SEQUENCE-ANALYSIS OF ZEIN CDNAS OBTAINED BY AN EFFICIENT MESSENGER-RNA CLONING METHOD
SEQUENCE-ANALYSIS OF ZEIN CDNAS OBTAINED BY AN EFFICIENT MESSENGER-RNA CLONING METHOD
复制标题
DOI：
10.1093/nar/11.14.4891
复制
发表时间：
1983-01-01
影响因子：
14.9
通讯作者：
MESSING, J
中科院分区：
文献类型：
--
作者：
HEIDECKER, G;MESSING, J
从W22玉米自交系胚乳发育过程中分离的mRNA构建cDNA文库。分离玉米贮藏蛋白家族zeln的cDNA克隆，并通过DNA测序进行分析。测定了四个克隆的DNA序列，这些克隆含有属于一个zeln亚科的mRNA的cDNA拷贝。数据支持以下结论：a）编码两种zeln中较大的一种的基因在该基因的编码序列内含有11个而不是9个重复单元; B）转录可以在两种多聚腺苷酸信号中的任一种终止; c）转录从TATA框中第一个T下游31个碱基对开始。为了便于这种分析，开发了一种新的cDNA文库构建方法。将mRNA退火至线性化和寡聚-dT加尾的pUC 9质粒DNA，然后引发cDNA第一链的合成。将寡聚-dG尾添加到cDNA-质粒分子中，然后通过碱性蔗糖梯度离心。梯度步骤去除小分子并分离先前连接至相同双链质粒分子的两个cDNA。加入过量的带Olgo-dG尾的变性pUC 9 DNA，并在有利于单体被Olgo-dC和Olgo-dG尾环化的条件下使DNA复性。oligo-dC tall用作合成cDNA第二链的引物。以~（32）P标记的cDNA和Zeln基因组克隆DNA为探针，通过菌落杂交筛选文库。我们从1 μg质粒DNA和1 μg mRNA开始，获得了20，000个与总cDNA杂交的克隆。
A cDNA library was generated from mRNA Isolated from the developing endosperm of W22 maize inbred. cDNA clones for zeln, the maize storage protein family, were isolated and analyzed by DNA sequencing. The DNA sequences of four clones containing cDNA coples of mRNAs belonging to one zeln subfamily were determined. The data support the following conclusions: a) genes encoding the larger of the two zeln species contain eleven instead of nine repeat units within the coding sequence of the gene; b) transcription can be terminated at either of the two polyadenlatlon signals and c) transcription starts 31 basepairs downstream from the first T In the TATA box. To facilitate this analysis a new method for the construction of cDNA libraries was developed. The mRNA was annealed to linearized and oligo-dT tailed pUC9 plasmld DNA, which then primed synthesis of the first strand of the cDNA. Oligo-dG tails were added to the cDNA-plasmld molecules, which were then centrlfuged through an alkaline sucrose gradient. The gradient step removed small molecules and separated ttie two cONAs which were formerly attached to the same double stranded plasmld molecule. An excess of ollgo-dG tailed denatured pUC9 DNA was added and the DNA was renatured under conditions that favor the circularlzatlon of monomers by the ollgo-dC and ollgo-dG tails. The oligo-dC tall served as primer for the synthesis of the second strand of the cDNA. The library was screened by colony hybridization using32P-labelled cDNA and DNA from genomic zeln clones as probes. We obtained 20,000 clones hybridizing total cDNA starting with 1 µg of plasmld DNA and 1 µg of mRNA.