ORGANIZATION OF ANIMAL CELL NUCLEI

动物细胞核的组织

• 批准号: 2021997
• 负责人: JOSEPH G. GALL
• 金额: $ 34.49万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2021997
• 关键词: DNA binding protein; DNA replication; Hydrozoa; RNA biosynthesis; Tetrahymena; Urodela; Xenopus; alternatives to animals in research; autoradiography; cell nucleus; complementary DNA; cytogenetics; egg /ovum; extracellular matrix; gel electrophoresis; gene expression; genetic manipulation; genetic regulatory element; genetic transcription; genetic translation; histones; mitochondrial DNA; molecular cloning; monoclonal antibody; nonhistone nucleoprotein; nucleic acid hybridization; nucleic acid sequence; oogenesis; protein sequence; proteins; radiotracer; transcription termination; transport proteins; transposon /insertion element

The proposed studies deal with nuclear proteins and their assembly into macromolecular complexes with RNA on the chromosomes and in other nuclear organelles. Advantage will be taken of the giant nucleus and giant lampbrush chromosomes (LBCs) of the amphibian oocyte. Monoclonal antibodies will be produced against nuclear proteins, whose subnuclear distribution will be determined by immunofluorescence. The extraordinary morphological detail in LBCs allows antigen detection within single transcription units or in defined subnuclear particulates. The antibodies will be used to recover cDNA clones from expression libraries. Protein sequences derived from the cDNA clones will permit deductions about the role(s) of the proteins in nuclear function. In vitro transcriptions made from the cDNA clones will be injected into amphibian oocytes, and the translation products (polypeptides) will be followed biochemically and cytologically from their site of synthesis in the cytoplasm through the nuclear envelope to their final destination on the chromosomes or elsewhere in the nucleus. By appropriate in vitro alteration of the injected transcripts we will determine what amino acid sequences are necessary for transport across the nuclear envelope and for targeting to the final subnuclear destination. Studies will also be conducted on the transcription of the 5S gene sequences on LBCs by in situ nucleic acid hybridization. Finally studies will be carried out on a novel selfcleaving RNA transcribed from a short, repeated sequence of newt DNA, with emphasis on the possible existence of a ribonucleoprotein particle. All of the proposed studies deal with how RNA produced by the chromosomes becomes associated with nuclear proteins in order to carry out basic cellular functions (RNA processing, storage, and transport). Knowledge of such functions is fundamental for understanding cell metabolism in both the normal and diseased state.

拟议的研究涉及核蛋白及其组装 大分子复合物与染色体上的RNA和其他核分子复合物 细胞器。 将对巨型和巨型的优势获取优势 两栖动物卵母细胞的lampbrush染色体（LBC）。 单克隆 将对核蛋白产生抗体，其亚核 分布将由免疫荧光确定。 非凡 LBC中的形态学细节允许单一抗原检测 转录单元或定义的亚核微粒。 抗体 将用于从表达文库中恢复cDNA克隆。 蛋白质 源自cDNA克隆的序列将允许扣除有关 蛋白质在核功能中的作用。 进行体外转录 从cDNA克隆中，将注入两栖动物卵母细胞，并将其注入 翻译产品（多肽）将在生化和 从其在细胞质中的合成部位从细胞学上通过 核包络到其在染色体上的最终目的地或 核中的其他地方。 通过适当的体外改变 注入的转录本我们将确定哪些氨基酸序列是 穿过核信封的运输和目标 最终的亚核目的地。 还将对 原位核酸对LBC的5s基因序列的转录 杂交。 最后，研究将对小说进行 从短的，重复的newt dna序列转录的自我剖析的RNA， 强调核糖核蛋白颗粒的可能存在。 所有拟议的研究都涉及染色体产生的RNA 与核蛋白相关，以进行基本 细胞功能（RNA处理，存储和运输）。 知识 这种功能对于理解两者的细胞代谢是基础 正常和患病状态。