CHARACTERIZATION OF THE GENE PRODUCTS OF THE ETS LOCUS

人ETS 1原癌基因蛋白已从T细胞中分离出来， 免疫亲和色谱法测定细胞系CEM，并确认其同一性 通过N-末端氨基酸测序。 p51和p42 ETS 1亚型反应， 使用针对细菌表达的ETS 1的单克隆抗体 本发明还涉及针对羧基末端13个氨基的寡肽， 人ETS 1蛋白的氨基酸。 p42人ETS 1不与 针对人ETSI的外显子VII的抗体，表明其是 缺少外显子VII的选择性剪接mRNA的产物。 P48和 人ETSI的p39同种型显示来源于p51 通过-SH的共价修饰来表达人ETS 1的p42亚型 由蛋白酶抑制剂（N（α）-对甲苯磺酰基-L-赖氨酸氯甲基 酮（TLCK）。 复性的人ETS 1显示具有DNA 与PEA-3（AGGAAGT）基序的序列特异性结合;该复合物可以 通过电泳迁移率变动分析（EMSA）观察。 的 纯化的ETSI延迟了与纯化的ETSI完全相同大小的复合物， 从T细胞白血病细胞制备的核提取物中延迟 线，CEM。 形成ETS 1需要减少的ETSI。 PEA-3复合物， 但用N-乙基马来酰胺或TLCK修饰ETSI-SH基团， 不抑制ETS1.PEA-3复合物的形成。 ETSI.PEA-3 TLCK修饰的ETSI形成的复合物的迁移率比TLCK修饰的ETSI低， 用未修饰的ETSI形成的复合物。 从用一种免疫的小鼠制备六种单克隆抗体， 细菌表达的人ETS 2蛋白。 这些抗体特别 识别两种人类ETS 2编码的蛋白质，p56和p54，但未能识别 与鸡、小鼠、大鼠、牛或猴蛋白反应，表明 抗体识别对人ETS 2特异的表位 蛋白 ETS 2蛋白的生物化学分析将是 通过这些单克隆抗体的发展， 作为领域特异性探针和工具， 在异源表达系统中检测人ETS 2蛋白。

The human ETS1 proto-oncogene proteins have been isolated from the T-cell line, CEM, by immunoaffinity chromatography and their identity confirmed by N-terminal amino acid sequencing. The p5l and p42 ETS1 isoforms react with monoclonal antibodies directed against a bacterially-expressed ETS1 protein and to an oligopeptide directed to the carboxyl-terminal 13 amino acids of the human ETS1 protein. The p42 human ETS1 does not react with an antibody directed to exon VII of the human ETSI, indicating that it is the product of alternatively-spliced mRNA lacking exon VII. The p48 and the p39 isoforms of the human ETSI are shown to be derived from the p5l and p42 isoforms of the human ETS1 by the covalent modification of -SH groups by the protease inhibitor (N(alpha)-p-tosyl-L-lysine chloromethyl ketone (TLCK). The renatured human ETS1 was shown to have DNA sequence-specific binding to the PEA-3 (AGGAAGT) motif; this complex can be observed by electrophoretic mobility shift assays (EMSA). The purified ETSI retards a complex which is exactly the same size complex as is retarded from nuclear extracts prepared from the T-cell leukemia cell line, CEM. Reduced ETSI is required to form the ETS1. PEA-3 complex, but modification of the ETSI -SH groups by N-ethylmaleiamide or by TLCK does not inhibit formation of the ETS1.PEA-3 complex. The ETSI.PEA-3 complex formed with TLCK-modified ETSI has a slower mobility than does the complex formed with unmodified ETSI. Six monoclonal antibodies were prepared from mice immunized with a bacterially-expressed human ETS2 protein. These antibodies specifically recognize the two human ETS2-encoded proteins, p56 and p54, but failed to react with chicken, mouse, rat, bovine, or monkey proteins, suggesting that the antibodies recognize epitopes specific to the human ETS2 protein. The biochemical analysis of the ETS2 protein will be facilitated by the development of these monoclonal antibodies, which may be useful as both domain-specific probes and tools for specifically detecting the human ETS2 protein in heterologous expression systems.