Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase

Mitotic cell division involves the equal segregation of all chromosomes during anaphase. The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome. Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells. Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions. We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other. Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction. These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII.     

Bacterial DNA segregation: its motors and positional control

A model for DNA segregation in bacteria is proposed which involves not merely growth of the cell membrane and wall, as previously assumed, but also the active movement of one of the two chromosome sister origins by a DNA helicase enzyme and of the chromosome termini and the bulk of the chromosomes by supercoiling tension exerted by DNA gyrase. This provides a unified mechanism for DNA chromosome movement in prosthecate budding bacteria as well as for bacteria that undergo binary fission. The positional control of DNA segregation and the plane of cell division depend, I suggest, on four things: (1) the attachment of the daughter chromosome termini to the cell wall in a position adjacent to the new cell poles at about the time of septation, (2) the displacement of the parental chromosome terminus from this attachment site by the mobile origin, which attaches itself instead to the wall at that point, (3) the movement of the chromosome terminus to a new location in between the daughter origins by the tension of supercoiling, and (4) the determination of the location of the future septum at the position occupied by the chromosome terminus at the time of septal initiation; septum-initiation proteins are postulated to achieve this by binding directly or indirectly to the chromosome terminus. This mechanism automatically ensures ordered DNA segregation in rapidly growing bacteria with more than two sister origins of replication.     

Circular ribosomal DNA and ribosomal DNA: replication in somatic amphibian cells

Pulse labelled rDNA from cultured somatic cells of Xenopus laevis was examined by electron microscope autoradiography. The pattern of replication closely resembles that of bulk chromosomal DNA and differs considerably from rDNA synthesis during amplification in the oocyte. - About 0.15% of the rDNA molecules in the purified preparations were circular. The presence of interlocked circles of equal size indicates that the circles are not in vitro cyclization artefacts, but may represent free rRNA genes. A low frequency of circles was also seen in Xenopus blood rDNA. Their stability in high concentration of formamide suggests that they too did not arise after DNA extraction.     

Neurodegeneration-associated instability of ribosomal DNA

Homologous recombination (HR)-mediated instability of the repetitively organized ribosomal DNA (rDNA) has been proposed as a mediator of cell senescence in yeast triggering the DNA damage response. High individual variability in the content of human rDNA suggests that this genomic region remained relatively unstable throughout evolution. Therefore, quantitative real-time polymerase chain reaction was used to determine the genomic content of rDNA in post mortem samples of parietal cortex from 14 young and 9 elderly individuals with no diagnosis of a chronic neurodegenerative/neurological disease. In addition, rDNA content in that brain region was compared between 10 age-matched control individuals and 10 patients with dementia with Lewy bodies (DLB) which involves neurodegeneration of the cerebral cortex. Probing rRNA-coding regions of rDNA revealed no effects of aging on the rDNA content. Elevated rDNA content was observed in DLB. Conversely, in the DLB pathology-free cerebellum, lower genomic content of rDNA was present in the DLB group. In the parietal cortex, such a DLB-associated instability of rDNA was not accompanied by any major changes of cytosine–phosphate–guanine methylation of the rDNA promoter. As increased cerebro-cortical rDNA content was previously reported in Alzheimer's disease, neurodegeneration appears to be associated with instability of rDNA. The hypothetical origins and consequences of this phenomenon are discussed including possibilities that the DNA damage-induced recombination destabilizes rDNA and that differential content of rDNA affects heterochromatin formation, gene expression and/or DNA damage response. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.     

Heterogeneity of pumpkin ribosomal DNA

The ribosomal DNA (rDNA) of Cucurbita pepo L. has been found to consist of tandemly arrayed repeat units, most of which are 10 kilobases in length. Thirty-six repeat units, cloned into the HindIII site of pACYC 177, fall into seven classes which differ from each other in length and/or nucleotide sequence. Most of the heterogeneity occurs in noncoding portions of the repeat unit although there is some nucleotide sequence variation in the coding portion as well. Heterogeneity of base modification was observed in genomic rDNA of which two examples are: (a) all of the repeat units have three BamHI sites, one of which is unavailable for restriction in about half of the units and (b) all of the CCGG sites except one are methylated at the internal cytidine in many of the units; a second site is unmethylated in some of the units and in a very few units a third site remains unmethylated.     

The ribosomal DNA of Calliphora erythrocephala; an analysis of hybrid plasmids containing ribosomal DNA

We have analysed the ribosomal DNA of Calliphora erythrocephala, a Dipteran fly of the same sub-order as Drosophila melanogaster, through a series of rDNA² fragments cloned in a plasmid vector. We have mapped the sites for eight restriction enzymes within these plasmids, and positioned the regions coding for the 18 S and 28 S rRNAs within the maps of selected plasmids using the S₁ endonuclease mapping procedure of Berk & Sharp (1977). This analysis establishes that some rDNA cistrons of C. erythrocephala contain an “intron” (Gilbert, 1978) which interrupts the 28 S coding region at the same position as that of D. melanogaster rDNA. Two introns of 2.85 kilobases in length and part of a longer, sequence-related variant were isolated in these cloned fragments. Restriction enzyme site analysis and preliminary hybridization data indicate that the 2.85 kb intron of C. erythrocephala is largely unrelated in sequence to the two classes of D. melanogaster rDNA introns.     

DNA segregation by bacterial actin homologs

The bacterial actin homolog ParM catalyzes segregation of plasmid DNA in E. coli. Recent studies now suggest a model in which ParM forms actin-like filaments between two plasmid molecules, thereby providing the driving force for plasmid DNA separation.     

Two distinct types of mitochondrial DNA segregation in mouse-rat hybrid cells. Stochastic segregation and chromosome-dependent segregation

Two distinct patterns of mitochondrial DNA (mtDNA) segregation were found in different mouse-rat hybrid cell lines. On mouse-rat hybrid cell line, H2, retained complete sets of chromosomes and mtDNAs of both mouse and rat. Even after cultivation for about one year after cloning, the H2 cell population still retained both parental mtDNAs. However, when mtDNAs of H2 subclones were examined, it was found that some individual cells in the H2 cell population contained only mouse or only rat mtDNA, although they still retained complete sets of both kinds of parental chromosomes. This type of mtDNA segregation, named stochastic segregation, is bidirectional and may be caused by the repetition of random sharing of mouse and rat mtDNAs with daughter cells. This segregation occurred spontaneously during long-term cultivation. The second type of mtDNA segregation, named chromosome-dependent segregation, was found in the other mouse-rat hybrid cell lines that segregated either mouse or rat chromosomes. In these hybrid cells, chromosomes and mtDNA of the same species co-segregated. This second type of segregation is unidirectional. The types of mtDNA segregation appear to depend on the stability of the parental chromosomes in the hybrid cells. When both mouse and rat chromosomes retain stably, mtDNA shows stochastic segregation. On the contrary, when either species of chromosomes is segregated from the cells, mtDNA shows chromosome-dependent segregation.     

Heterogeneity in cucumber ribosomal DNA

Summary Restriction and hybridization analysis of cucumber native ribosomal (r) DNA purified from actinomycin-D/CsCl gradients suggested that the repeat units were heterogeneous in both length and sequence. Several full length rDNA repeat units were cloned and five are described which account for all the EcoR I and Xba I fragments present in native DNA. One of a number of BamH I sites found in the clones is not found in a proportion of native rDNA because of base modification. Restriction maps are described for the representative clones and aligned with R-loop maps obtained from electron microscope analysis of each type of repeat unit hybridized under R-loop conditions to pure 18S and 25S rRNAs. The major heterogeneity is explained by differences in length of the external spacer region and by a proportion of the repeat units showing a restriction fragment length polymorphism on EcoR I digestion. The regions coding for 18S and 25S rRNA are uninterrupted and highly conserved.     

Replicon size of yeast ribosomal DNA

Summary The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9Kbp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 m long, on chromosome XII. Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined. We have used immobilised rRNA as a probe to examine the size of radioactively labelled rDNA replicons resolved on alkaline sucrose gradients. The replicons were found to be uniformly sized, about 5 repeat units in length, and groups of 4 adjacent replicons may be activated simultaneously. These observations suggest that replicon initiation events are not determined solely by the recognition of specific DNA sequences that function as origins of replication.     

The ribosomal DNA plasmids of entamoeba

In most organisms, the nuclear ribosomal RNA (rRNA) genes are highly repetitive and arranged as tandem repeats on one or more chromosomes. In Entamoeba, however, these genes are located almost exclusively on extrachromosomal circular DNA molecules with no clear evidence so far of a chromosomal copy. Such an uncommon location of rRNA genes may be a direct consequence of cellular physiology, as suggested by studies with Saccharomyces cerevisiae mutants in which the rDNA is extrachromosomal. In this review, Sudha Bhattacharya, Indrani Som and Alok Bhattacharya summarize current knowledge on the structural organization and replication of the Entamoeba rDNA plasmids. Other than the rRNAs encoded by these molecules, no protein-coding genes (including ribosomal protein genes) are found on any of them. They are unique among plasmids in that they do not initiate replication from a fixed origin but use multiple sites dispersed throughout the molecule. Further studies should establish the unique biochemical features of Entamoeba that lead to extrachromosomal rDNA.     

The evolution of eukaryotic ribosomal DNA

Mutations occur randomly throughout the ribosomal DNA (rDNA) sequence. Molecular drive (unequal crossing-over, gene conversion, and transposition) spreads these variations through the multiple copies of rDNA. Forces of selection act upon the variants to favor and fix them or disfavor and eliminate them. Selection has not permitted changes in regions within rRNA vital for its function; these sequences are evolutionarily conserved between diverse species. Possible functions for some of these conserved sequences are discussed. The secondary structure of rRNA is also highly conserved during evolution. However, eukaryotic rRNA is larger than prokaryotic rRNA due to blocks of "expansion segments". Arguments are put forward that expansion segments might not play any functional role. Other examples are reviewed of rDNA sequence insertion or deletion, including introns and the internal transcribed spacer 2.     

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA (M_r = 2.7 × 10⁶) is much smaller than HeLa or L-cell 45 8 rRNA (M_r = 4.7 × 10⁶), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA.Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments.Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.     

Prokaryotic DNA segregation by an actin-like filament

The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments with properties expected for a force-generating protein. Filament formation depended on the other components encoded by par, ParR and the centromere-like parC region to which ParR binds. Mutants defective in ParM ATPase exhibited hyperfilamentation and did not support plasmid partitioning. ParM polymerization was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.     

Gimap3 regulates tissue-specific mitochondrial DNA segregation

Mitochondrial DNA (mtDNA) sequence variants segregate in mutation and tissue-specific manners, but the mechanisms remain unknown. The segregation pattern of pathogenic mtDNA mutations is a major determinant of the onset and severity of disease. Using a heteroplasmic mouse model, we demonstrate that Gimap3, an outer mitochondrial membrane GTPase, is a critical regulator of this process in leukocytes. Gimap3 is important for T cell development and survival, suggesting that leukocyte survival may be a key factor in the genetic regulation of mtDNA sequence variants and in modulating human mitochondrial diseases.     

Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

Heterogeneity in Chinese hamster ribosomal DNA

A discrete heterogeneity has been detected in Chinese hamster ribosomal DNA after Eco R1 digestion of total DNA followed by a Southern transfer and hybridization with [¹²⁵I]18S or [¹²⁵I]28S ribosomal RNA. Digestion with Eco R1 produces three fragments, 4.3, 6.0 and 9.5×10⁶ daltons respectively, which hybridize with 18S RNA. The smallest fragment also hybridizes with 28S RNA. Either length heterogeneity or sequence heterogeneity (i.e. presence of an additional Eco R1 site in some of the rDNA molecules) must be invoked to account for the two larger Eco R1 fragments that contain 18S but not 28S sequences. Eco R1 and Hind III maps, consistent with either length or sequence heterogeneity, are presented. The data at this time, however, do not distinguish between the two alternatives.     

The segregation of DNA in epithelial stem cells

It has recently been suggested that stem cells may invariably keep, from one division to the next, the daughter DNA molecules that contain the older of the two parental strands—that is, they may retain a complete set of “immortal strands,” through successive cell divisions (Cairns, 1975). We can test this hypothesis by labeling either the old immortal strands at the time the stem cells are created or the newly synthesized strands during subsequent divisions of the stem cells. In the former case, the stem cells should become permanently labeled; in the latter case, they should eliminate their label on their second division.Experiments of this sort have been conducted with the tongue papilla under steady state conditions and with the regenerating small intestinal crypts. The results clearly show that by far most of the multiplying cells in tongue and intestinal epithelium segregate their DNA “randomly” at mitosis. Nevertheless, the results, though far from conclusive, suggest that there are a small number of cells (1–5 in the stem cell region of each crypt and one at the base of each column of cells in the tongue) that selectively segregate their old and new DNA strands in the expected way. Thus in the immortal strand labeling experiments, there are a few labeled cells that retain their label for up to 4 weeks; conversely, in the new strand labeling experiments, a few cells appear to rid themselves of label after intervals equivalent to approximately two cell cycles.