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.
