Hirose F, Yamaguchi M, Kuroda K, Omori A, Hachiya T, Ikeda M, Nishimoto Y, Matsukage A
Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan.
J Biol Chem. 1996 Feb 16;271(7):3930-7. doi: 10.1074/jbc.271.7.3930.
DREF, a transcription regulatory factor which specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication-related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respectively. Deletion analysis of bacterially expressed DREF fused with glutathione S-transferase (GST-DREF) indicated that a part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase alpha promoter in vitro. Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related genes. Immunocytochemical analysis demonstrated the presence of DREF polypeptide in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences.
DREF是一种转录调节因子，它能特异性地结合DNA复制相关基因的启动子激活元件DRE（DNA复制相关元件），已从果蝇Kc细胞的核提取物中纯化至同质。利用基于部分氨基酸序列合成的引物，通过逆转录聚合酶链反应方法分离出DREF的cDNA，并随后筛选cDNA文库。根据cDNA的核苷酸序列推断，DREF是一种由701个氨基酸残基组成的多肽，分子量为80,096，它包含三个分别富含碱性氨基酸、脯氨酸和酸性氨基酸的特征区域。对与谷胱甘肽S-转移酶融合的细菌表达的DREF（GST-DREF）进行缺失分析表明，N端碱性氨基酸区域的一部分（16-115个氨基酸）负责与DRE的特异性结合。针对GST-DREF融合蛋白制备了一种多克隆抗体和四种单克隆抗体。这些抗体在体外特异性抑制DNA聚合酶α启动子的转录。使用Kc细胞进行的共转染实验表明，DREF蛋白的过量表达克服了zerknüllt基因产物对增殖细胞核抗原基因启动子的抑制作用。这些结果证实DREF是DNA复制相关基因的反式激活因子。免疫细胞化学分析表明，在第八个核分裂周期后，细胞核中存在DREF多肽，这表明DREF的核积累对于携带DRE序列的DNA复制相关基因的合子协调表达很重要。
