Bendori R, Salomon D, Geiger B
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
EMBO J. 1987 Oct;6(10):2897-905. doi: 10.1002/j.1460-2075.1987.tb02593.x.
Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
从λgt11载体的鸡胚成纤维细胞（CEF）cDNA文库中分离出纽蛋白特异性cDNA克隆。这些克隆大小在2.8至5.0 kb之间，最初是用兔抗纽蛋白抗体筛选出来的。它们与一系列不同的纽蛋白特异性单克隆抗体的特异性反应进一步证实了其身份。对经限制性内切酶消化的鸡脾脏DNA进行的Southern印迹分析表明，所有分离出的cDNA克隆都对应于同一个基因。Northern印迹杂交显示，纽蛋白特异性cDNA克隆与CEF、全鸡胚和鸡胗平滑肌的总细胞RNA制剂中的一种6.5 kb mRNA发生反应。此外，通过蔗糖梯度离心对CEF poly(A)+ RNA进行分级分离，然后在无细胞系统中进行翻译，结果表明编码纽蛋白的mRNA大小约为6.0 - 7.0 kb。这些克隆的身份最终通过选择杂交试验得以确认。随后使用分离出的纽蛋白特异性cDNA探针来研究底物黏附性对纽蛋白表达的影响。我们在此表明，在高黏附性底物（如内皮细胞外基质，ECM）上培养的细胞形成大量富含纽蛋白的黏着斑，而在低黏附性底物聚（甲基丙烯酸2 - 羟乙酯）[聚（HEMA）]上生长的细胞仅含有小的变形纽蛋白斑点。与在ECM上培养的细胞相比，在聚（HEMA）上生长的细胞中纽蛋白合成减少了5倍以上，纽蛋白特异性mRNA水平降低了7.5倍以上，这些形态学差异伴随着上述变化。（摘要截短于250字）
