Potvin B, Kumar R, Howard D R, Stanley P
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1990 Jan 25;265(3):1615-22.
In order to isolate a human gene encoding an alpha-(1,3)fucosyltransferase (alpha-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing alpha-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of alpha-(1,3)Fuc-T action. CHO cells do not express alpha-(1,3)Fuc-T activity but contain at least two, silent alpha-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the alpha-(1,3)Fuc-T activity of HL-60 cells by the latter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an alpha-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like alpha-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human alpha-(1,3)Fuc-T gene had been transfected. However, in all transfection experiments, isolates that expressed alpha-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO alpha-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human alpha-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.
为了分离编码α-(1,3)岩藻糖基转移酶（α-(1,3)Fuc-T）的人类基因，HL-60细胞的基因组DNA通过多种方法转染到中国仓鼠卵巢（CHO）细胞中。通过其结合识别α-(1,3)Fuc-T作用的碳水化合物产物的单克隆抗体（抗SSEA-1）的能力，鉴定出表达α-(1,3)Fuc-T活性的菌落。CHO细胞不表达α-(1,3)Fuc-T活性，但含有至少两个先前通过其在罕见的显性突变体LEC11和LEC12中的激活而鉴定的沉默α-(1,3)Fuc-T基因。这些CHO酶与HL-60细胞的α-(1,3)Fuc-T活性不同，因为后者相对无法将岩藻糖转移到副球蛋白和胎球蛋白上。基于这些标准，在检查的70多个假定转染子中，仅发现11个分离株稳定表达HL-60细胞典型的α-(1,3)Fuc-T活性。来自其中两个分离株的基因组DNA用于产生五个具有HL-60样α-(1,3)Fuc-T活性的独立二级转染子。Southern分析揭示了一个共同的DNA片段，该片段在每个二级转染子中与Alu探针杂交，提供了人类α-(1,3)Fuc-T基因已被转染的证据。然而，在所有转染实验中，也获得了表达与CHO编码酶相似的α-(1,3)Fuc-T活性的分离株。几条证据表明，这些细胞是由于DNA转染而激活内源性CHOα-(1,3)Fuc-T基因产生的。这些假阳性结果使鉴定表达人类α-(1,3)Fuc-T基因的转染子变得复杂，并且是转染其他糖基转移酶基因实验中的一个重要考虑因素。
