THE INTRANUCLEAR ORGANIZATION OF NORMAL, HEMIZYGOTIC AND EXCISION-DEFICIENT RIBOSOMAL-RNA GENES DURING DEVELOPMENTAL AMPLIFICATION IN TETRAHYMENA-THERMOPHILA

In the ciliated protozoan, Tetrahymena thermophila, the diploid germin al micronucleus contains two allelic copies of the gene for ribosomal RNA (rDNA). During genesis of new somatic macronuclei the germline rDN A gene is excised by developmentally programmed chromosome breakage an d preferentially amplified to similar to 9,000 copies. We have studied this process by fluorescence in situ hybridization. We find that init ially rDNA amplification is restricted to two separate and highly conf ined regions of the nucleus. Analysis of nuclei that are hemizygous fo r the rDNA locus reveals that each focus of hybridization is derived f rom a single allele of the rDNA. As rDNA amplification progresses thes e two foci of hybridization disperse and spread throughout the macronu cleus, eventually forming similar to 100-500 new nucleoli. These event s are correlated with morphologically distinct developmental stages. W e investigated the amplification of the C3 allele of the rDNA that con fers a replication advantage over the B allele during vegetative propa gation, and find no evidence for preferential amplification of the C3 early in rDNA maturation. We also show that the rmm 11 rDNA mutant all ele, which is defective for developmentally programmed rDNA excision, can be amplified during the two-foci stage in mutant homozygotes and h eterozygotes, but fails to amplify further and disperse into multiple nucleoli. These data indicate that amplification of the rmm 11 allele is not delayed during the initial rounds of amplification, and suggest that efficient excision is not required for this amplification to occ ur. We propose that rDNA amplification is a two-step process. First, t he two rDNA alleles are independently amplified, while allelic copies remain closely associated. Later, copies of the rDNA disperse and are further amplified, presumably because rDNA excision has occurred, gene rating fully mature rDNA minichromosomes that are able to replicate to high copy number.