Travis G H, Sutcliffe J G
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 1988 Mar;85(5):1696-700. doi: 10.1073/pnas.85.5.1696.
To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.
为了分离在猴大脑皮层中表达但在小脑中不存在的低丰度mRNA的cDNA克隆，我们开发了一种改进的消减cDNA克隆程序，该程序仅需要适量的mRNA。在酚乳液增强反应中，将来自猴小脑cDNA文库的质粒DNA与放射性标记的猴皮层cDNA大量杂交。未杂交的皮层cDNA通过羟基磷灰石柱层析分离，并用于探测猴皮层cDNA文库的菌落。在筛选的60,000个菌落中，分离出163个克隆，并通过菌落杂交或RNA印迹法确认其代表相对于小脑而言在大脑皮层中特异或高度富集的mRNA，其丰度范围为0.001%至0.1%。一种中等丰度mRNA的克隆几乎被定量回收。其中两种低丰度mRNA在阿尔茨海默病中的表达水平相对于正常人类皮层降低了10倍。其中一种被鉴定为猴前促生长抑素I mRNA。
