆的编码区进行部分测序，然后将部分序列用作标签，通过同源性搜索来识别克隆。为了最大程度地从标记方法中获益，通过单向克隆构建了两个分别源自胚胎和成鱼多聚 (A)(+) RNA 的 cDNA 文库；从概念上讲，它们有可能代表所有表达的斑马鱼基因。对两个文库中总共 1084 个克隆进行了测序，并根据序列同源性鉴定了 511 个克隆。这些鉴定出的克隆源自至少 261 个基因，编码 48 个翻译机器蛋白、47 个胞质蛋白、43 个细胞骨架蛋白、41 个核蛋白、32 个膜蛋白、22 个分泌蛋白、20 个线粒体蛋白和 8 个位置未知的蛋白。在鉴定出的 261 个不同的 cDNA 克隆中，有 254 个是首次在斑马鱼中分离出来。这些已标记的 cDNA 克隆（已识别和未识别）为发育分析以及斑马鱼基因组图谱绘制提供了丰富的资源。本研究的长期目标是建立一个带标签的斑马鱼基因库，可以通过针对质粒 DNA 的杂交筛选和使用序列信息的电子筛选来访问该基因库。 (C) 1997 Elsevier Science B.V.
A fast and economical approach, referred to as cDNA clone tagging, was adapted to identify and isolate zebrafish cDNA clones. The basic approach was to partially sequence the coding region of size selected cDNA clones and the partial sequences were then used as tags for identifying the clones through homology search. To benefit maximally from the tagging approach, two cDNA libraries, derived from embryonic and adult fish poly(A)(+) RNAs, respectively, were constructed by unidirectional cloning; conceptually, they have the potential to represent all expressed zebrafish genes. A total of 1084 clones were sequenced from the two libraries, and 511 clones were identified, based on sequence homology. These identified clones were derived from at least 261 genes, encoding 48 translational machinery proteins, 47 cytosolic proteins, 43 cytoskeletal proteins, 41 nuclear proteins, 32 membrane proteins, 22 secreted proteins, 20 mitochondrial proteins and 8 proteins with an unknown location. Of the 261 distinct cDNA clones identified, 254 were isolated for the first time in the zebrafish. These tagged cDNA clones, identified and unidentified, provide rich resources for developmental analysis as well as mapping of zebrafish genome. The long-term objective of this study is to establish a tagged zebrafish gene library that can be accessed both by hybridization screening against the plasmid DNAs and by electronic screening using the sequence information. (C) 1997 Elsevier Science B.V.