Bora N S, Post T W, Atkinson J P
Howard Hughes Medical Institute, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110.
J Immunol. 1991 Apr 15;146(8):2821-5.
An RFLP was found in the DNA of 25 unrelated persons, two families, and five cell lines that correlated with their membrane cofactor protein phenotype. If restricted with HindIII, DNA derived from upper band predominant protein (U) phenotypes had a band at 2 kb, whereas DNA of lower band predominant (L) phenotypes had a 4-kb band. The equal band protein phenotype, in which equal quantities of the two species are expressed, had bands at both 4 and 2 kb. The polymorphic HindIII site was localized to an intron within the membrane cofactor protein gene between exon 1 (codes for 5'UT/signal peptide) and exon 2 (codes for the first short consensus repeat). Using the polymerase chain reaction (PCR), sequences around this site were amplified and a single band of 260 bp was produced. In the U phenotype, the PCR product was restricted with HindIII into 200- and 60-bp fragments. In the L phenotype, there was no change in the size of 260 bp upon restriction with HindIII. For the equal band protein phenotype, the PCR product was partially cleaved. The 260-bp PCR product was subcloned and sequenced. DNA from the U phenotype demonstrated an intact HindIII site (AAGCTT), whereas in the DNA of the L phenotype, this site was altered because a "G" was substituted for a "C" (AAGGTT).
在25名无亲缘关系的个体、两个家族以及5个细胞系的DNA中发现了一种限制性片段长度多态性（RFLP），它与它们的膜辅助因子蛋白表型相关。用HindIII酶切时，来自上带优势蛋白（U）表型的DNA在2 kb处有一条带，而下带优势（L）表型的DNA有一条4 kb的带。等量带蛋白表型，即两种蛋白等量表达，在4 kb和2 kb处都有带。多态性的HindIII位点定位于膜辅助因子蛋白基因中第1外显子（编码5'非翻译区/信号肽）和第2外显子（编码第一个短共有重复序列）之间的一个内含子内。使用聚合酶链反应（PCR）扩增该位点周围的序列，产生了一条260 bp的单带。在U表型中，PCR产物用HindIII酶切后产生200 bp和60 bp的片段。在L表型中，用HindIII酶切后260 bp的大小没有变化。对于等量带蛋白表型，PCR产物被部分切割。将260 bp的PCR产物进行亚克隆并测序。U表型的DNA显示有一个完整的HindIII位点（AAGCTT），而在L表型的DNA中，该位点发生了改变，因为一个“G”取代了一个“C”（AAGGTT）。
