SYNTHESIS AND STRUCTURE OF AVIAN TUMOR VIRUS DNA

禽肿瘤病毒DNA的合成和结构

• 批准号: 3174364
• 负责人: RAMAREDDY V GUNTAKA
• 金额: $ 8.47万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 1987-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3174364
• 关键词: DNA directed RNA polymerase; avian sarcoma virus; cell free system; gene expression; genetic manipulation; genetic mapping; genetic transcription; host organism interaction; nucleic acid structure; oncogenes; oncogenic virus; tissue /cell culture; virus DNA; virus genetics; virus replication

Retroviruses replicate through a DNA intermediate which covalently associates with host chromosomal DNA. Many steps in the synthesis, structure, integration and expression of viral DNA are not completely understood. By using recombinant DNA techniques we wish to study some of these events in avian sarcoma virus-infected cells. The direction and temporal relationship of (-) and (+) strands will be examined by using cloned viral DNA fragments as probes. The structure of various circular DNA intermediates and the role of host cell in the formation of circles will be assessed. Specific nucleotide sequences in the viral DNA that are involved in the integrative recombination will be determined. This will be approached by creating deletions in the inverted repeats of the LTRs. The wild type and mutated DNA will be introduced into permissive cells by gene transfer techniques, virus produced by the cells will be used to re-infect chicken QT6 cells and viral DNA synthesis and integration will be monitored. Whether integration of viral DNA is mandatory for gene expression will also be studied by maintaining viral DNA in an episomal state using bovine papilloma DNA vector. The requirement of a unique sequence in the viral genome, generally referred to as NT (nontranslatable), in virus replication will be studied by constructing deletions and by analyzing for new transcripts specific for this region. The role of TATA box and the upstream regulatory sequences in the LTR on the efficiency of transcription initiation will be determined. These studies will be approached by site directed mutagenesis and by introducing specific deletions using recombinant DNA techniques. More specifically, oligonucleotide primers with altered bases will be synthesized and the restriction fragments containing these base changes will be put back into pATV-8 and the effect of these altered nucleotides on gene expression will be determined. The significance of the U3 sequence downstream from the CAAT box (-170) and upstream from TATA box (-30) will be studied by replacing this sequence with a nonspecific sequence from pBR DNA. Finally, the mechanism by which Pr180 is translated from 35S mRNA will be studied. We would like to determine whether a splice, to remove the amber codon between gag and pol genes, is involved in the genesis of Pr180 or whether other mechanisms such as ribosome stuttering at the gag-pol junction is responsible for the synthesis of low levels of Pr180.

逆转录病毒通过DNA中间体复制， 与宿主染色体DNA结合。 合成中的许多步骤， 病毒DNA结构、整合和表达并不完全 明白 通过使用重组DNA技术，我们希望研究一些 禽肉瘤病毒感染细胞中的这些事件。 的方向和 （-）和（+）链的时间关系将通过使用 克隆的病毒DNA片段作为探针。 各种圆形结构 DNA中间体与宿主细胞在环形成中的作用 将予以评估。 病毒DNA中的特定核苷酸序列 将确定参与整合重组的基因。 这将是 通过在LTR的反向重复序列中产生缺失来接近。 的 野生型和突变的DNA将通过基因导入到允许细胞中， 转移技术，由细胞产生的病毒将被用于重新感染 鸡QT 6细胞和病毒DNA的合成和整合， 监测。 病毒DNA的整合是否是基因的强制性 还将通过将病毒DNA保持在游离体中来研究表达 使用牛乳头状瘤DNA载体。 一个独特的需求 病毒基因组中的一段序列，通常称为NT （不可翻译），在病毒复制中将通过构建 缺失和分析该区域特异性的新转录本。 TATA盒及其上游调控序列在LTR中的作用 将确定转录起始的效率。 这些 研究将通过定点诱变和引入 使用重组DNA技术进行特异性缺失。 更具体地说， 将合成具有改变的碱基的寡核苷酸引物， 含有这些碱基变化的限制性片段将被放回 pATV-8和这些改变的核苷酸对基因表达的影响将 被确定。 U3序列下游的重要性 CAAT箱（-170）和TATA箱（-30）上游将通过以下方式进行研究： 用来自pBR DNA的非特异性序列替换该序列。 最后， 将研究Pr 180从35 S mRNA翻译的机制。 我们想确定是否有一个剪接，删除琥珀密码子 在gag和pol基因之间，参与Pr 180的发生，或者是否 其他机制如gag-pol连接处的核糖体口吃， 负责合成低水平的Pr 180。