Davis J, Scherer M, Tsai W P, Long C
J Virol. 1976 May;18(2):709-18. doi: 10.1128/JVI.18.2.709-718.1976.
A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.
一种灵敏的硝酸纤维素滤膜分析法用于检测和纯化来自劳氏肉瘤病毒且保留生物学功能的DNA结合蛋白，该方法可测定125I标记的单链小牛胸腺DNA的保留情况。通过在寡聚（dT）-纤维素柱和DEAE-生物凝胶柱上连续纯化，以及在10%至30%甘油梯度中离心，已将依赖RNA的DNA聚合酶与第二种病毒体DNA结合蛋白分离。该蛋白与DNA的结合受NaCl浓度的强烈影响，但在较宽的温度或pH范围内活性变化很小。经甘油梯度纯化后，该蛋白的聚丙烯酰胺凝胶电泳显示出一条主要条带，分子量约为9800。该蛋白与单链大肠杆菌DNA、小牛胸腺DNA或70S C型病毒RNA的结合能力相当。未标记的大肠杆菌或小牛胸腺单链DNA以及70S鼠类或猫类病毒RNA能非常有效地抑制其与125I单链小牛胸腺DNA的结合。需要大量得多的双链DNA才能产生同等程度的抑制。因此，该蛋白显示出对单链DNA或病毒RNA的优先结合。
