STRUCTURE AND FUNCTION OF NUCLEOLAR, NONHISTONE PROTEINS

该研究的目的是分析其结构并阐明 两种主要的核仁非核糖体非组蛋白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。 它们含有大量 酸性磷酸化区域，它们存在于核仁前核糖体 颗粒，并且它们对银具有高亲和力。 蛋白质C23位于 位于染色体的核仁组织者区域，并与DNA结合， 对18 S表达区上游序列的明显偏好。 大约15%的蛋白质C23的序列和主要的位置， 碎片已经确定。 本研究的主要目的是 完成C23蛋白的测序。 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 来自C23蛋白质信使RNA的互补DNA。 cDNA将是 将其克隆到适当的载体中，将对DNA进行测序， 将从DNA序列推导出序列。 的位置 大约35个磷酰基将通过自动化蛋白质 测序 类似的方法将应用于蛋白质B23;即， 部分蛋白质测序、cDNA克隆和DNA测序。 额外 与这两种蛋白质的结构和功能相关的研究将是 进行：（1）确定蛋白C23的DNA结合结构域 通过测定蛋白质的大片段的DNA结合活性， 蛋白质C23-DNA复合物的蛋白水解; DNA结合结构域将 通过自动蛋白质测序鉴定;（2）天然分子量 将通过沉降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 圆二色性;（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 和光交联。 这些研究将有助于长期 目的是了解核仁的结构和功能。 因为 核仁对生物化学和形态学非常敏感 肿瘤过程和药物治疗的改变，这些研究 有助于阐明疾病分子机制， 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.