期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1974
卷号：71
期号：3
页码：984-988
DOI：10.1073/pnas.71.3.984
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：{phi}X174 RF (replicative form) II DNA, labeled in vivo with [methyl-3H]thymidine, was isolated from Escherichia coli polA (DNA polymerase I-deficient) and polA+ cells during RF replication. [32P]dCMP was incorporated into the gaps present in the RF II DNA with [[α]-32P]dCTP and T4 DNA polymerase. Sedimentation in alkaline sucrose gradients revealed that much of the incorporated 32P was present in a heterogeneous collection of fragments shorter than unit length. Inclusion of polynucleotide ligase in the gap-filling reaction increased the average size of the 32P-labeled fragments. Gel electrophoresis of the products formed by digestion of the 32P-labeled RF II molecules with the restriction nuclease, endonuclease R, indicated that in the population of RF II molecules gaps could occur anywhere in the genome. Competition-annealing experiments provided evidence that the majority of the label incorporated into gaps was present in the minus strand. RF II molecules isolated from polA+ cells were enriched for gaps in a unique region of the genome in comparison with RF II molecules isolated from polA cells. The presence of multiple gaps in the minus strand implies that it is synthesized by a discontinuous mechanism during {phi}X RF replication.
关键词：DNA polymerase ; restriction nuclease ; competition hybridization ; gap-filling ; pol A
