Domains of Rinderpest virus phosphoprotein involved in interaction with itself and the nucleocapsid protein.

The yeast two-hybrid system was used to identify domains involved in specific in vivo interactions between the Rinderpest virus (RPV) phosphoprotein (P) and nucleocapsid protein (N). N and P genes were cloned in both the yeast GAL4 DNA-binding and GAL4 activation domain vectors, which enabled analysis of self and interprotein interactions. Mapping of the domain of P protein involved in its association with itself revealed that the COOH-terminal 32 amino acids (316-347) that forms a part of the highly conserved coiled coil region is important for interaction. In addition, just the coiled coil region of RPV P protein fused to the DNA-binding domain and activation domain of GAL4 was found to be sufficient to bring about activation of the beta-galactosidase reporter. Similarly, mapping of the domains of P protein involved in its interaction with N protein revealed that NH2-terminal 59 amino acids and COOH-terminal 32 amino acids (316-347) involved in P-P interaction are simultaneously required for association with N protein. Interestingly, a P protein mutant with just the NH2-terminal 59 amino acids and the coiled coil domain with all other P protein regions deleted retained its ability to interact with N protein. Furthermore, we were able to show N and P protein interaction in vitro using recombinant N and P proteins expressed in Escherichia coli, demonstrating the existence of direct physical interaction between the two proteins.     

HBV Pre S1蛋白在酵母细胞中的转录激活功能

目的 探索应用双杂交体系统克隆与乙型肝炎病毒（HBV）Pre S1蛋白结合的肝细胞受体的可行性。方法 构建编码HBV Pre S1全序列或部分序列与酵母蛋白GAL 4 DNA结合区域融合蛋白的酵母表达质粒，应用这些质粒转化酵母报道菌株SFY526，检测β－半乳糖苷酶活性作为酵母中转录激活的指标。构建编码Pre S1全序列与GAL4 DNA结合区域融合蛋白的哺乳动物细胞表达质粒，与CAT报道质粒共转染肝癌细胞系Huh-7，使用薄层层析法检测细胞提取物的氯霉素乙酰转移酶活性。结果 全序列Pre S1蛋白与GAL4 DNA结合区域的融合蛋白在酵母细胞中呈现转录激活功能，其转录激活序列被定位于Pre S1第21－47氨基酸之间。全序列Pre S1蛋白与GAL 4 DNA结合区域的融合蛋白在哺乳动物细胞中未呈现转录激活功能。结论 HBV Pre S1蛋白在酵母细胞中的转录激活功能，限制了研究人员应用酵母双杂交体系统克隆与Pre S1结合的HBV受体。然而，Pre S1蛋白在哺乳动物细胞未呈现转录激活功能。哺乳动物细胞双杂交体系统可能是克隆与Pre S1蛋白作用的肝细胞受体的可行途径。     

The regulatory protein NIT4 that mediates nitrate induction inNeurospora crassa contains a complex tripartite activation domain with a novel leucine-rich,acidic motif

Expression ofnit-3 andnit-6, the structural genes which encode nitrate reductase and nitrite reductase inNeurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys₆/Zn₂ zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo inN. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.     

双向杂交系统：研究蛋白质间相互作用的新手段

双向杂交系统：研究蛋白质间相互作用的新手段王巍（中国医学科学院基础医学研究所医学分子生物学国家重点实验室，北京１００００５）ＡｂｓｔｒａｃｔＴｈｅｔｗｏ－ｈｙｂｒｉｄｓｙｓｔｅｍｉｓａｙｅａｓｔｇｅｎｅｔｉｃａｓａｙｆｏｒｄｅｔｅｃｔｉｎｇｐｒｏｔｅ...     

Two distinct regions of the BPV1 E1 replication protein interact with the activation domain of E2

Papillomavirus E1 and E2 proteins co-operation in viral DNA replication is mediated by protein-protein interactions that lead to formation of an E1-E2 complex. To identify the domains involved, portions of the two proteins were expressed as fusions to the DNA-binding protein LexA or the transactivation domain of VP16 and analyzed by the yeast two-hybrid system. The C-terminal 266 amino acids of BPV1 E1 (E1C266) interacted strongly with E2 in the yeast system and in a mammalian two-hybrid assay. VP16-E1C266 interacted with a region encompassing amino acids 1-200 of the transactivation domain of E2 that was fused to LexA. The interaction between E1 full length and E2 was clearly observed only when E1 was expressed as LexA-E1 chimera. In addition, we found that in the LexA context also the N-terminal region encompassing the first 340 amino acids of E1 (E1N340) interacted with E2 full length. The interactions of E1N340 and E1C266 with E2 were confirmed also by in vitro binding studies. These observations demonstrate that two distinct regions of E1 mediate the interaction with E2 in vivo.