Genetic Analysis of Silenced Chromatin in Neurospora

真核基因组不仅仅是基因的总和。基因的染色体背景是重要的，某些染色体部分，包括着丝粒，端粒和rDNA区域，在细胞中起结构和编码作用。一些证据表明，着丝粒，端粒和rDNA区域的染色质结构比大多数染色体区域更密集，这种“异染色质”染色质导致基因沉默。女性失活的X染色体是异染色质导致基因沉默的一个例子。着丝粒、端粒和rDNA提供了很好的模型来识别染色质的替代形式之间的关键差异。迄今为止，已研究异染色质区域的生物体（主要是酵母和果蝇）之间正在出现相似之处，但也发现了显着差异。目前的项目将扩大我们的理解沉默的特点异染色质序列的优势，利用一个模式的真核生物，丝状真菌粗糙脉孢菌。这项研究建立在这种生物体发育良好的遗传学和最近获得的几乎完整的基因组序列的基础上。有效的遗传学方法将被用来确定细胞中导致异染色质形成和正常功能的重要参与者。通过在携带沉默基因nst-1突变的菌株中进行同源重组，将报告基因置于脉孢菌染色体的端粒、着丝粒和rDNA区域。所得菌株将用作转化宿主以测试参与异染色质沉默的候选基因。该项目的这一部分利用了显性的转录后沉默过程，quelling，以有效地筛选大量的基因。最初将筛选通过与其他生物体的基因同源性鉴定的约30个候选基因。此外，新的沉默基因将寻求在一个非冗余的测序cDNA克隆集。显示参与沉默的证据的基因将使用RIP（重复诱导的点突变）选择性地破坏。沉默的报告基因也将用于选择通过插入诱变产生的新的沉默突变体。突变的DNA将被分离和测序，以确定沉默基因。最后，着丝粒，端粒和rDNA染色质将在野生型脉孢菌和沉默突变体的特点。将使用遗传和物理方法来鉴定沉默突变体的缺陷的性质。从这个项目中获得的知识应该既提高了我们对基因沉默的具体机制的理解，又阐明了各种真核生物中正常和异常染色体行为的机制。除了其科学价值外，该项目还将用于培训学生，并促进脉孢菌作为功能基因组学实用模型系统的使用。

Eukaryotic genomes are more than just the sum of their genes. The chromosomal context of genes is important and certain chromosomal parts, including the centromeres, telomeres and rDNA regions, serve structural as well as coding roles in the cell. Several lines of evidence indicate that the chromatin structure of centromeric, telomeric and rDNA regions is more condensed than most chromosomal regions and that this "heterochromatic" chromatin causes gene silencing. The inactivated X chromosome in women represents an example of gene silencing by heterochromatin. Centromeric, telomeric, and rDNA provide excellent models to identify critical differences between alternative forms of chromatin. Parallels are emerging among organisms whose heterochromatic regions have been studied to date (principally yeasts and Drosophila) but significant differences have also been detected. The current project will broaden our understanding of the silencing that characterizes heterochromatic sequences by taking advantage of a model eukaryote, the filamentous fungus Neurospora crassa. The study builds on the well developed genetics of this organism and the recent availability of the nearly complete sequence of its genome. Efficient genetic approaches will be used to identify the important players in the cell that result in the formation, and normal function of, heterochromatin. Reporter genes will be placed in telomeric, centromeric and rDNA regions of Neurospora chromosomes by homologous recombination in a strain bearing a mutation in a silencer gene, nst-1. The resulting strains will be used as transformation hosts to test candidate genes for involvement in heterochromatin silencing. This part of the project takes advantage of a dominant post-transcriptional silencing process, quelling, to efficiently screen a large number of genes. Approximately 30 candidate genes identified by homology to genes of other organisms will be screened initially. In addition, novel silencer genes will be sought in a non-redundant set of sequenced cDNA clones. Genes showing evidence of involvement in silencing will be selectively disrupted using RIP (repeat-induced point mutation). Silenced reporter genes will also be used to select new silencing mutants generated by insertional mutagenesis. The mutated DNA will be isolated and sequenced to identify silencer genes. Finally, centromeric, telomeric and rDNA chromatin will be characterized in wildtype Neurospora and in silencing mutants. Both genetic and physical methods will be used to identify the nature of the defect(s) of the silencing mutants. Knowledge gained from this project should both improve our understanding of specific mechanisms of gene silencing and shed light on mechanisms responsible for normal and abnormal chromosome behavior in a variety of eukaryotes. In addition to its scientific merit, this project will serve to train students and to advance the use of Neurospora as a practical model system for functional genomics.