Replication and meiotic transmission of yeast ribosomal RNA genes.

Abstract

The yeast Saccharomyces cerevisiae has ap proximately 120 genes for the ribosomal RNAs (rDNA) which are organized in tandem within chromosomal DNA. These multiple-copy genes are homogeneous in sequence but can undergo changes in copy number and topology. To (ii) Electron microscopy of meiotic cells indicates that a portion of the parental nucleolus is excluded from the four haploid spores which independently generate new nucleoli (9, 10). A possible extrapolation from this observation is that rDNA units are discarded during meiosis and are replaced at a later time. (iii) Strains monosomic for chromosome I have fewer rDNA units than diploids (11,12), and amplification of the rDNA back to the normal level occurs during vegetative growth (13-15). (iv) Circular extrachromosomal copies of rDNA, which are one or a few units in length, are found at low frequencies in vegetative yeast cells (16,17). Extrachromosomal circular rDNA molecules are found in many other systems, including Xenopus oocytes, where amplification of rDNA occurs by a rolling circle form of replication (18). The circular rDNA molecules found in yeast may reflect a mode of replication that is responsible for the normal duplication of rDNA or for the amplification of rDNA units after meiosis. These observations imply a flexibility of number and topology for yeast rDNA, which may reflect important aspects of its replication and inheritance.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6739We have examined some features of rDNA metabolism in S. cerevisiae that are relevant to the replication and inheritance of the individual rDNA units. We find that all rDNA units are transmitted through meiosis to the haploid generation and that each unit serves as a template for one replication event in each S phase of the mitotic cell cycle.EXPERIMENTAL PROCEDURES Density Transfer Experiments. The strain used was a diploid uracil auxotroph, A364A D5 U-A+ (called D5). The 13C glucose/(15NH4)2S04 medium and the cell transfer and cell lysis procedures have been described (19). CsCl equilibrium centrifugation was carried out in a Spinco VTi65 rotor. Lysates (1.0 ml, about 3 X 108 cells) were added to 1.23 g of CsCl, and enough CsCl solution (1.23 g of CsCl per 1 ml of 10 mM Tris-HCI/100 mM EDTA, pH 8) was added to fill 5.3-ml VTi65 heat-seal tubes. Gradients were formed by centrifugation at 30,000 rpm for 60 hr and then collected by bottom puncture into microtiter plates with fractions of 0.11 ml. The distribution of total DNA in the gradients was determined by sampling each fraction for alkali-stable, acid-precipitable 3H cpm.Quantitative Spot Hybridization. The density distribution in CsCl gradients of the repeated rRNA genes was determined by hybridizing 32P-labeled, nick-translated cloned DNA fragments to samples of each gradient fraction that had been spotted on nitrocellulose sheets. A portion...

Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication.

“…rDNA repeats have been reported to replicate early in S phase in CHO cells (1,13), while they have been reported to replicate late in S phase in kangaroo rat cells (18). Other workers claim that rDNA is replicated throughout the S phase in murine erythroleukemia cells (15), in HeLa cells (2), and in yeast cells (6).…”

“…Of these, the center or "origin" of 26 mapped in or near the nontranscribed spacer. 6. Schematic representation of replication bubbles mapped to transcribing rDNA repeats represented by hatched regions.…”

“…The yeast S. cerevisiae is an organism which is particularly suited for such a study because it contains only 1.5 x 104 kb of DNA (29), equivalent to about four times the DNA of E. coli. This yeast has a short cell cycle of 1.5 to 2 h at maximum growth rates (6), and cells can easily be synchronized. This should maximize the number of replication structures seen and allow the observation of patterns of replicon opening throughout S phase.…”

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

“…The published data indicate that a diploid genome can be replicated faster than a haploid genome (2,13,14,16). The experiments, however, were carried out under different growth conditions and in strains with different genetic backgrounds.…”

To fire or not to fire: origin activation in Saccharomyces cerevisiae ribosomal DNA

“…Despite their apparent sequence identity, only about 20% of the ARSs in the rDNA are active in any given S phase (Saffer and Miller 1986;Brewer and Fangman 1988;Linskens and Huberman 1988). Activation of origins in only a subset of rDNA repeats helps explain why rDNA replication occurs throughout the entire S phase (Brewer et al 1980). Although replication is initially bidirectional, when the leftward-moving fork encounters the RFB, it stops.…”