The genetic control of nucleolus formation in wheat

The wheat variety Chinese Spring has four pairs of nucleolus organisers of known rDNA content. The genetic control of these has been investigated in root tip cells by cytologically scoring the number of nucleoli per cell in (a) aneuploid derivatives each having a different dosage of a particular chromosome or chromosome arm and (b) in substitution lines where nucleolus organiser chromosomes have been replaced by homologues possessing different amounts of rDNA. It has been assumed that nucleolus organiser activity is correlated with nucleolus size and thus with the presence of a cytologically visible nucleolus. Those nucleolus organisers on chromosomes 1A and 5D, which together possess only 10% of the rDNA form a visible nucleolus only infrequently in the presence of the larger nucleolus organisers on chromosomes 1B and 6B. When a major pair of organisers on chromosomes 1B or 6B is deleted, the smaller nucleolus organisers form a visible nucleolus more frequently. Similarly, when the major nucleolus organisers are replaced by organisers with less rDNA, the smaller nucleolus organisers form visible nucleoli more frequently. When a small nucleolus organiser is replaced by one with much more rDNA, a larger nucleolus is formed. These and other findings lead to the general conclusions that there is a frequently, but not invariably, seen correlation between rRNA gene number and nucleolus size. However the relative size of the nucleolus formed depends principally upon the proportion of the total active rRNA genes in the cell which are localised at the nucleolus organiser in question. Varying the dosage of at least 13 non nucleolus organiser chromosomes also resulted in changes in the number of visible nucleoli per cell. This implies the genetic control of individual nucleolus organisers is complex. Inclusion in the wheat genome of the nucleolus organiser chromosome from Aegilops umbellulata, causes suppression of the wheat nucleolus organisers, the Aegilops umbellulata organiser remaining active. This suppression is similar to that observed in many interspecific plant and animal hybrids.     

Amounts of ribosomal DNA inAllium (Liliaceae)

Eight species ofAllium subgen.Allium sect.Allium have been studied at the cytological level by means of karyological analyses and at the biochemical level with regard to the proportions of ribosomal DNA. All the species have a basic genome of x = 8.A. sativum, A. commutatum, A. ampeloprasum, andA. vineale possess approximately 0.050% rDNA and two nucleolus organizer regions per basic chromosome set.A. sphaerocephalon andA. arvense have two nucleolus organizers, andA. amethystinum three nucleolus organizers per haploid (n = x) genome: the three species possess approximately 0.075% rDNA.A. acutiflorum has five nucleolus organizer regions per haploid genome and 0.121% rDNA. An attempt to relate these differences with functional and ecological characteristics indicates that evolutionary variation of rDNA proportions is not casual. Such data also can help to define systematic affinities and circumscribe infrageneric taxa.     

Ribosomal gene amplification does not occur in the oocytes of Locusta migratoria

It has been suggested that Locusta migratoria amplifies its ribosomal RNA genes in the growing oocytes (Kunz (1967) Chromosoma20, 332–370). Cloned ribosomal DNA of L. migratoria was used to analyze rDNA structure and number. The rDNA is localized on three chromosome pairs in six nucleolus organizers. It was found that all structural variants of the rRNA genes which have been described previously are represented in the same relative amounts in DNA from isolated oocytes as in somatic cells. Furthermore, the rRNA gene number is not increased in oocyte DNA, i.e., amplification does not occur. Therefore, the large number of multiple nucleoli seen in the growing oocytes has to be interpreted as the fully extended and fully active set of chromosomal rRNA genes. The total rRNA gene number was determined by dot blot hybridization to be about 3300 genes/haploid genome.     

Karyotyping of Brassica oleracea L. based on rDNA and Cot-1 DNA fluorescence in situ hybridization

To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.     

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity.     

Genetic mapping of 18s ribosomal RNA-related loci to mouse Chromosomes 5, 6, 9, 12, 17, 18, 19, and X

The organization of ribosomal RNA genes (rDNA) in the genome of the mouse varies significantly from one strain to another, but has been shown to follow the pattern of clusters of tandem repeats located at chromosome ends, often associated with cytological nucleolus organizer regions. The number of copies of the repeat unit at each locus also varies. A probe for the 18S ribosomal RNA sequence on Southern blots reveals both high copy number bands and fainter bands indicative of low repeat number. We have mapped a number of newly identified low-copy-number rDNA loci in C57BL/6J, in addition to placing some of the NOR-associated rDNA repeats on the Jackson interspecific backcross (BSS) map. We suggest that additional low-copy-number loci may remain to be mapped, and that the evolution of rDNA loci in the genome may include the proliferation of single copies by retroinsertion or other mechanisms. Received: 23 February 1996 / Accepted: 29 July 1996     

Supernumerary heterochromatic segments associated with the nucleolar chromosomes of Pyrgomorpha conica (Orthoptera) contain methylated rDNA sequences

The two nucleolus organizing chromosome pairs of the grasshopper Pyrgomorpha conica can carry a proximal supernumerary heterochromatic segment. We employed different cytological techniques to characterize and analyze the possible origin of this segment. The supernumerary segment and the nucleolus organizing regions (NORs) show similar responses after C-banding plus either Giemsa or acridine orange, and chromomycin A₃/distamycin A staining to detect GC-rich chromosome regions. Fluorescence in situ hybridization with a biotinylated rDNA probe demonstrated that the segment originated by amplification of the rDNA genes. However, as the silver staining indicates, the ribosomal genes present in the segment are not active since no nucleolus is formed. The use of in situ digestion with the isoschizomeric MspI and HpaII restriction endonucleases and subsequent Giemsa, ethidium bromide or chromomycin A₃/distamycin A staining, suggests that the segment has been inactivated by DNA methylation.     

The genes for ribosomal RNA in diploid and polytene chromosomes of Drosophila melanogaster

The proportion of the Drosophila genome coding for ribosomal RNA was examined in DNA from both diploid and polytene tissues of Drosophila melanogaster by rRNA-DNA hybridization. Measurements were made on larvae with one, two, three and four nucleolus organizer regions per genome. In DNA from diploid tissues the percent rDNA (coding for 28S and 18S ribosomal DNA) was found to be in proportion to the number of nucleolus organizers present. The number of rRNA genes within a nucleolus organizer therefore does not vary in response to changes in the number of nucleolus organizers. On the other hand, in DNA from cells with polytene chromosomes the percent rDNA remained at a level of about 0.1% (two to six times lower than the diploid values), regardless of either the number of nucleolus organizers per genome or whether the nucleolus organizers were carried by the X or Y chromosomes. This independence of polytene rDNA content from the number of nucleolus organizers is presumably due to the autonomous polytenization of this region of the chromosome. When the rDNA content of DNA from whole flies is examined, both the rDNA additivity of the diploid cells and the rDNA independence of polytene cells will affect the results. This is a possible explanation for the relative rDNA increase known to occur in X0 flies, but probably not for the phenomenon of rDNA magnification. — In further studies on DNA from larval diploid tissues, the following findings were made: 1) the Ybb-chromosome carries no rDNA; 2) flies carrying four nucleolus organizers do not tend to lose rDNA, even after eleven generations, and 3) the nucleolus organizer on the wild type Y chromosome may have significantly less rDNA than does that on the corresponding X chromosome.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

A new role of the rDNA and nucleolus in the nucleus--rDNA instability maintains genome integrity

The nucleolus is a region of the nucleus with high protein density and it acts as a ribosome factory. The nucleolus contains a distinct region of the genome, the ribosomal RNA gene repeats (rDNA) that supply ribosomal RNA (rRNA) molecules. The rDNA is the most-abundant gene and occupies a large part of the genome, for example, there are thousands of rDNA copies in the genomes of plant cells. Therefore, it is natural to suppose that the condition of the rDNA, such as its stability, might affect cellular functions. Here I would like to propose a new model regarding the roles of the rDNA and nucleolus. The key point of this model is that they act to preserve genome stability and trigger aging.     

Nucleolus size varies with sex,ploidy and gene dosage in insects

The nucleolus constitutes a cytologically visible phenotype for ribosomal DNA (rDNA). Nucleolar size, as determined by silver staining, is a good indicator of cell proliferation rate and biosynthetic activity. Nevertheless, the relationship between rDNA content and sexual dimorphism for nucleolar size is not well documented. In the present study, the impact of sex and ploidy level on nucleolar size is investigated in three haplo/diploid and three diplo/diploid species of insect. Nucleolar sizes are found to be proportional to ploidy level in the haplo/diploid hymenopterans Trypoxylon albitarse and Nasonia vitripennis. Conversely, in the ant Messor barbarus, nucleolar sizes are larger in haploid males (winged) than diploid females (apterous). Among the diplo/diploid species, evidence for gene dosage compensation on nucleolar activity is suggested by the absence of sex differences in Drosophila simulans, a species in which rDNA is limited to the X chromosome. By contrast, in the grasshopper Stenobothrus festivus, another species with rRNA genes restricted to the X chromosome, the size of the nucleolus is significantly larger in females than in males. Additionally, in the grasshopper Chorthippus parallelus, where rDNA is distributed evenly on several autosomes of males and females, the females also show larger nucleoli than males. In both grasshopper species, the magnitude of the female/male ratio for nucleolus area is very similar to the body size ratio, suggesting that body size, as well as sex, ploidy, gene dosage and physiological activity, may be an important determinant of nucleolus area.     

Alteration of the Tetrahymena Genome During Nuclear Differentiation*†

Synopsis.
The DNA of the macro- and the micronucleus of Tetrahymena thermophila has been compared by various biochemical methods. It became evident from their thermal denaturation temperatures and buoyant densities that the 2 DNAs were very similar in overall composition. Small differences were detected when the sequence complexities of these DNAs were compared by DNA renaturation studies. The studies suggested that ˜ 10% of the micronuclear genome was lost or underrepresented in the macronucleus. Comparison of individual gene levels revealed further differences. By using the technic of gene cloning a micronuclear sequence was isolated which hybridized only with micronuclear, but not with macronuclear DNA. These results indicated the occurrence of elimination or underreplication of this sequence in the macronucleus. Gene amplification was also shown to occur. In the micronucleus only a single copy of rDNA was found integrated into the chromosome. During macro-nuclear development, amplification was observed to occur, and the amount of rDNA to increase, until there were ˜ 200 copies per haploid genome in the mature macronucleus. all of them extrachromosomal and palindromic. The 3rd case of alteration involved a simple repeated sequence, (CCCCAA)n, present in the termini of rDNA and also in many other locations of the genome. Restriction endonuclease digestion studies revealed drastic differences in the organization of the repeats between macro-and micronucleus. These differences may be interpreted as the results of chromosome fragmentation which occurs at every cluster of the repeats during macronuclear development. The relationship between this event and gene amplification and elimination is discussed.     

The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs

The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is required to maintain the integrity of the rDNA repeats. Sgs1p is a member of the RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1, and the human BLM and WRN genes. These genes encode proteins which are essential to maintenance of genomic integrity and which share a highly conserved helicase domain. Here we show that recombinant Sgs1p helicase efficiently unwinds guanine-guanine (G-G) paired DNA. Unwinding of G-G paired DNA is ATP- and Mg2+-dependent and requires a short 3' single-stranded tail. Strikingly, Sgs1p unwinds G-G paired substrates more efficiently than duplex DNAs, as measured either in direct assays or by competition experiments. Sgs1p efficiently unwinds G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to prevent telomere-telomere interactions which can lead to chromosome non-disjunction. The rDNA is G-rich and has considerable potential for G-G pairing. Diminished ability to unwind G-G paired regions may also explain the deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated aging characteristic of yeast strains that lack Sgs1 as well as humans deficient in the related WRN helicase.