Cowell I G, Willmore E, Chalton D, Marsh K L, Jazrawi E, Fisher L M, Austin C A
The Medical School, University of Newcastle, Newcastle-upon-Tyne, NE2 4HH, United Kingdom.
Exp Cell Res. 1998 Sep 15;243(2):232-40. doi: 10.1006/excr.1998.4150.
We have analyzed the subcellular distribution of the beta isoform of human topoisomerase II using both isoform-specific antisera and an epitope-tagging approach. Previous immunocytochemical studies have yielded differing results with one reporting this isoform to be predominantly nucleolar. Later studies seem to refute this finding, as do our results with isoform-specific antisera reported here. Epitope tagging minimizes potential complications arising from the use of anti-topoisomerase II antisera that may recognize epitopes that are modified or masked in vivo and could lead to misleading results in immunocytochemical studies. A second strength of this approach is that it allowed a comparison with similarly tagged control proteins (derived from the nucleolar transcription factor UBF) that were known to localize unambiguously to the cytoplasmic, nucleoplasmic, or nucleolar compartments. We report that the C-terminal domain of topoisomerase IIbeta fused to a beta-galactosidase tag localizes to the nucleus (but not the nucleolar compartment) and that this is indistinguishable from the localization of native topoisomerase IIbeta detected by isoform-specific antisera. Further analysis revealed that the nuclear localization determinant lies within the 116-residue C-terminal tail of human topoisomerase IIbeta.
我们使用同工型特异性抗血清和表位标记方法分析了人类拓扑异构酶IIβ同工型的亚细胞分布。先前的免疫细胞化学研究得出了不同的结果，其中一项报告称这种同工型主要位于核仁。后来的研究似乎反驳了这一发现，我们在此报告的同工型特异性抗血清的结果也是如此。表位标记最大限度地减少了使用抗拓扑异构酶II抗血清可能产生的潜在并发症，这些抗血清可能识别在体内被修饰或掩盖的表位，并可能在免疫细胞化学研究中导致误导性结果。这种方法的另一个优点是，它可以与已知明确定位于细胞质、核质或核仁区室的类似标记的对照蛋白（源自核仁转录因子UBF）进行比较。我们报告说，与β-半乳糖苷酶标签融合的拓扑异构酶IIβ的C末端结构域定位于细胞核（但不是核仁区室），这与通过同工型特异性抗血清检测到的天然拓扑异构酶IIβ的定位没有区别。进一步分析表明，核定位决定因素位于人类拓扑异构酶IIβ的116个残基的C末端尾巴内。
