Visualizing Genetic Activity in Normal and Mutant Yeast

拟议研究的长期目标是更好地理解核糖体高度调控生产的分子机制，核糖体比任何其他细胞事件都与细胞的生长状态直接相关。在快速生长的酵母细胞（本文使用的模型系统）中，60%的总转录用于RNA聚合酶I合成核糖体RNA，核糖体的合成速度为每分钟2000个。因此，有必要协调和严格调节这些基因，它们使用了细胞资源的很大一部分。尽管我们对参与Pol I转录的分子已经了解了很多，但在我们的理解上仍有很大的漏洞。这在很大程度上是由于Pol I系统的不同寻常的特性，在该系统中，一个多基因家族是由一个专门用于该目的的聚合酶转录的，因此通过调节活性基因的数量或调节这些基因的活性水平来调节是可能的。尽管使用遗传、分子和生化技术很难区分这两个水平的调节，但通过电子显微镜（EM）方法可以获得这些信息。因此，提出的研究的直接目标是通过应用Miller染色质扩散技术直接可视化活性rDNA基因和非活性核仁染色质，为酵母rRNA转录研究增加现有的可用工具库。这种方法很简单，结合了酵母遗传学的力量和图片的力量。EM方法将用于（目标1）表征Pol I转录上调和下调的正常模式，例如通过典型的生长曲线和营养状况的改善，（目标2）通过可视化转录来确定Pol I转录因子和Pol I增强子在正常转录模式中的作用，因为它们被耗尽或突变。和（Aim 3）确定染色质结构和rDNA沉默对Pol I转录的贡献，方法是定位rDNA染色质中的组蛋白修饰，并在没有已知具有核仁作用的组蛋白去乙酰化酶的情况下观察rRNA转录。该方法有望回答这个重要的多基因家族中基因调控的基本问题，这些问题无法通过生化和遗传方法解决，但可以通过直接可视化轻松明确地解决。考虑到rRNA合成与细胞核仁大小和生长速度之间的直接正相关关系，并且这三者在癌细胞中都被上调，rRNA合成的上调似乎可能是肿瘤发生的一个促进因素。此外，最近发现的核仁在衰老中的作用可能会被提出的实验进一步阐明。

The long term goal of the proposed research is to better understand molecular mechanisms in the highly regulated production of ribosomes, which more than any other cellular event is directly correlated with the growth status of the cell. In a rapidly growing yeast cell (Saccharomyces cerevisiae), which is the model system used herein, 60 percent of total transcription is devoted to the synthesis of ribosomal RNA by RNA polymerase I and ribosomes are made at a rate of 2000 per minute. Thus, it is necessary to coordinately and tightly regulate these genes, which use a significant fraction of the cell's resources. Although a great deal is known about the molecules that participate in Pol I transcription, there are still large holes in our understanding. Much of this is due to the unusual properties of the Pol I system, in which a multi-gene family is transcribed by a polymerase that is solely devoted to that purpose, such that regulation is possible either by adjusting the number of active genes or by adjusting the activity level of those genes. Although it is difficult to distinguish between these two levels of regulation using genetic, molecular and biochemical techniques, the information is accessible to an electron microscopic (EM) approach. Thus, the immediate goal of the proposed research is to add to the current repertoire of available tools for the study of yeast rRNA transcription by applying the Miller chromatin spreading technique for the direct visualization of active rDNA genes and inactive nucleolar chromatin. The approach is straightforward and combines the power of yeast genetics with the power of a picture. The EM approach will be used to (Aim 1) characterize normal patterns of up- and down-regulation of Pol I transcription, such as across a typical growth curve and as nutritional status is improved, (Aim 2) determine the role of Pol I transcription factors and the Pol I enhancer to normal patterns of transcription by visualizing transcription as they are being depleted or when mutated, and (Aim 3) determine the contribution of chromatin structure and rDNA silencing to Pol I transcription by localizing histone modifications in rDNA chromatin and by visualizing rRNA transcription in the absence of histone deacetylases known to have a nucleolar role. The approach holds promise to answer fundamental questions regarding gene regulation in this important multi-gene family that have eluded biochemical and genetic approaches but can easily and unambiguously be addressed by direct visualization. Given the direct positive correlation between rRNA synthesis, nucleolar size and growth rate of a cell, with all three being up-regulated in cancer cells, it seems likely that up-regulation of rRNA synthesis is a contributing factor to tumorigenesis. Furthermore, the recently uncovered role of the nucleolus in aging may be elucidated further by the proposed experiments.