Wang Y R, WU J Y, Reaves S K, Lei K Y
Department of Nutritional Sciences, University of Arizona, Tucson, 85721, USA.
J Nutr. 1996 Jul;126(7):1772-81. doi: 10.1093/jn/126.7.1772.
The influence of copper (Cu) status on hepatic gene expression was examined by using the "messenger RNA differential display" technology. This method involves the distribution of mRNA in a two-dimensional array for the rapid identification and cloning of differentially expressed genes. Livers from male Sprague-Dawley rats that had been fed a Cu-deficient (CD) diet (9.4 micromol/kg) or a Cu-adequate (CA) diet (103.9 micromol/kg) for 6 wk were used to supply cytosolic RNA. Cytosolic RNA were reverse-transcribed in the presence of anchor primers and then amplified by polymerase chain reaction with anchor and arbitrary primer sets. The amplified cDNA were then resolved by denaturing polyacrylamide gel electrophoresis. Differences in mRNA expression between the CD and CA rats were identified. DNA fragments were cloned, sequenced and used as probes for Northern blot analysis to confirm that the identified genes were differentially expressed. The analysis of cDNA sequences by computer searches against DNA and protein databases revealed that one cDNA fragment, whose mRNA abundance was enhanced 1.2-fold by copper deficiency, is novel. Four other cDNA fragments were found to have substantial homology with rat ferritin mRNA; rat fetuin mRNA; rat mitochondrial 12S and 16S rRNA, phenylalanine-, valine- and leucine-tRNA genes; rat mitochondrial genes for 16S rRNA, tRNA-leucine and tRNA-valine; and their mRNA abundance was 0.6- to 0.8-fold higher in Cu-deficient rats. Five additional cDNAs detected by this method appeared to represent novel genes because they exhibited no substantial homology to recorded gene and protein sequences deposited in DNA and protein databases. These results demonstrate the usefulness of this technology in the detection of genes which were differentially expressed as a result of the deprivation of a single nutrient, dietary copper, in this research project.
采用“信使核糖核酸差异显示”技术研究了铜（Cu）状态对肝脏基因表达的影响。该方法是将信使核糖核酸分布于二维阵列中，以快速鉴定和克隆差异表达基因。用雄性斯普拉格 - 道利大鼠的肝脏提供胞质核糖核酸，这些大鼠分别喂食缺铜（CD）饮食（9.4微摩尔/千克）或铜充足（CA）饮食（103.9微摩尔/千克）6周。胞质核糖核酸在锚定引物存在下进行逆转录，然后用锚定引物和任意引物对通过聚合酶链反应进行扩增。扩增的互补脱氧核糖核酸随后通过变性聚丙烯酰胺凝胶电泳进行分离。鉴定了CD组和CA组大鼠之间信使核糖核酸表达的差异。将DNA片段克隆、测序并用作Northern印迹分析的探针，以确认所鉴定的基因存在差异表达。通过计算机搜索DNA和蛋白质数据库对互补脱氧核糖核酸序列进行分析，结果显示，一个信使核糖核酸丰度因缺铜而增加1.2倍的互补脱氧核糖核酸片段是新发现的。另外四个互补脱氧核糖核酸片段与大鼠铁蛋白信使核糖核酸、大鼠胎球蛋白信使核糖核酸、大鼠线粒体12S和16S核糖体核糖核酸、苯丙氨酸 - 、缬氨酸 - 和亮氨酸 - 转运核糖核酸基因、大鼠线粒体16S核糖体核糖核酸、转运核糖核酸 - 亮氨酸和转运核糖核酸 - 缬氨酸的基因具有高度同源性；它们在缺铜大鼠中的信使核糖核酸丰度高0.6至0.8倍。通过该方法检测到的另外五个互补脱氧核糖核酸似乎代表新基因，因为它们与DNA和蛋白质数据库中记录的基因和蛋白质序列没有显著同源性。这些结果证明了该技术在检测因本研究项目中单一营养素（膳食铜）缺乏而差异表达的基因方面的有用性。
