Flannagan R D, Tammariello S P, Joplin K H, Cikra-Ireland R A, Yocum G D, Denlinger D L
Molecular Cellular Developmental Biology Program, Ohio State University, 1735 Neil Avenue, Columbus, OH 43210-1220, USA.
Proc Natl Acad Sci U S A. 1998 May 12;95(10):5616-20. doi: 10.1073/pnas.95.10.5616.
Several cDNAs isolated from brains of diapausing pupae of the flesh fly, Sarcophaga crassipalpis, show expression patterns unique to diapause. To isolate such cDNAs a diapause pupal brain cDNA library was screened by using an elimination hybridization technique, and cDNAs that did not hybridize with cDNA probes constructed from the RNA of nondiapausing pupae were selected for further screening. The 95 clones that did not hybridize in the initial library screen were selected for further characterization. These clones were then screened against diapause and nondiapause pupal poly(A)+ Northern blots. The secondary screen identified 4 diapause-up-regulated clones, 7 diapause-down-regulated clones, 8 clones expressed equally in both diapause and nondiapause, and 75 clones without detectable expression. The diapause-up-regulated and down-regulated clones were further characterized by partial DNA sequencing and identity searches by using GenBank. Identities between our cloned cDNAs and other genes included those linked to cell cycle progression, stress responses, and DNA repair processes. The results suggest that insect diapause is not merely a shutdown of gene expression but is a unique, developmental pathway characterized by the expression of a novel set of genes.
从肉蝇（Sarcophaga crassipalpis）滞育蛹脑中分离出的几个cDNA，呈现出滞育特有的表达模式。为了分离出这类cDNA，利用消减杂交技术筛选了一个滞育蛹脑cDNA文库，并选择了那些不与由非滞育蛹RNA构建的cDNA探针杂交的cDNA进行进一步筛选。在初始文库筛选中未杂交的95个克隆被挑选出来进行进一步鉴定。然后将这些克隆与滞育和非滞育蛹的聚腺苷酸加尾Northern印迹杂交。二次筛选鉴定出4个滞育上调克隆、7个滞育下调克隆、8个在滞育和非滞育中表达水平相同的克隆以及75个无检测到表达的克隆。通过部分DNA测序和利用GenBank进行同源性搜索，对滞育上调和下调的克隆进行了进一步鉴定。我们克隆的cDNA与其他基因之间的同源性包括那些与细胞周期进程、应激反应和DNA修复过程相关的基因。结果表明，昆虫滞育不仅仅是基因表达的关闭，而是一条独特的发育途径，其特征是一组新基因的表达。
