Targeting of Integration

为了观察诱变元件对靶位的异常偏好，我们构建了一株酿酒葡萄球菌，将URA3基因置于III号染色体上首选甘酰trna （suf16）上游的基因组中。这种结构的结构是这样的，正确靶向suf16上游的一个元素会破坏URA3，从而允许细胞在FOA存在的情况下存活。利用修复缺陷型大肠杆菌，我们在GAL1启动子的控制下随机诱变了一个含有Ty1元件的质粒，并含有逆转录转位指示剂his3AI。大约10,000个突变质粒通过在缺乏组氨酸的培养基上存活来筛选转位，通过在含有FOA的培养基上无法存活来筛选异常靶向，以及通过在含有菜豆氨酸（精氨酸渗透酶的有毒类似物）的培养皿上存活来筛选染色体复制的缺失。通过对提取的基因组DNA进行PCR，进一步分析suf16上游Ty1插入的候选基因。该筛选产生了31个足够有兴趣的突变体，以保证测序。测序结果显示，突变分散在整个元件中，落在移码位点、蛋白酶、n端锌结合域和整合酶活性位点域内。然而，在整合酶的c端结构域中发现了一些突变，这是一个未知功能的区域。此外，一些突变落在逆转录酶的n端区域。虽然假设靶向决定因素存在于逆转录酶而不是整合酶中可能是违反直觉的，但最近的一些观察表明，在蛋白质水解裂解形成成熟蛋白质后，逆转录酶和整合酶在功能上（如果不是物理上）仍然存在关联，这导致了靶向决定因素可能由多个位点组成的猜测。这些突变菌株现在已经被转移到佐治亚大学。

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.