Johnston B H, Ohara W, Rich A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1988 Apr 5;263(10):4512-5.
We have analyzed, at nucleotide resolution, the progress of the B-to-Z transition as a function of superhelical density in a 2.2-kilobase plasmid containing the sequence d(C-A)31.d(T-G)31. The transition was monitored by means of reactivity to two chemical probes: diethyl pyrocarbonate, which is sensitive to the presence of Z-DNA, and hydroxylamine, which detects B-Z junctions. At a threshold negative superhelical density between about 0.048 and 0.056, hyper-reactivity to diethyl pyrocarbonate appears throughout the CA/TG repeat and remains as the superhelical density is further increased. However, there is no reactivity characteristic of B-Z junctions until the superhelical density reaches 0.084, when single cytosines at each end of the repeat become hyper-reactive to hydroxylamine. A two-dimensional gel analysis of this system by others (Haniford, D. B., and Pulleyblank, D. E. (1983) Nature 302, 632-634) indicates that only about half of the 62 base pairs of the CA/TG repeat undergo the initial transition at omega = 0.056. Our results indicate that this region of Z-DNA is free to exist anywhere along the CA/TG repeat and is probably constantly in motion. Well defined B-Z junctions are seen only when there is sufficient supercoiling to convert the entire CA/TG sequence to Z-DNA. The implications for possible B-Z transitions in chromosomal domains of different sizes are discussed.
我们在核苷酸分辨率水平上，分析了在一个含有序列d(C-A)31.d(T-G)31的2.2千碱基质粒中，B型到Z型转变过程随超螺旋密度的变化情况。通过对两种化学探针的反应性来监测这种转变：焦碳酸二乙酯，它对Z-DNA的存在敏感；以及羟胺，它能检测B-Z交界处。在大约0.048至0.056之间的阈值负超螺旋密度下，整个CA/TG重复序列对焦碳酸二乙酯出现超反应性，并随着超螺旋密度进一步增加而持续存在。然而，直到超螺旋密度达到0.084时，重复序列两端的单个胞嘧啶才对羟胺变得超反应，在此之前没有B-Z交界处的反应特征。其他人对该系统进行的二维凝胶分析（哈尼福德，D. B.，和普利布兰克，D. E.（1983年）《自然》302，632 - 634）表明，在ω = 0.056时，CA/TG重复序列的62个碱基对中只有约一半经历了初始转变。我们的结果表明，这个Z-DNA区域可以在CA/TG重复序列的任何位置自由存在，并且可能一直在运动。只有当有足够的超螺旋将整个CA/TG序列转化为Z-DNA时，才能看到明确的B-Z交界处。本文讨论了不同大小染色体结构域中可能的B-Z转变的意义。
