MAB PROBES OF DOUBLE-HELIX STRUCTURE FUNCTION

双螺旋结构功能的MAB探针

• 批准号: 3294838
• 负责人: DAVID A ZARLING
• 金额: $ 23.11万
• 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/3294838
• 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鉴定特定的单抗 识别细胞表面独特结构或构象的抗体 双螺旋RNA或DNA序列，并使用这些单抗作为实验 免疫化学证明研究双链核酸结构和 功能。我们将研究核仁、细胞核和细胞质的双重- 研究链状核酸的合成、转运、稳定性和 活细胞中双链RNA的功能。显微注射免疫球蛋白 针对与核酸特异结合的RNA或DNA双链 活体内将继续被我们的小组用于探测核酸 活体人体细胞的代谢和功能(如免疫抗体 抑制细胞增殖)。具体目标是：(1) 制备和鉴定新型抗独特型高亲和力小鼠单抗 生物上重要的双链RNA和DNA的决定因素。 重点将放在A-RNA和B-DNA双链以及N-ACO-AFF 加引子序列。(2)利用这些新的和现有的试验和 用自身免疫性抗核酸单抗定量检测血吸虫抗体的原位分布 RNA和DNA双螺旋上的独特决定因素，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，我们还建议本地化并识别MAb 免疫电子显微镜的反应靶标。(3)研究双链 活细胞中的RNA和DNA功能。胞质和/或核 这些抗核酸单抗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。 这些分析是理解现场分布的基础 以及对独特的天然和/或化学修饰致癌物的识别 双工序列。这些免疫细胞学研究的结果将 极大地促进了我们对结构-功能关系的了解 双螺旋。