EUKARYOTIC CHROMOSOME REPLICATION

复制是DNA作为分子的生物学作用的核心。 继承。DNA复制的控制确保了染色体 以及时和准确的方式复制。所有可用的证据都表明 该控件在复制开始时在 个体起源以高密度散布在真核细胞的染色体上。 我们打算研究的是复制起始点的控制。 我们对这些控制的理解很差，主要是因为 复制来源本身一直难以捉摸。最近，两名敏感的 凝胶电泳法已经被开发出来用于鉴定 染色体DNA。我们对酿酒酵母的研究有 揭示了起源的启动活动在很大程度上取决于 在他们的染色体背景下。语境控制的三个方面 本课程将考察酵母的原产地使用情况。(I)原点位于 紧密的串联阵列--在质粒多聚体和rDNA基因座上--许多 不使用潜在的原产地。不活动的原因是什么？ 是否存在间距限制？那么，活跃的起源是如何选择的呢？ (Ii)在转录活性rDNA重复序列中的复制启动 发生在非转录间隔区，复制是单向的 移动转录方向的活动叉子。这是不是 其他染色体区域的转录活性会影响起源用途吗？ 进化是否有利于允许复制的起始点的放置 分叉通过活跃转录的基因跟随转录复合体， 而不是与他们正面相撞？(Iii)确定染色体来源 近端粒(染色体的物理末端)在S后期被激活 相较于位于内部站点的原点。端粒的什么特征 影响起始点激活时间，并且某些起始点处于非活动状态，因为 它们与端粒的接近程度？我们解决这些问题的方法包括 在酵母质粒中进行定向替换和改变 并使用我们最近开发的二维凝胶技术来鉴定 并估计它们的激活效率。这个 这些问题的答案将使我们更好地理解 正常细胞和慢性粒细胞白血病患者染色体复制的调节 改变生长特性，如癌细胞。

Replication is central to the biological role of DNA as the molecule of inheritance. The control of DNA replication ensures that chromosomes are duplicated in a timely and precise way. All available evidence indicates that controls operate at the point of initiation of replication at individual origins scattered at high density over eukaryotic chromosomes. It is the control of replication origins that we propose to investigate. Our understanding of these controls is very poor, primarily because replication origins themselves have been elusive. Recently, two sensitive gel electrophesis techniques have been developed for identifying origins in chromosomal DNA. Our studies in the yeast Saccharomyces cerevisiae have revealed that the initiation activity of origins depends to a large extent on their chromosomal context. Three aspects of contextual control of origin use in yeast will be examined. (I) When origins are located in close tandem arrays--in plasmid multimers and in the rDNA locus--many potential origins are not used. What is responsible for the inactivity? Are there spacing constraints? And, how are the active origins chosen? (II) Replication initiation in the transcriptionally active rDNA repeats occurs in the non-transcribed spacer and replication is unidirectional with the active fork moving the transcriptional direction. Does the transcriptional activity of other chromosomal regions influence origin use? Has evolution favored the placement of origins that permits replication forks to follow transcription complexes through actively transcribed genes, rather than colliding head-on with them? (III) Chromosomal origins located near telomeres (the physical ends of chromosomes) are activated later in S phase than origins located at internal sites. What feature of telomeres influences origin activation times, and are some origins inactive because of their proximity to telomeres? Our approach to these questions involves making directed replacements and alterations in yeast plasmids and chromosomes and using our recently developed 2-D gel technique to identify active origins and to estimate the efficiency of their activation. The answers to these questions will lead to a greater understanding of the regulation of chromosome replication in normal cells and in those with altered growth properties, such as cancer cells.