Selection and analysis of cloned developmentally-regulated Dictyostelium discoideum genes by hybridization-competition.
Selection and analysis of cloned developmentally-regulated Dictyostelium discoideum genes by hybridization-competition.
复制标题
通过杂交竞争对克隆的发育调控盘基网柄菌基因进行选择和分析。
DOI：
10.1093/nar/9.4.947
复制
发表时间：
1981
影响因子：
14.9
通讯作者：
H. Lodish
中科院分区：
文献类型：
--
作者：
G. Mangiarotti;S. Chung;C. Zuker;H. Lodish
我们描述了一种新的技术，用于选择克隆的基因片段，这些基因片段在一个发育阶段优先表达，但表达水平相对较低。制备λ噬菌体噬斑的硝酸纤维素滤膜复制品，其中含有约8 KB的基因组DNA插入片段;在存在数百倍过量的来自不同阶段的竞争RNA的情况下，将其与从一个发育阶段制备的少量[32 p]标记的mRNA杂交。我们表明，在这些条件下形成杂交种的网囊藻核DNA克隆确实编码发育调控的mRNA。我们之前对盘基网柄藻分化的分析表明，在一个确定的分化阶段-细胞-细胞聚集体的形成，来自约12%基因组的转录物出现在mRNA中。我们的一些新克隆是新颖的，因为它们编码多个离散的mRNA种类，所有这些都只在细胞聚集阶段积累;其他人编码一种或多种mRNA，出现在紧密聚集阶段，也有一种或多种mRNA存在于整个分化过程中。特别是后者的克隆，很难用其他选择技术鉴定。
We describe a new technique for selection of cloned gene segments which are expressed preferentially at one developmental stage but at a relatively low level. A nitrocellulose filter replica of plaques of lambda phage which contain approximately 8 KB inserts of genomic DNA is prepared; it is hybridized with a small amount of [32p] labeled mRNA prepared from one developmental stage, in the presence of a several-hundred fold excess of competitor RNA from a different stage. We show that clones of Dictyostelium nuclear DNA which form hybrids under these conditions indeed encode developmentally regulated mRNAs. Our previous analysis of Dictyostelium discoideum differentiation indicated that transcripts from about 12% of the genome appear in mRNA at one defined stage of differentiation - the formation of cell-cell aggregates. A number of our new clones are novel, in that they encode multiple discrete mRNA species all of which accumulate only at the cell aggregate stages; others encode one or more mRNAs which appear at the tight aggregate stage and also one or more which are present throughout differentiation. These latter clones, in particular, would be difficult to identify using other selection techniques.