MINICHROMOSOMES INTRODUCED IN CELL NUCLEI

我们的目标是了解染色质的结构如何参与蛋白质的合成。 基因表达和复制的调控。 几个具体问题 将通过使用组装在DNA上的微型染色体来询问 注入青蛙卵母细胞和卵子。 首先，特殊的“动态”微型染色体的结构将被 研究了 这些微型染色体携带不受约束的DNA超螺旋， 被认为代表转录活性染色质。 动态 微型染色体将从传统的“静态” 微型染色体，它们的结构将通过核酸酶来表征 通过电泳鉴定蛋白质组成， 通过电子显微镜观察。 利用体积小的优势 的5S RNA基因，进一步的努力将致力于分离和分析， 一小部分的动态微型染色体， 转录。 第二，将研究动态微型染色体的复制， 关于它们是否相对于所述细胞被优先复制， 传统的，静态的微型染色体，以及动态结构是否可以 直接繁殖到后代微型染色体。 有效 在5S DNA的预形成复合物上组装动态微型染色体， 转录因子TFIIIA将被用来研究这种可能性。 第三，核小体位置在失活的、静态的携带有 将检查5S RNA或组蛋白H4的基因， 假设基因失活可能是由于阻断了 启动子序列通过核小体核心。 携带32P的微型染色体 在独特限制性位点标记将用于定位核小体位置。

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.