Sekiguchi J M, Kmiec E B
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Mol Gen Genet. 1989 Dec;220(1):73-80. doi: 10.1007/BF00260859.
Using an in vitro chromatin assembly system, we analyzed the influence of DNA superhelicity on the development of transcriptionally active minichromosomes. Plasmid DNA molecules containing either a Xenopus borealis 5S RNA gene or an X. laevis methionine tRNA gene were utilized as templates for the assembly of chromatin. Both plasmids were processed into active minichromosomes if introduced as supercoiled molecules into the extract (S-150). The degree of superhelicity is a determining factor in the assembly of active chromatin. Molecules containing varying superhelical densities were processed into minichromosomes with different transcriptional activities. The absence of supercoils leads to the assembly of chromatin with substantially lower transcriptional activity. Assembled minichromosomes are stable enough to be isolated by sucrose gradient centrifugation while retaining their transcriptional phenotype. The formation of nucleosomes with a periodic spacing occurred with the same efficiency and to the same degree regardless of the initial DNA topology. Hence, a determining factor in the development of transcriptionally active chromatin may be the initial superhelicity of the DNA molecule to which activator (trans-acting factors) or repressor (histones) proteins bind. Once the chromatin assembly process has begun, the transcriptional activity of the resulting minichromosome may already have been determined.
利用体外染色质组装系统，我们分析了DNA超螺旋对转录活性微型染色体形成的影响。含有北极爪蟾5S RNA基因或非洲爪蟾甲硫氨酸tRNA基因的质粒DNA分子被用作染色质组装的模板。如果将这两种质粒作为超螺旋分子引入提取物（S-150）中，它们都会被加工成活性微型染色体。超螺旋程度是活性染色质组装的一个决定性因素。含有不同超螺旋密度的分子被加工成具有不同转录活性的微型染色体。没有超螺旋会导致组装出转录活性显著较低的染色质。组装好的微型染色体足够稳定，可以通过蔗糖梯度离心分离出来，同时保留其转录表型。无论初始DNA拓扑结构如何，具有周期性间距的核小体的形成效率和程度都是相同的。因此，转录活性染色质形成的一个决定性因素可能是激活剂（反式作用因子）或阻遏物（组蛋白）蛋白所结合的DNA分子的初始超螺旋。一旦染色质组装过程开始，所产生的微型染色体的转录活性可能就已经被确定了。
