NEUROSPORA RIBOSOMAL DNA: ORGANIZATION AND REGULATION

该提案涉及研究组织的项目和 真核生物核糖体DNA(RDNA)的功能性表达 粗枝脉孢菌就是一个例子。第一个也是主要的项目是研究 猪瘟病毒复制株rRNA重复单位数的调控 含有过量或缺失rRNA重复单位的新孢子菌 与野生型相比。在无性繁殖时， 重复单位减少(缩小)或增加(放大)，如 适当，直到接近通配类型数为止。去放大化 可能是由于复制的破裂或重组事件造成的 例如同源染色单体之间不等交换、不等交换 姐妹染色单体之间，或染色质内缺失。放大倍率 可能来自特定rDNA序列的选择性复制，或者来自 不等姐妹染色单体互换。基因分析将被用于 确定重复单位调节的机制，主要通过使用 将单拷贝基因整合到rDNA中的转化细胞 作为一种遗传标记。各种实验，使用 重组缺陷突变体，异核体，抑制因子 大分子过程和核糖体生物合成突变体计划 研究生物体如何感知异常数量的重复单位。 关于rDNA甲基化是否在正常的菌株中改变的问题， 增加或减少重复单元的数量也将得到解决。 第二个项目涉及到通过限制映射和 RDNA重复单元内感兴趣区域的DNA测序。这些 区域包括已证实的区域中的非转录间隔区 限制酶切位点多态，可能的甲基化敏感区，以及 RDNA和非核糖体DNA之间可能的连接。第三个项目 利用菌株间rDNA限制性内切酶切点多态性进行实验 旨在研究rRNA基因遗传分离的机制。 在减数分裂和有丝分裂期间。拟议中的实验应该有助于 提供大量新信息，特别是在管制复制方面 多基因家族中的数目及其进化和意义 重复阵列中的表达和间隔区序列。此外，还有 这些研究与目前对 肿瘤发生的分子过程，包括基因研究 扩增和DNA甲基化。

This proposal involves projects for studying the organization and functional expression of the ribosomal DNA (rDNA) of eukaryotes, as exemplified by Neurospora crassa. The first and main project is a study of the regulation of rRNA repeat unit number in duplication strains of Neuorspora that contain either an excess or deficit of rRNA repeat units compared with the wild type. Upon vegetative propagation, the number of repeat units is decreased (demagnified) or increased (magnified), as appropriate, until the wildtype number is approximated. Demagnification could result from breakdown of the duplication or from recombination events such as unequal exchange between homologous chromatids, unequal exchange between sister chromatids, or intrachromatid deletions. Magnification could result from selective replication of specific rDNA sequences, or from unequal sister chromatid exchange. Genetic analysis will be used to determine the mechanism of repeat unit regulation, principally by using transformed cells in which a single copy gene is integrated into the rDNA as a genetic marker. A variety of experiments, using recombination-deficient mutants, heterokaryons, inhibitors of macromolecular processes, and ribosome biosynthesis mutants are planned to investigate how the organism senses an abnormal number of repeat units. The question of whether rDNA methylation is altered in strains with normal, increased, or decreased numbers of repeat units will also be addressed. The second project involves a detailed analysis by restriction mapping and DNA sequencing of regions of interest within the rDNA repeat unit. These regions include nontranscribed spacer in the area of a demonstrated restriction site polymorphism, possible methylation-sensitive regions, and a possible junction between rDNA and non-ribosomal DNA. The third project exploits the interstrain rDNA restriction site polymorphism in experiments designed to study the mechanism of genetic segregation of the rRNA genes during meiosis and mitosis. The proposed experiments should contribute substantial new information, particularly in regard to regulation of copy number in multi-gene families and the evolution and significance of expressed and spacer sequences in repeated arrays. In addition, there are possible correlations between these studies and current investigations into the molecular processes of oncogenesis, including studies of gene amplification and DNA methylation.