CONSTRUCTION, CHARACTERIZATION, AND UTILIZATION OF CELL-LINES WHICH INDUCIBLY EXPRESS THE ADENOVIRUS DNA-BINDING PROTEIN
CONSTRUCTION, CHARACTERIZATION, AND UTILIZATION OF CELL-LINES WHICH INDUCIBLY EXPRESS THE ADENOVIRUS DNA-BINDING PROTEIN
复制标题
DOI：
10.1016/0042-6822(92)90900-a
复制
发表时间：
1992-10-01
期刊：
影响因子：
3.7
通讯作者：
KLESSIG, DF
中科院分区：
文献类型：
--
作者：
BROUGH, DE;CLEGHON, V;KLESSIG, DF
为了进一步了解多功能腺病毒 DNA 结合蛋白 (DBP) 内的结构与功能关系，需要更多样化的突变体集合。先前构建了表达 DBP 的细胞系 (gmDBP)，以补充 DBP 阴性突变体的病毒生长。然而，他们不允许严重缺陷的病毒形成噬菌斑。由于有效的突变体构建依赖于所需突变病毒的空斑分离作为最后一步，因此构建了额外的 gmDBP 细胞系，其允许所有 DBP 阴性突变体形成空斑。在这里，我们描述了 12 个新的 gmDBP 细胞系的构建和表征。通过使用表达 DBP 的细胞系组合有效构建新的缺陷突变体 H5in804，证明了这些细胞系的实用性。 H5in804 突变在野生型蛋白质的羧基末端添加了 22 个氨基酸。 H5in804 的表征表明，它复制病毒 DNA 的能力发生了改变。 DNA 合成的抑制很可能是由于 H5in804 DBP 结合 ssDNA 的能力降低所致。
To further our understanding of structure-function relationships within the multifunctional adenovirus DNA binding protein (DBP) a more diverse collection of mutants is necessary. DBP-expressing cell lines (gmDBP) were previously constructed that complemented DBP-negative mutants for viral growth. However, they did not allow severely defective viruses to form plaques. Since efficient mutant construction is reliant on plaque isolation of the desired mutant virus as a final step, additional gmDBP cell lines were constructed which allow all DBP-negative mutants to form plaques. Here we describe the construction and characterization of 12 new gmDBP cell lines. The utility of these lines was demonstrated by the efficient construction of a new defective mutant, H5in804, using a combination of DBP-expressing lines. The H5in804 mutation adds 22 amino acids at the carboxyl end of an otherwise wild type protein. Characterization of H5in804 revealed that it was altered in its ability to replicate viral DNA. The depression of DNA synthesis most probably results from a reduced ability of H5in804 DBP to bind ssDNA.