MINICHROMOSOMES INTRODUCED IN CELL NUCLEI

我们的目标是了解染色质的结构是如何参与 基因表达和复制的调控。几个具体问题 将通过使用在DNA上组装的微型染色体来询问 注射在青蛙卵母细胞和卵子中。 首先，特殊的动态微染色粒的结构将是 学习。这些微小的染色体携带着不受限制的DNA超螺旋，并且 被认为代表转录能力强的染色质。动态 微染色体将从常规的“静态”中分离出来 微染色体，它们的结构将通过核酸酶来表征 消化，通过蛋白质组成的电泳鉴定，以及 通过电子显微镜观察。利用小号的优势 在5S RNA基因中，将进一步努力分离和分析一种 实际参与的动态微染色体的一部分 抄写。 其次，动态微染色体的复制将用 关于它们是否相对于 常规的、静态的微染色体，以及动态结构是否可以 直接繁殖到后代微染色体。高效的 动态微染色体在5S DNA和5S DNA预制复合体上的组装 转录因子TFIIIA将被用来研究这种可能性。 第三，不活跃的、静止的微染色体携带的核小体位置 将检测5S RNA或组蛋白H4的基因，以检测 假设基因失活可能是由于阻断了 启动子序列由核小体核心组成。携带32P的微小染色体 唯一限制位点的标记将用于绘制核小体位置图。

Our goal is to understand how the structure of chromatin is involved in the regulation of gene expression and replication. Several specific questions will be asked by employing minichromosomes that are assembled on DNA injected in frog oocytes and eggs. Firstly, the structure of the peculiar "dynamic" minichromosomes will be studied. These minichromosomes carry unconstrained DNA supercoils, and are thought to represent transcriptionally competent chromatin. Dynamic minichromosomes will be separated from the conventional "static" minichromosomes, and their structure will be characterized by nuclease digestions, by electrophoretic identification of protein composition, and by electron microscopic observations. Taking advantage of the small size of the 5S RNA gene, further efforts will directed to isolate and analyze a fraction of dynamic minichromosomes that are actually engaged in transcription. Secondly, replication of dynamic minichromosomes will be studied with respect to whether they are preferentially replicated relative to the conventional, static minichromosomes, and whether the dynamic structure can be directly propagated to the progeny minichromosomes. The efficient assembly of dynamic minichromosomes on the preformed complex of 5S DNA and transcription factor TFIIIA will be exploited to examine such a possibility. Thirdly, nucleosome positions in inactive, static minichromosomes carrying a gene for either 5S RNA or histone H4 will be examined in order to test the hypothesis that the gene inactivation could be a result of blockage of promoter sequences by a nucleosome core. Minichromosomes carrying 32P label at a unique restriction site will be used to map nucleosome positions.