Otterson G A, Kratzke R A, Lin A Y, Johnston P G, Kaye F J
NCI-Navy Oncology Branch, Bethesda, Maryland 20889.
Oncogene. 1993 Apr;8(4):949-57.
We have isolated cDNA and genomic clones for the human retinoblastoma binding protein 1 (RBP1) gene, and have identified alternative splicing of RBP1 clustered within a 207-nucleotide internal exon. Three of the predicted RPB1 peptides share amino-terminal and carboxy-terminal domains, while a fourth species encodes a distinct carboxy-terminal domain. Functional analysis of these peptides demonstrated that they are capable of precipitating retinoblastoma (RB) protein in vitro from K562 cell lysates, but cannot bind to mutant RB protein. However, each of the RBP1 peptides differed within an internal exon that contains potential casein kinase II and p34cdc2 phosphorylation sites. Immunoblot analysis using polyclonal alpha-RBP1 antiserum revealed that the RBP1 protein is expressed in a wide range of cell lines of differing histologic type and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis predominantly as a 200-kDa protein. Immunohistochemical analysis using the alpha-RBP1 antiserum demonstrated a distinct nuclear staining pattern that was eliminated when the antiserum was preabsorbed with RBP1 peptide. The RBP1 gene encodes a widely expressed 200-kDa nuclear protein and undergoes alternative splicing that predicts a family of RB-binding peptides.
我们已经分离出人类视网膜母细胞瘤结合蛋白1（RBP1）基因的cDNA和基因组克隆，并确定了RBP1的可变剪接集中在一个207个核苷酸的内部外显子内。预测的三种RPB1肽具有共同的氨基末端和羧基末端结构域，而第四种肽编码一个独特的羧基末端结构域。对这些肽的功能分析表明，它们能够在体外从K562细胞裂解物中沉淀视网膜母细胞瘤（RB）蛋白，但不能与突变的RB蛋白结合。然而，每个RBP1肽在一个包含潜在酪蛋白激酶II和p34cdc2磷酸化位点的内部外显子内存在差异。使用多克隆α-RBP1抗血清的免疫印迹分析显示，RBP1蛋白在多种不同组织学类型的细胞系中表达，并且在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上主要以200 kDa的蛋白形式迁移。使用α-RBP1抗血清的免疫组织化学分析显示出独特的核染色模式，当抗血清用RBP1肽预先吸收时，这种模式消失。RBP1基因编码一种广泛表达的200 kDa核蛋白，并经历可变剪接，预测出一个RB结合肽家族。
