Tsai-Pflugfelder M, Liu L F, Liu A A, Tewey K M, Whang-Peng J, Knutsen T, Huebner K, Croce C M, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Proc Natl Acad Sci U S A. 1988 Oct;85(19):7177-81. doi: 10.1073/pnas.85.19.7177.
Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.
通过两种独立方法鉴定出了两个编码人类DNA拓扑异构酶II的重叠cDNA克隆。一种方法是，用一种混合寡核苷酸探针与噬菌体λ中的人类cDNA文库进行杂交筛选，该探针编码一段在酵母和果蝇DNA拓扑异构酶II中发现的七个氨基酸序列；另一种方法是，在λgt11表达载体中的另一个人类cDNA文库中筛选能被人类DNA拓扑异构酶II特异性兔抗体识别的抗原决定簇的表达。从这些克隆以及通过使用克隆的人类TOP2基因序列作为探针鉴定出的几个其他克隆中确定了人类DNA拓扑异构酶II基因的完整编码序列。克隆序列与mRNA和基因组DNA之间的杂交表明，人类酶由单拷贝基因编码。通过将克隆片段与中期染色体进行原位杂交以及与一组保留人类染色体子集的小鼠-人类杂交细胞系进行杂交分析，将该基因定位到了17号染色体的q21-22区域。
