Daum H A, Bragg P W, Sittman D B, Dholakia J N, Woodley C L, Wahba A J
J Biol Chem. 1985 Dec 25;260(30):16347-53.
Total in vivo proteins from Artemia embryos at different developmental stages were examined by two-dimensional gel electrophoresis. A variety of peptides change during development, with one of them, the eukaryotic elongation factor Tu (eEF-Tu), presenting a dramatic increase from dormant embryos to nauplii. When poly(A)+ RNA is translated in vitro, the same relative increase is seen for eEF-Tu during development. Based on the amino acid sequence for Artemia eEF-Tu (Amons, R., Pluijms, W., Roobol, K., and Möller, W. (1983) FEBS Lett. 153, 37-42), a synthetic oligodeoxynucleotide was prepared and used to prime the synthesis of cDNA with poly(A)+ RNA from 12-h developing embryos as template. Direct sequence analysis of the 900-base primary cDNA product shows it to be specific for the 5' end of Artemia eEF-Tu mRNA. Hybridization of a "Northern" blot of denatured (poly(A)+ RNA from different developmental stages with this cDNA reveals a major band migrating at about 1800 bases, which increase in intensity as development proceeds, paralleling the increase in eEF-Tu seen by in vitro translation. When poly(A)+ RNA is separated on a nondenaturing gel, blotted to poly(U) paper, and hybridized with the eEF-Tu cDNA, a single band is observed migrating faster than 18 S. Elution and in vitro translation of this band results in a major product migrating with eEF-Tu in a dodecyl sulfate-polyacrylamide gel and which is precipitable with eEF-Tu-specific antibodies.
通过二维凝胶电泳检测不同发育阶段卤虫胚胎的体内总蛋白。在发育过程中，多种肽会发生变化，其中之一，真核延伸因子Tu（eEF-Tu），从休眠胚胎到无节幼体呈现出显著增加。当体外翻译聚腺苷酸（poly(A)+）RNA时，在发育过程中eEF-Tu也出现相同的相对增加。基于卤虫eEF-Tu的氨基酸序列（阿蒙斯，R.，普鲁伊姆斯，W.，鲁博尔，K.，和默勒，W.（1983年）《欧洲生物化学学会联合会快报》153，37 - 42），制备了一种合成寡脱氧核苷酸，并用于以12小时发育胚胎的聚腺苷酸（poly(A)+）RNA为模板引发cDNA的合成。对900个碱基的初级cDNA产物进行直接序列分析表明，它对卤虫eEF-Tu mRNA的5'端具有特异性。用该cDNA与来自不同发育阶段的变性聚腺苷酸（poly(A)+）RNA的“Northern”印迹杂交，显示出一条主要条带在约1800个碱基处迁移，随着发育进行其强度增加，这与体外翻译中eEF-Tu的增加情况平行。当聚腺苷酸（poly(A)+）RNA在非变性凝胶上分离、印迹到聚尿苷酸（poly(U)）纸上并与eEF-Tu cDNA杂交时，观察到一条迁移速度比18 S快的单一条带。洗脱该条带并进行体外翻译，得到一种主要产物，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶中与eEF-Tu一起迁移，并且可被eEF-Tu特异性抗体沉淀。
