Genetic Analysis of Silenced Chromatin in Neurospora

真核生物的基因组不仅仅是它们基因的总和。基因的染色体环境很重要，某些染色体部分，包括着丝粒、端粒和rDNA区域，在细胞中起着结构和编码的作用。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，这种异染色质导致基因沉默。女性失活的X染色体代表了异染色质基因沉默的一个例子。着丝粒、端粒和rDNA提供了极好的模型来识别不同形式的染色质之间的关键差异。到目前为止，已经研究了异染色区的生物(主要是酵母和果蝇)之间出现了相似之处，但也检测到了显着的差异。目前的项目将扩大我们对沉默的理解，通过利用一种模式真核生物，丝状真菌粗面脉孢子菌来表征异染色质序列。这项研究建立在这种生物发达的遗传学和最近获得的几乎完整的基因组序列的基础上。有效的遗传方法将被用来识别细胞中导致异染色质形成和正常功能的重要角色。报告基因将通过同源重组放置在脉孢子虫染色体的端粒、着丝粒和rDNA区域，在携带沉默基因nst-1突变的菌株中。得到的菌株将被用作转化宿主，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一种占主导地位的转录后沉默过程--抑制，以有效地筛选大量基因。大约30个与其他生物基因同源的候选基因将被初步筛选出来。此外，还将在一组无冗余的测序的cdna克隆中寻找新的沉默基因。显示参与沉默的证据的基因将使用RIP(重复诱导点突变)选择性地干扰。沉默的报告基因也将被用于选择插入突变产生的新的沉默突变体。突变的DNA将被分离和测序，以识别沉默基因。最后，着丝粒、端粒和rDNA染色质将在野生型脉孢子虫和沉默突变体中得到表征。将使用遗传和物理方法来鉴定沉默突变体缺陷(S)的性质。从这个项目中获得的知识应该会提高我们对基因沉默的具体机制的理解，并有助于阐明导致各种真核生物的正常和异常染色体行为的机制。除了它的科学价值，这个项目还将用于培训学生，并促进脉孢子菌作为实用的功能基因组学模型系统的使用。

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.