Nucleolus and chromosome relationships at pachynema in four Scarabaeoidea (Coleoptera) species with various combinations of NOR and sex chromosomes

Nucleolus organizer regions (NORs) and nucleolus locations were studied after silver staining in spermatocytes at pachynema from four beetle species selected for their various combinations of sex chromosomes. Their karyotypic formulae were: 18,neoXY (Dorcus parallelipipedus); 25,X (Passalus unicornis) and 20,Xyp (Cetonia aurata and Protaecia (Potosia) opaca). NORs were located in the short arms of a unique acrocentric autosome pair in the first three and in intercalary position in a sub-metacentric autosome pair in the last species. Silver staining gave remarkably more consistent results in pachytene than in mitotic spreads, enabling the detection of both NORs and nucleoli, and also better results in embryo than in spermatogonial metaphases. At pachynema the NORs were elongated, roughly in proportion to the number of nucleoli, which always remained associated with NOR. Nucleoli were not recurrently associated with sex chromosomes, except in P. unicornis, at late pachynema. In C. aurata and P. opaca the sex body was recurrently associated with acrocentric short arms and metacentric telomeres, respectively. Even in these simple situations, with NORs located in a single autosome pair, the number of nucleoli and their relationships with sex chromosomes varied strongly from species to species. These variations appear to be largely determined by the chromosome rearrangements which have occurred during evolution, which makes extrapolations and generalizations quite hazardous. In D. parallelipipedus pachytene cells a quasi-systematic and transient fusion between the terminal heterochromatin of two sub-metacentrics was detected. Other chromosome bivalents could also be occasionally associated, but not the NOR carrier one. A strong enhancement of DAPI or quinacrine mustard staining was observed at the fusion point.     

Unusually high amount of inactive ribosomal DNA in the grasshopper Stauroderus scalaris

Fluorescence in-situ hybridization (FISH) was employed to determine the chromosomal location of the ribosomal DNA cistrons in spermatocytes of two populations of the grasshopper Stauroderus scalaris. The results showed that paracentromeric C-bands, which in this species constitute about 50% of the total chromatin, contain substantial amounts of rDNA in all chromosomes. However, silver impregnation showed the presence of a single active nucleolus organizing region (NOR) in chromosome 3 of primary spermatocytes, indicating an extremely high amount of silent rDNA across the whole genome of this species in the two geographically distant populations analysed. The significance of such an unusual phenomenon is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The very long telomeres in <Emphasis Type="Italic">Sorex granarius</Emphasis> (Soricidae,Eulipothyphla) contain ribosomal DNA

Two closely related shrew species, Sorex granarius and Sorex araneus, in which Robertsonian rearrangements have played a primary role in karyotype evolution, present very distinct telomere length patterns. S. granarius displays hyperlong telomeres specifically associated with the short arms of acrocentrics, whereas telomere lengths in S. araneus are rather short and homogenous. Using a combined approach of chromosome and fibre FISH, modified Q-FISH, 3D-FISH, Ag-NOR staining and TRF analysis, we carried out a comparative analysis of telomeric repeats and rDNA distribution on chromosome ends of Sorex granarius. Our results show that rDNA sequences forming active nuclear organizing regions are interspersed with the long telomere tracts of all short arms of acrocentrics. These observations suggest that the major rearrangements that gave rise to today’s karyotype in S. granarius were accompanied by a profound reorganization of chromosome ends, which comprised extensive amplification of telomeric and rDNA repeats on the short arms of acrocentrics and finally contributed to the stabilization of telomeres. This is the first time that such telomeric structures have been observed in any mammalian species.     

Chromosomal Location and Nucleotide Sequences of 5S Ribosomal DNA of Two Cyprinid Species (Osteichthyes, Pisces)

5S ribosomal DNAs (rDNAs) from two cyprinid species, Acheilognathus tabira subsp. 1 and Cyprinus carpio, were isolated and sequenced. Tandemly arranged rDNAs were 179 bp in A. tabira and 204 bp in C. carpio. The non-transcribed spacer region elucidates the size difference of 5S rDNA between the two species. Fluorescence in-situ hybridization (FISH) localized 5S rDNAs to the short arms of two pairs of chromosomes in A. tabira and two to four pairs in C. carpio. Subsequent analysis demonstrated NORs in one pair of chromosomes in both species. Both the NOR and 5S rDNA are carried by a chromosome pair in A. tabira, but they are located on different chromosomes separately in C. carpio. Karyotype evolution by tetraploidy seems complex in cyprinid species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spontaneous in vitro neoplastic evolution of cultured Chinese hamster cells. Nucleolus organizing region activity

Silver staining to demonstrate active nucleolus organizing regions (NORs) was performed at four different stages of the spontaneous tumorigenic progression in vitro of Chinese hamster WCHE/5 cells. The number of active NORs increased for fully transformed, highly tumorigenic, late passage cells. The increase of NOR material was due to additional NOR-bearing chromosomes or chromosome arms, i.e., trisomy 5, trisomy 8, and the marker chromosome i(3q). Intermediate stages of the neoplastic evolution showed changing patterns of NOR activity, but not an overall increase. We postulate that the increase of active rDNA enhances cell growth and provides undefined selective advantage, and that this supports our previous conclusion that selectable karyotype changes provide competitive advantages rather than being essential for neoplastic evolution in vitro.     

Ribosomal DNA and the nucleolus in the context of genome organization

The nucleolus constitutes a prominent nuclear compartment, a membraneless organelle that was first documented in the 1830s. The fact that specific chromosomal regions were present in the nucleolus was recognized by Barbara McClintock in the 1930s, and these regions were termed nucleolar organizing regions, or NORs. The primary function of ribosomal DNA (rDNA) is to produce RNA components of ribosomes. Yet, ribosomal DNA also plays a pivotal role in nuclear organization by assembling the nucleolus. This review is focused on the rDNA and associated proteins in the context of genome organization. Recent advances in understanding chromatin organization suggest that chromosomes are organized into topological domains by a DNA loop extrusion process. We discuss the perspective that rDNA may also be organized in topological domains constrained by structural maintenance of chromosome protein complexes such as cohesin and condensin. Moreover, biophysical studies indicate that the nucleolar compartment may be formed by active processes as well as phase separation, a perspective that lends further insight into nucleolar organization. The application of the latest perspectives and technologies to this organelle help further elucidate its role in nuclear structure and function.

     

Mapping of rRNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe

Summary The major rRNA genes of the fission yeast Schizosaccharomyces pombe were mapped on chromosome III by plasmid integration. The integration vector YIp33 containing S. cerevisiae LEU2 gene was combined with the S. pombe rDNA. Since LEU2 complements S. pombe leu1 deficiency, it could be used as the genetic marker for integration. The 10.4 kb rDNA repeat contained ARS sequence, and therefore 2.4 kb and 0.7 kb subfragments not containing ARS were subcloned into YIp33 and transformed leu1 S. pombe cells to Leu⁺. Genetic analyses of the transformants indicated that the integrated rDNA resides in the long arm of the shortest chromosome III, tightly linked to ade5 (1.4 cM). This result is consistent with our previous finding that the DAPI-stained smallest chromosomes were associated with the nucleolus (Umesono et al. 1983).Abbreviations ARS autonomously replicating sequence - DAPI 4,6-diamidino-2-phenylindole - kb kilo base pairs - rDNA DNA segment containing ribosomal RNA genes - rRNA ribosomal RNA     

Breakpoint within the nucleolus organizer region resulting in a reciprocal translocation t(4;14)(q21;p12)

Reciprocal translocations involving a break in the nucleolus organizer region (NOR) are rare. A balanced translocation in a mother and her fetus with breakpoints in the NOR at 14p12 and on the long arm of a chromosome 4 at band 4q21 is described. The rearrangement was characterized by Ag‐NOR staining, multiplex fluorescence in situ hybridization (M‐FISH), and FISH with rDNA probes. This and other cases with breakpoints within NORs are discussed. Am. J. Med. Genet. 92:264–268, 2000. © 2000 Wiley‐Liss, Inc.     

Reproductive outcome in 3 families with a satellited chromosome 4 with review of the literature

We describe 3 families segregating for a translocation of the nucleolus organizer region (NOR) onto chromosome 4. Review of previously reported cases of translocations involving NOR and chromosome 4 shows that these translocations may be associated with variable reproductive outcomes. We provide evidence that imprinting is not the mechanism responsible for the variable reproductive outcomes in the case of satellited 4p chromosomes; this may offer indirect support for a ribosomal gene position effect. Translocated ribosomal genes may influence the expression of neighboring genes and could explain the variable phenotypes in individuals with satellited nonacrocen-tric chromosomes. We recommend that prenatal counseling of individuals with satellited nonacrocentric chromosomes should be cautious. © 1995 Wiley-Liss, Inc.     

Post-zygotic modifications and intra- and inter-individual nucleolar organizing region variations in fish: report of a case involvingLeporinus friderici

Silver nitrate staining, a rapid and efficient method, has proven to be excellent for nucleolar organizing region (NOR) studies in fish. Some fish appear to have only two NOR-bearing chromosomes in their karyo-type, whereas others probably have several. In the present study we analyzed the NORs ofLeporinus friderici, a species that, on the basis of previous studies, has been considered as representative of species with NORs carried by a single chromosome pair. The analyses were performed by a combination of three methods,i.e. silver nitrate staining, staining with the GC-specific fluorochrome chromomycin A₃, andin situ hybridization with digoxigenin-labeled probes. The results showed that, although more frequent and conspicuous in a single chromosome pair, the NORs of this species are present in multiple chromosomes. Intra- and inter-individual variations observed by the three methods strongly suggest the occurrence of post-zygotic modifications involving NORs. NOR identification in fish, almost exclusively performed by the silver nitrate method, is currently being re-evaluated by methods such as chromomycin A₃ staining andin situ hybridization, which may provide important information leading to a better understanding of chromosome evolution in these animals.     

Triploid origin of the gibel carp as revealed by 5S rDNA localization and chromosome painting

5S ribosomal DNA (rDNA) was isolated and sequenced from the gibel carp Carassius auratus gibelio with 162 chromosomes and crucian carp Carassius auratus with 100 chromosomes, and fluorescent probes for chromosome localization were prepared to ascertain the ploidy origin and evolutionary relationship between the two species. Using fluorescence in-situ hybridization (FISH), major 5S rDNA signals were localized to the short arms of three subtelocentric chromosomes in the gibel carp and to the short arms of two subtelocentrics in the crucian carp. In addition, some minor signals were detected on other chromosomes of both species. Simultaneously, six chromosomes were microdissected from the gibel carp metaphase spreads using glass needles, and the isolated chromosomes were amplified in vitro by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Significantly, when the DOP-PCR-generated probes prepared from each single chromosome were hybridized, three same-sized chromosomes were painted in each gibel carp metaphase, whereas only two painted chromosomes were observed in each crucian carp metaphase spread. The data indicate that gibel carp is of triploid origin in comparison with diploid crucian carp.     

Tracking chromosome dynamics in live yeast cells: coordinated movement of rDNA homologs and anaphase disassembly of the nucleolus during meiosis

A prerequisite for determination of chromosome dynamics in live cells is development of a method for staining or marking the chromosome of interest. We describe here a unique chromosome-tracking system that differentially marks two large chromosome segments from homologs in the budding yeast Saccharomyces cerevisiae. Using yeast genetics and the special features at the repetitive ribosomal RNA (rRNA) gene cluster, we incorporated arrays of the tet operator and the lac operator into each repeat of the two rDNA homologs by homologous recombination. Expression of tet repressor-fused green fluorescent protein and lac repressor-fused red fluorescent protein in engineered cells led to the differential labeling of rDNA homologs. Using live-cell three-dimensional fluorescence microscopy, we showed that homologs undergo contraction and expansion cycles in an actin-dependent manner during meiosis and that chromosome mobility appears to be correlated with nuclear positioning. Our observations further revealed that, in contrast to mitosis, in meiosis the yeast nucleolus, the site of rRNA processing, was disassembled upon anaphase onset, suggesting a differential regulation of the rDNA array during meiotic chromosome segregation. Because rRNA genes are highly conserved, a similar chromosome-engineering approach may be adaptable in other eukaryotes for functional assays of chromosome organization in live cells.     

Reprogramming of rye rDNA in triticale during microsporogenesis

To test the hypothesis that interspecific genomic and chromosome interactions leading to nucleolar dominance could be reprogrammed in meiosis, we compared the expression of distinct nucleolar organizing region (NOR) loci in hexaploid triticale root tip meristematic cells, pollen mother cells and young pollen grains. Interphase and metaphase cells were silver stained to quantify nucleoli and active NOR loci respectively. A marked difference in the ribosomal RNA gene activity of each locus was observed when different types of cells were compared: in somatic and pollen mother cells, rRNA gene activity was mainly restricted to major wheat NORs (1B and 6B) with only a small contribution from rye NORs (1R). In contrast, in young pollen grains, all NORs present, including the 1R NORs, were consistently active. The expression of all NORs just after meiosis is considered to be a consequence of meiotic reprogramming of rye origin rDNA. Gene reprogramming mediated by the resetting of methylation patterns established early in embryogenesis is suggested to be responsible for the differential expression of the NORs of rye origin in distinct developmental stages of triticale.     

Linkage group-chromosome correlations in Sordaria macrospora: Chromosome identification by three dimensional reconstruction of their synaptonemal complex

Summary Reconstruction of serially sectioned zygotene and pachytene nuclei has allowed, by measuring the lengths of synaptonemal complexes, an assignment of the 7 linkage (LG) groups to the 7 chromosomes in the fungus Sordaria macrospora. The 7 LG have been established using 19 mutants showing low second division segregation frequencies. Eight chromosomal rearrangements mapped on the 7 LG were used to identify the chromosomes involved. The following one to one assignment of the 7 LG to specific chromosomes was obtained: LG a: chromosome (chr) 1, LG b: chr5, LG c: chr6, LG d: chr7, LG e: chr4, LG f: chr3 and LG g: chr2 (the chromosome carrying the nucleolus organizer region).Abbreviations LG linkage group - SC synaptonemal complex - SDS second division segregation - NOR nucleolus organizer region     

Integration of cytogenetic and genetic linkage maps of <Emphasis Type="Italic">Lotus japonicus</Emphasis>, a model plant for legumes

To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.     

Genetic load caused by variation in the amount of rDNA in a wasp

Extensive variation in the size of the short (heterochromatic) arm of chromosome 14 was found in the wasp Trypoxylon (Trypargilum) albitarse. Ten different variants were differentiated by size and C-banding pattern. Fluorescent in-situ hybridization (FISH) revealed that ribosomal DNA in this species is clustered in the darkly C-banded parts of the heterochromatic short arm of chromosome 14. On this basis, we got an indirect estimate of the amount of rDNA from the area of these dark C-bands. The significant absence in males of the three chromosome variants with lower amounts of rDNA indicates that these three variants are lethal in this sex, and suggests the existence of a threshold marking the minimum amount of rDNA which is tolerable in haploidy. This implies about 4% genetic load in the population caused by variation in rDNA amount. This revised version was published online in July 2006 with corrections to the Cover Date.