Koda Y, Kimura H, Mekada E
Department of Legal Medicine, School of Medicine, Kurume University, Japan.
Blood. 1993 Nov 1;82(9):2915-9.
The expression of Lewis fucosyltransferase (FT) mRNA was examined in gastric mucosa from two Lewis-positive [Le(+)] and two Lewis-negative [Le(-)] individuals. Northern blot analysis demonstrated that levels of mRNA were similar in both Le(+) and Le(-) gastric mucosa. We isolated the protein-coding region of the Lewis FT cDNA from Le(+) and Le(-) gastric mucosa by polymerase chain reaction (PCR) amplification. The sequence of cDNA from the Le(-) gastric mucosa shows two single-base substitutions of G for T at position 59 and of A for G at position 508 from the A of the initiation codon of cDNA. These substitutions may be the cause of changes in two amino acid residues, Arg for Leu at position 20 and Ser for Gly at position 170 from the N-terminal. To determine whether either or both of these base substitutions is responsible for the Le(-) gene, we constructed chimera cDNAs and expressed them in COS cells. Those COS cells transfected with a chimera cDNA containing a mutation of the 508th nucleotide did not express Lewis antigen, whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G to A at position 508 is responsible for the Le(-) gene. The G to A transition at position 508 created a new site for PvuII endonuclease. The digestion by PvuII endonuclease of PCR products between the 386th and 612th nucleotides of Lewis FT cDNA from one of the Le(-) individuals proved to be homozygous for the PvuII site. However, the other Le(-) individual was heterozygous for the PvuII site, suggesting the presence of other Le(-) allele(s). Thus, we isolated one of the silent Lewis genes (le).
在两名Lewis阳性[Le(+)]和两名Lewis阴性[Le(-)]个体的胃黏膜中检测了Lewis岩藻糖基转移酶（FT）mRNA的表达。Northern印迹分析表明，Le(+)和Le(-)胃黏膜中的mRNA水平相似。我们通过聚合酶链反应（PCR）扩增从Le(+)和Le(-)胃黏膜中分离出Lewis FT cDNA的蛋白质编码区。来自Le(-)胃黏膜的cDNA序列显示，从cDNA起始密码子的A开始，在第59位有两个单碱基取代，即G取代T，在第508位有A取代G。这些取代可能是导致两个氨基酸残基发生变化的原因，从N端起，第20位的Leu被Arg取代，第170位的Gly被Ser取代。为了确定这些碱基取代中的一个或两个是否是Le(-)基因的原因，我们构建了嵌合cDNA并在COS细胞中表达。用含有第508个核苷酸突变的嵌合cDNA转染的那些COS细胞不表达Lewis抗原，而用含有第59个核苷酸突变的嵌合cDNA转染的那些细胞表达Lewis抗原，这表明第508位从G到A的单碱基变化是Le(-)基因的原因。第508位的G到A转换产生了一个新的PvuII内切酶位点。来自其中一名Le(-)个体的Lewis FT cDNA第386位和第612位核苷酸之间的PCR产物经PvuII内切酶消化后，被证明对PvuII位点是纯合的。然而，另一名Le(-)个体对PvuII位点是杂合的，这表明存在其他Le(-)等位基因。因此，我们分离出了一个沉默的Lewis基因（le）。
