NEUROSPORA RIBOSOMAL DNA: ORGANIZATION AND REGULATION

这项建议涉及研究该组织的项目， 真核生物的核糖体DNA（rDNA）的功能性表达，如 例如粗糙脉孢菌（Neurosporacrassa）。 第一个也是主要的项目是研究 重复菌株中rRNA重复单元数目调控 含有过量或缺乏rRNA重复单位的新孢子菌 与野生型相比。 在营养繁殖时， 重复单位减少（缩小）或增加（放大），如 适当，直到野生型数量接近。 缩小率 可能是由复制的破坏或重组事件引起的 例如同源染色单体之间不均等交换，不均等交换 姐妹染色单体之间，或者染色单体内缺失。 放大率 可能是由于特定rDNA序列的选择性复制，或 姐妹染色单体交换不等。 基因分析将用于 确定重复单元调节的机制，主要通过使用 单拷贝基因整合到rDNA中的转化细胞 作为遗传标记。 各种各样的实验， 重组缺陷突变体，异核体， 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， 还将讨论重复单元数目的增加或减少。 第二个项目涉及限制性酶切图谱的详细分析， rDNA重复单元内感兴趣区域的DNA测序。 这些 区域包括非转录间隔区， 限制性位点多态性，可能的甲基化敏感区域，和 rDNA和非核糖体DNA之间可能的连接。 第三个项目 在实验中利用菌株间rDNA限制性位点多态性， 旨在研究rRNA基因的遗传分离机制， 在减数分裂和有丝分裂期间。 拟议的实验应有助于 大量新资料，特别是关于复制品管制的资料 多基因家族中的数目及其进化和意义 重复阵列中的表达序列和间隔序列。 此外还有 这些研究与目前对以下问题的调查之间可能存在的相关性： 肿瘤发生的分子过程，包括基因的研究 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.