MAB PROBES OF DOUBLE-HELIX STRUCTURE FUNCTION

双螺旋结构功能的MAB探针

• 批准号: 3294834
• 负责人: DAVID A ZARLING
• 金额: $ 20.72万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-10-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294834
• 关键词: DNA replication; RNA biosynthesis; acetylaminofluorene; adduct; antibody formation; antibody specificity; chemical carcinogen; chemical structure function; conformation; covalent bond; fluorescence microscopy; gel electrophoresis; immunocytochemistry; immunoelectron microscopy; immunologic techniques; laboratory mouse; monoclonal antibody; nucleic acid metabolism; nucleic acid sequence; radiotracer; tissue /cell culture

Our goals are to continue to produce and characterize specific monoclonal antibodies (Mabs) that recognize unique structures or conformations on double-helical RNA or DNA sequences and to use these Mabs as experimental immunochemical proves to study duplex nucleic acid structures and functions. We will investigate nucleolar, nuclear, and cytoplasmic double- stranded nucleic acids and study the synthesis, transport, stability, and functions of double-stranded RNa in living cells. Microinjected IgGs directed against RNA or DNA duplexes that specifically bind nucleic acids in vivo will continue to be used by our group to probe nucleic acid metabolism and functions in living human cells (e.g., immune antibody inhibition of cell multiplication). The specific aims are to: (1) Generate and characterize new high-affinity mouse Mabs against unique determinants on biologically important double-stranded RNAs and DNAs. Emphasis will be placed on A-RNA and B-DNA duplexes as well as on N-AcO-AFF adducted sequences. (2) Use these new and existing experimental and autoimmune anti-nucleic-acid Mabs to quantitate the in situ distribution of unique determinants on RNA and DNA double helices, including N-AcO-AAF adducted sequences in fixed cells, primarily by quantitative fluorescence laser microscopy. Also, we propose to localize and identify the Mab reactive targets by immunoelectronmicroscopy. (3) Study double-stranded RNA and DNA functions in living cells. Cytoplasmic and/or nuclear microinjection of these anti-nucleic-acid Mab probes will be used to determine their effects on nucleic acid metabolism and cell functions. These analyses are fundamental to understanding the in situ distribution and recognition of unique natural and/or chemical-carcinogen-modified duplex sequences. The results of these immunocytological studies will significantly advance our knowledge of structure-function relationships of double helices.

我们的目标是继续生产和表征特异性单克隆抗体， 抗体（Mab）识别上的独特结构或构象， 双螺旋RNA或DNA序列，并使用这些单克隆抗体作为实验 免疫化学证明研究双链核酸结构， 功能协调发展的 我们将研究核仁，核，和细胞质双- 链核酸和研究的合成，运输，稳定性， 双链RNA在活细胞中的功能。 显微注射IgG 针对特异性结合核酸的RNA或DNA双链体 我们的小组将继续使用体内探针来探测核酸 在活的人类细胞中的代谢和功能（例如，免疫抗体 抑制细胞增殖）。 具体目标是：（1） 生成并表征新的高亲和力小鼠单抗， 生物学上重要的双链RNA和DNA的决定因素。 重点将放在A-RNA和B-DNA双链体以及N-AcO-AFF上 内收序列 (2)使用这些新的和现有的实验和 自身免疫性抗核酸单克隆抗体，以定量 RNA和DNA双螺旋上的独特决定簇，包括N-AcO-AAF 固定细胞中的加合序列，主要通过定量荧光 激光显微镜 此外，我们建议定位和鉴定单克隆抗体， 免疫电镜下的反应性靶点。 (3)研究双链 RNA和DNA在活细胞中发挥作用。 细胞质和/或细胞核 这些抗核酸Mab探针的显微注射将用于 确定它们对核酸代谢和细胞功能的影响。 这些分析是了解原位分布的基础 并识别独特的天然和/或化学致癌物修饰 双链体序列 这些免疫细胞学研究的结果将 大大提高了我们对结构-功能关系的认识， 双螺旋