Targeting of Integration

整合目标

• 批准号: 8157775
• 负责人: David J. Garfinkel
• 金额: $ 26.63万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157775
• 关键词: Active Sites; Binding; C-terminal; Canavanine; Cell Survival; Cells; Chromosomal Duplication; Chromosomes; DNA; Elements; Escherichia coli; Failure; Genes; Genome; Genomics; Histidine; Integrase; Mutation; N-terminal; Periodicity; Plasmids; Proteins; RNA Polymerase III; RNA-Directed DNA Polymerase; Reaction; Recombinant DNA; Retrotransposition; Saccharomyces cerevisiae; Site; Transfer RNA; Universities; Zinc; analog; arginine permease; falls; glycine-tRNA; interest; mutant; preference; promoter; protease N; repaired

To observe aberrant target site preference by mutagenized elements, we constructed a S. cerevisiae strain in which the URA3 gene was placed in the genome upstream of a preferred glycyl-tRNA (suf16) on chromosome III. The configuration of this construction is such that correct targeting of an element upstream of the suf16 disrupts the URA3 thereby permitting survival of cells in the presence of FOA. Using repair-deficient Escherichia coli, we randomly mutagenized a plasmid containing the Ty1 element under the control of the GAL1 promoter, and containing the retrotransposition indicator, his3AI. Approximately 10,000 mutagenized plasmids were screened for transposition by survival on media lacking histidine, for aberrant targeting by failure to survive on media containing FOA, and absence of chromosomal duplications by the ability to survive on plates containing canavanine, the toxic analogue of arginine permease. Candidates from this initial screen were further analyzed by PCR of extracted genomic DNA for Ty1 insertions upstream of the suf 16. This screen generated 31 mutants of sufficient interest to warrant sequencing. Sequencing results showed mutations dispersed throughout the element, falling within the frameshift site, protease, the N-terminal zinc binding domain and active site domain of integrase. However, several mutations were found within the C-terminal domain of integrase, a region of no known function. Also, several mutations fell within the N-terminal region of reverse transcriptase. Although it may be counterintuitive to hypothesize that the targeting determinant resides in reverse transcriptase rather than integrase, a number of recent observations that reverse transcriptase and integrase remain functionally, if not physically, associated after proteolytic cleavage to form mature proteins, leads to speculation that the targeting determinant may be comprised of multiple sites. These mutant strains have now been relocated to the University of Georgia.

为了观察突变元件对靶位点的偏好性，我们构建了一个S。酿酒酵母菌株，其中URA 3基因位于染色体III上优选的甘氨酰-tRNA（suf 16）的基因组上游。这种结构的构造使得suf 16上游元件的正确靶向破坏URA 3，从而允许细胞在FOA存在下存活。使用修复缺陷型大肠杆菌，我们随机诱变的质粒含有的GAL 1启动子的控制下的Ty 1元件，并含有反转录转座指示剂，his 3AI。通过在缺乏组氨酸的培养基上存活来筛选约10，000个诱变质粒的转座，通过在含有FOA的培养基上不能存活来筛选异常靶向，以及通过在含有刀豆氨酸（精氨酸通透酶的毒性类似物）的平板上存活的能力来筛选染色体重复的缺失。通过提取基因组DNA的PCR进一步分析来自该初始筛选的候选物在suf 16上游的Ty 1插入。该筛选产生了31个足够感兴趣的突变体以保证测序。测序结果表明，突变分散在整个元件，属于移码位点，蛋白酶，N-末端锌结合结构域和整合酶的活性位点结构域。然而，在整合酶的C-末端结构域内发现了几个突变，这是一个功能未知的区域。此外，几个突变落在逆转录酶的N-末端区域内。尽管假设靶向决定簇存在于逆转录酶而不是整合酶中可能是违反直觉的，但最近的许多观察结果表明，逆转录酶和整合酶在蛋白水解切割形成成熟蛋白质后，即使不是物理上的，也保持功能上的关联，这导致推测靶向决定簇可能由多个位点组成。这些突变株现已被转移到格鲁吉亚大学。