DIFFERENTIAL COMPARTMENTALIZATION OF PLASMID DNA MICROINJECTED INTO XENOPUS-LAEVIS EMBRYOS RELATES TO REPLICATION EFFICIENCY
DIFFERENTIAL COMPARTMENTALIZATION OF PLASMID DNA MICROINJECTED INTO XENOPUS-LAEVIS EMBRYOS RELATES TO REPLICATION EFFICIENCY
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DOI：
10.1128/mcb.11.1.299
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发表时间：
1991-01-01
影响因子：
5.3
通讯作者：
BENBOW, RM
中科院分区：
文献类型：
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作者：
MARINI, NJ;BENBOW, RM
显微注射非洲爪蟾胚胎后，由相同质粒形成的环状质粒DNA分子和线性多联体表现出明显不同的命运。在这份报告中，我们定量证明，只有少数的小，环状DNA分子复制（平均= 14%），从受精到囊胚发育阶段。在所有检测浓度下，极少数分子（约1%）在这些多个细胞周期内经历一轮以上的DNA合成。此外，与内源性染色质不同，大多数环状模板对微球菌核酸酶的切割具有抗性。核酸酶抗性的程度与复制和未复制的模板相似。环状分子被隔离在膜隔室（核）中，而不是形成具有异常大小或间距的核小体，显然赋予了核抗性。相比之下，大多数线性连接的DNA分子（来自显微注射的单体质粒DNA的端到端连接）在同一时期经历了至少两轮DNA复制。线性多联体也表现出微球菌核酸酶消化模式相似的内源性染色质，但判断其未能坚持在胚胎发生的后期阶段，很可能被复制和维持extrachromosomally。因此，我们建议，模板的大小和构象决定复制的效率，通过指导DNA注射后细胞内的一个特定的隔室显微注射的质粒DNA。模板依赖性区室化可以由内源性细胞核内相对于细胞核区室的差异定位或由依赖于模板构型的超分子组装过程（例如，与核基质或核被膜结合）。
Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibits strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1 %) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar to both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nucleus resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogeneous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).