SYNTHESIS AND STRUCTURE OF AVIAN TUMOR VIRUS DNA

逆转录病毒通过一种DNA中间体进行复制，该中间体共价 与宿主染色体DNA相关。合成过程中的许多步骤， 病毒DNA的结构、整合和表达还不完全 明白了。通过使用重组DNA技术，我们希望研究一些 这些事件发生在禽肉瘤病毒感染的细胞中。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。各种圆形的结构 DNA中间体与宿主细胞在环状结构形成中的作用 将会被评估。病毒DNA中的特定核苷酸序列 将确定参与整合重组的公司。这将是 通过在LTRS的反向重复序列中创建删除来处理。这个 野生型和突变的DNA将通过基因导入允许细胞 转移技术中，细胞产生的病毒将被用来再次感染 鸡QT6细胞与病毒DNA的合成与整合 被监视着。基因是否必须整合病毒DNA 还将通过在上体中保持病毒DNA来研究其表达 使用牛乳头状瘤DNA载体。独一无二的 病毒基因组中的序列，通常指的是NT (不可翻译)，在病毒复制中将通过构建 并通过分析该区域特有的新的转录产物。 TATA盒和上游调控序列在LTR中的作用 转录启动的效率将被确定。这些 研究将通过定点突变和通过引入 利用重组DNA技术进行特定的缺失。更确切地说， 将合成碱基改变的寡核苷酸引物，并 包含这些碱基更改的限制片段将被放回 PATV-8和这些改变的核苷酸对基因表达的影响 要下定决心。U3序列下游的意义 CAAT箱(-170)和TATA箱(-30)的上游将研究 用来自pBR DNA的非特异性序列替换该序列。最后， 我们将对Pr180从35S mRNA翻译的机制进行研究。 我们想确定是否有剪接，以去除琥珀色密码子 在Gag和Poll基因之间，与Pr180的发生有关，或者 其他机制，如核糖体在Gag-Poll连接处的口吃是 负责低水平Pr180的合成。

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.