STRUCTURE AND FUNCTION OF NUCLEOLAR, NONHISTONE PROTEINS

拟议研究的目的是分析结构并阐明 两种主要核仁非核糖体、非组蛋白蛋白B23的功能 (Mr 38,000)和C23(Mr 110,000)。这两种蛋白质被认为是 组织核仁的各种成分。它们含有很高的 酸性、磷酸化区域，发现于核仁胸前 颗粒，它们对银有很高的亲和力。蛋白质C23被定位 在染色体的核仁组织者区域，并将DNA与 明显偏爱18个S表达区域上游的序列。 大约15%的蛋白质C23序列和主要的 碎片已经确定。这项研究的主要目标是 完成蛋白质C23的序列测定。这将通过使用已知的 合成用于生产的寡核苷酸引物的序列信息 从蛋白质C23信使RNA中提取互补DNA。该基因将被命名为 在适当的载体中克隆，DNA将被测序，氨基酸将被 序列将从DNA序列中推断出来。的位置。 大约35个磷酸基团将由自动蛋白质确定 测序。类似的方法将应用于蛋白质B23；即， 蛋白质部分测序、cDNA克隆和DNA测序。其他内容 与这两种蛋白质的结构和功能相关的研究将在 (1)确定蛋白质C23的DNA结合域 通过检测蛋白质的大片段的DNA结合活性和 蛋白质C23-DNA复合体的蛋白质分解；DNA结合域将是 蛋白质自动测序鉴定；(2)天然相对分子质量 将由沉积研究和化学交联确定；(3) 蛋白质C23及其片段的二级结构将通过 圆二色谱；以及(4)这些蛋白质与其他蛋白质的相互作用 大分子将通过使用可裂解的化学交联剂来确定 和照片交联剂。这些研究将有助于长期的 目的是了解核仁的结构和功能。因为 核仁对生化和形态变化高度敏感。 肿瘤过程和药物治疗的变化，这些研究 应有助于阐明疾病的分子机制和 化疗。

The aim of the proposed research is to analyze the structures and elucidate the functions of two major nucleolar nonribosomal, nonhistone proteins, B23 (Mr 38,000) and C23 (Mr 110,000). The two proteins are believed to organize the various components of the nucleolus. They contain highly acidic, phosphorylated regions, they are found in nucleolar preribosomal particles and they have a high affinity for silver. Protein C23 is located at the nucleolus organizer regions of chromosomes and binds DNA with an apparent preference for sequences upstream from the 18 S expressed region. Approximately 15% of the sequence of protein C23 and the location of major fragments have been determined. The major goal of this study is to complete the sequence of protein C23. This will be achieved by using known sequence information to synthesize oligonucleotide primers for production of complementary DNA from protein C23 messenger RNA. The cDNA will be cloned in appropriate vectors, the DNA will be sequenced and the amino acid sequence will be deduced from the DNA sequence. The location of the approximately 35 phosphoryl groups will be determined by automated protein sequencing. A similar approach will be applied to protein B23; i.e., partial protein sequencing, cDNA cloning and DNA sequencing. Additional studies related to the structure and function of these two proteins will be conducted: (1) the DNA binding domains of protein C23 will be determined by assaying large fragments of the protein for DNA binding activity and by proteolysis of the protein C23-DNA complex; DNA binding domains will be identified by automated protein sequencing; (2) the native molecular weight will be determined by sedimentation studies and chemical crosslinking; (3) the secondary structure of protein C23 and its fragments will be studied by circular dichroism; and (4) the interactions of these proteins with other macromolecules will be determined by use of cleavable chemical crosslinkers and photo crosslinking. These studies will contribute to the long range goal of understanding the structure and function of the nucleolus. Because the nucleolus is highly susceptible to biochemical and morphological alterations in neoplastic processes and in drug treatment, these studies should aid in elucidation of molecular mechanism of disease and chemotherapy.