Molecular analysis of a NOR site polymorphism in brown trout (Salmo trutta): organization of rDNA intergenic spacers.

Using restriction endonuclease mapping, we have analyzed the organization of rDNA (DNA coding for ribosomal RNA (rRNA)) units in the salmonid fish Salmo trutta, as an initial step toward understand the molecular basis of a nucleolar organizer region (NOR) site polymorphism detected in this species. The size of the rDNA units ranged between 15 and 23 kb, with remarkable variation both within individuals and between populations. Three regions of internal tandem repetitiveness responsible for this length polymorphism were located to the intergenic spacers. NOR site polymorphic individuals showed a higher number of length classes, in some cases forming a complete 1 kb fragment ladder. The amount of rRNA genes was as much as 8-fold higher in polymorphic individuals compared with standard individuals. All individuals from the most polymorphic population showed a 14-kb insertion of unknown nature in a small proportion (below 25%) of the 28S rRNA genes.     

Two-dimensional RFLP analyses reveal megabase-sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution

Eukaryotic genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNA (rRNA genes or rDNA) are virtually identical within a species, yet they evolve rapidly between species, a phenomenon known as concerted evolution. The mechanisms by which sequence homogenization and fixation of new rRNA gene variants occurs within a genome are not clear. In diploid Arabidopsis thaliana , approximately 1500 rRNA genes are tandemly arrayed at two nucleolus organizer regions, one on chromosome 2 ( NOR2 ), the other on chromosome 4 ( NOR4 ). This paper shows that NOR2 and NOR4 are similar in size, each spanning approximately 3.5–4.0 Mbp. Using two-dimensional mapping techniques involving a combination of pulsed-field and conventional gel electrophoresis, the distributions of four distinct rRNA gene variants at NOR2 and NOR4 have been determined. rRNA genes at NOR4 are homogeneous with respect to a Hin dIII site occurring once per gene. In contrast, fewer than 10% of the rRNA genes at NOR2 are Hin dIII-bearing variants. A single intergenic spacer length is found among rRNA genes at NOR2 but three classes of spacer length variants are present at NOR4 . The NOR4 variants are not intermingled with one another; instead, they are highly clustered over distances as large as 1.5 Mbp. These data suggest that in the concerted evolution of rRNA genes, homogenization is a consequence of local spreading of new rRNA gene variants.     

Molecular characterization of ribosomal gene variation within and among NORs segregating in specialized populations of chicken.

The molecular organization of the 18S, 5.8S, and 28S ribosomal RNA gene repeat units, located at the single nucleolus organizer region (NOR) locus in the chicken, was investigated in genetically distinct populations of research and commercial chickens. Substantial gene repeat variation within and among NORs was documented. Intact ribosomal gene repeat size ranged from 11 kb to over 50 kb. Unique combinations of ribosomal genes, of different size, were specific to particular populations. It was determined that the basis for the ribosomal gene repeat size variation was intergenic spacer (IGS) length heterogeneity. Interestingly, in different populations, the location of the variation that contributes to length heterogeneity was specific to particular IGS subregions. In addition to IGS variation, an inbred line of Red Jungle Fowl exhibited coding region variation. Ribosomal gene copy number variation was also studied, and line averages ranged from 279 to 368. Average rDNA array size (a function of copy number and gene repeat length) was calculated for each of the populations and found to vary over a range of two megabases, from 5 to 7 Mb.     

Structure and variation of the Nucleolus Organizer Region in turtles

Differential staining techniques were used to study the structure and variation of the NORs of 27 species of cryptodiran turtles. No species or individuals had more than a single pair of NORs. Extensive variation in NOR structure and chromosomal location was found among higher taxa and individual variation in NOR size was common. Thirty eight percent of all individuals studied were heterozygous for the size of the NOR. However, interspecific variation in chromosomal location and structure of the NOR within major taxa was relatively rare. It is concluded that ( I ) the pattern of variation of NORs is consistent with patterns of chromosomal evolution in turtles; (2) Turtles have only a single pair of NORs whereas other animals, such as some mammals, possess numerous NORs; (3) The heterochromatin associated with the NOR is involved in the structure of the nucleolus.     

Expression of ribosomal genes under the action of 5-azacytidine in the African green monkey RAMT cell line

The use of cytosine analogue--5-Azacytidine(5AzaC), derepression of ribosomal genes has been studied in one of organising chromosomes in the African green monkey RAMT cell line in which the nucleolar organizer region (NOR) in parental cells was active. The effect of 5AzaC on the functional state of NOR was assessed by the length of the secondary constriction in this chromosome and by the intensity of Ag-staining of NOR. 5AzaC was added to the cell culture at concentrations 2-16 M, either immediately after the cell passage or at the 24th h from the beginning of cell cultivation for the following 17-34 hours. As a control the cells cultivated in the absence of 5AzaC were used. Comparison of control cells with those treated with 5AzaC showed: 1) increase of the length of the second constriction in the chromosome with the initial inactive NOR in the 5AzaC--treated cells; 2) a marked increase of the intensity of NOR's Ag-staining in the same chromosome after incorporation of 5-AzaC into DNA. The conclusion about the methylation of cytosine bases in the DNA of ribosomal genes in one NOR organising chromosomes in RAMT cell line was made.     

Molecular and cytological characterization of ribosomal RNA genes in Chenopodium quinoa and Chenopodium berlandieri.

The nucleolus organizer region (NOR) and 5S ribosomal RNA (rRNA) genes are valuable as chromosome landmarks and in evolutionary studies. The NOR intergenic spacers (IGS) and 5S rRNA nontranscribed spacers (NTS) were PCR-amplified and sequenced from 5 cultivars of the Andean grain crop quinoa (Chenopodium quinoa Willd., 2n = 4x = 36) and a related wild ancestor (C. berlandieri Moq. subsp. zschackei (Murr) A. Zobel, 2n = 4x = 36). Length heterogeneity observed in the IGS resulted from copy number difference in subrepeat elements, small re arrangements, and species-specific indels, though the general sequence composition of the 2 species was highly similar. Fifteen of the 41 sequence polymorphisms identified among the C. quinoa lines were synapomorphic and clearly differentiated the highland and lowland ecotypes. Analysis of the NTS sequences revealed 2 basic NTS sequence classes that likely originated from the 2 allopolyploid subgenomes of C. quinoa. Fluorescence in situ hybridization (FISH) analysis showed that C. quinoa possesses an interstitial and a terminal pair of 5S rRNA loci and only 1 pair of NOR, suggesting a reduction in the number of rRNA loci during the evolution of this species. C. berlandieri exhibited variation in both NOR and 5S rRNA loci without changes in ploidy.     

Cytogenetic characterisation of the Caspian Pond Turtle,Mauremys caspica in Golestan and Mazandaran provinces,Iran (Reptilia: Testudines)

Cytogenetic characteristics of the Caspian Pond Turtle, Mauremys caspica, in Golestan and Mazandaran provinces in the northern part of Iran show that the chromosome number is 2n?=?52 and the arm number NF?=?78. The karyotype consisted of 9 metacentric (M), 1 submetacentric (SM), 3 subtelocentric (ST) and 13 telocentric (T) chromosome pairs. The Centromeric Index ranges from 11.79 to 45.68, the arm ratio between 1.18 and 7.47, the relative length between 1.60 and 11.46, and the length variation between 1.05 and 7.48. Average total length of the chromosomes is 65.27 µm. The largest chromosome in this species is a pair of the metacentric chromosome. Location of NOR was determined on chromosome pair no. 10.     

Cytogenetic characterization of Rhamdia quelen (Siluriformes, Heptapteridae) from the Bodoquena Plateau, Mato Grosso do Sul, Brazil

We made a cytogenetic study of the fish Rhamdia quelen collected from the Bodoquena Plateau, an isolated national park region in Mato Grosso do Sul State, Brazil. The diploid number was 2n = 58, with 36 metacentric + 16 submetacentric + 6 subtelocentric chromosomes. We found one to three B chromosomes, which were metacentric and submetacentric and of medium size, showing both intra- and interindividual variation. The nucleolus organizer region (NOR) was located in the terminal region of the short arm of submetacentric pair 20. Staining with CMA3 fluorochrome revealed the NOR location, while there was no evidence of fluorescent staining with DAPI. C banding revealed heterochromatin mainly in the terminal regions of the chromosome arms, including the NOR pair. In addition, metacentric pair 2 showed three heterochromatic blocks in the terminal portions and in the pericentromeric region. The B chromosomes appeared euchromatic. The CB + CMA3 staining combination demonstrated only one chromosome pair with fluorescence, probably the NOR-bearing one, while CB + DAPI gave various fluorescent signals, including metacentric pair 2, indicating that these heterochromatic regions are AT-rich in this population of R. quelen. The R. quelen population in this isolated region of Brazil is chromosomally distinct from that of other populations that have been studied.     

Subcongenic analysis of genetic basis for impaired development of invariant NKT cells in NOD mice

Reduced numbers and function of invariant NKT (iNKT) cells partially contribute to type 1 diabetes (T1D) development in NOD mice. Previous linkage analysis identified a genetic locus on chromosome 2 controlling numbers of thymic iNKT cells. Interestingly, this locus resides within the Idd13 region that distinguishes NOD mice from the closely genetically related, but strongly T1D-resistant NOR strain. Thus, we tested if a genetic variant that confers T1D resistance in NOR mice may do so by enhancing iNKT cell numbers. iNKT cells were enumerated by an α-GalCer analog loaded CD1d tetramer in NOD and NOR mice as well as in NOD stocks carrying NOR-derived congenic regions on chromosome 1, 2, or 4. Significantly, more thymic and splenic iNKT cells were present in NOR than NOD mice. The NOR-derived Idd13 region on chromosome 2 contributed the most significant effect on increasing iNKT cell numbers. Subcongenic analyses indicated that at least two genes within the Idd13 region regulate iNKT cell numbers. These results further define the genetic basis for numerical iNKT cell defects contributing to T1D development in NOD mice.     

Cytological and molecular evidence of deletion of ribosomal RNA genes in chromosome 6 of barley (Hordeum vulgare).

The barley chromosomal mutant T-35, in which only one pair of satellite chromosomes is apparent, was analyzed using a range of cytological and molecular techniques. Using conventional Feulgen staining, Giemsa and silver banding, in situ hybridization, and Southern blot analysis, unequivocal cytological and molecular evidence was obtained that T-35 is a homozygous deletion of rRNA genes residing in the nucleolus organizer region (NOR) of chromosome 6. According to the criteria of arm ratio and Giemsa-banding pattern of this chromosome, the deletion involved the whole NOR, one of the breakpoints being localized in the short arm proximally to the NOR-associated heterochromatic band, the other probably in the satellite of the chromosome. As a result of this deletion, an increased activity of the rRNA genes (as indicated by the size of the silver bands) on the other NOR-bearing chromosome (chromosome 7) was observed. The possible reasons for this phenomenon are discussed.     

Characterization of a Drosophila repeat mapping at the early-ecdysone puff 63F and present in many eucaryotic genomes

In this report we describe the nucleotide sequence of a 229 bp tandemly repeated sequence that hybridizes in situ to the early-ecdysone puff site 63F on salivary gland polytene chromosome 3 (Izquierdo, M., Arribas, C. and Alonso, C. (1981) Chromosoma 83, 363–366). Restriction analysis of genomic clones from the region indicates the existence of a minimum of 15 copies tandemly arranged at two separated sites, within the 63F puff region. The 229 basic units include conserved and variable segments and have two possible open-reading frames. A slight variation in the length of basic repeats was also observed. Some fly-stocks from Drosophila melanogaster contain particular RNA size classes complementary to the 63F repeat, while other RNAs remain constant in all stocks analyzed. A 5 kb fragment containing the repeat is present in many eucaryotic living beings, including plants and humans.     

Patterns of ribosomal gene variation in elite commercial chicken pure line populations

The nucleolus organizer region (NOR) encodes the tandemly repeated 18S, 5.8S and 28S ribosomal (r) RNA genes. The NORs of broiler and layer commercial chicken pure lines were studied to establish the type and extent of genetic variation at this important locus. The parameters studied were gene copy number, repeat size, and diversity of NOR-types. The populations were organized into three groups for analysis including brown-egg broiler (13 lines), brown-egg layer (six lines), and white-egg layer (eight lines). The ribosomal gene copy number average of the white-egg layer populations was significantly lower (329 genes) than that of the brown-egg layers (372 genes); the brown-egg broiler ribosomal gene average was intermediate (350 genes). The white-egg layer populations exhibited a ribosomal repeat unit average size of 36 kb, significantly different from the brown-egg layer and brown-egg broiler average repeat unit size of 32.5 and 33.9 kb, respectively. NOR array size was similar among the three groups (6 mb). The brown-egg broiler populations exhibited polymorphic NOR patterns, intra- and interline, whereas the white-egg layer populations were essentially monomorphic for NOR-type; brown-egg layers exhibited an intermediate level of NOR diversity. Some NOR array characteristics may be a function of breed origin as brown-egg commercial populations, both broilers and layers, have similar breed origins and exhibited similarities for predominant repeat unit size as compared with white-egg layer populations. However, the finding that brown-egg broiler lines typically exhibit a greater number of segregating NOR-types than brown-egg layer lines suggests that the selection schemes of broiler vs. layer pure line populations may also have influenced the degree of variation at this gene complex.     

New insights into the effects of extra nucleolus organizer regions

Summary Chromosome studies were carried out in four members of a sibship with a 15p+chromosome. Two carriers had normal offspring, one was unmarried, and the index case had three abortions and no live children. By means of different banding techniques, up to four satellites and four stalks could be observed on the abnormal chromosome. The Ag-I method showed from zero to four silver precipitates on the 15p+marker. Mean Ag-staining for 15p+and the ten acrocentric chromosomes were obtained in the carriers. Statistically significant differences between sibs were found. These results suggest the existence of: (a) An interindividual and intercellular variation of nucleolus organizer region (NOR) activity in man. (b) An optimal threshold of NOR activity, so that disturbances at the meiotic level could occur when it is exceeded. Our conclusions lead us to advise analysis of NOR activity in individuals with extra nucleolus organizer regions.     

Unusual primitive heteromorphic ZZ/ZW sex chromosomes in Proceratophrys boiei (Anura, Cycloramphidae, Alsodinae), with description of C-Band interpopulational polymorphism

We performed cytogenetic analyses on specimens from three population samples of Proceratophrys boiei from southeastern and northeastern Brazil. We stained chromosomes of mitotic and meiotic cells with Giemsa, C-banding and Ag-NOR methods. All specimens of P. boiei presented a karyotype with a full chromosome complement of 2n=22, metacentric and submetacentric. We observed the secondary constriction within the short arm of pair 8, which was in the same position of the nucleolus organizer region (NOR). NOR heteromorphism was observed within two specimens from the municipality of Mata de S?o Jo?o (northeastern Bahia State). The C-banding evidenced an unusual heterochromatic pattern in the genome of P. boiei. In the southern most population samples (S?o Paulo State), we observed large blocks of heterochromatin in the centromeric regions of all chromosomes, whereas the northernmost samples (Bahia State) presented a small amount of constitutive heterochromatin. We suppose that this geographic variation in heterochromatin quantities could be due to heterochromatinization of some chromosome regions in the genome of the S?o Paulo samples. Furthermore, females from S?o Paulo presented, within chromosome pair 1 from C-banded karyotypes, one homologous chromosome almost heterochromatic, whereas males had heterochromatin restricted to the centromeric region. This unusual heterochromatic arrangement led us to assume that P. boiei owns a ZZ/ZW type of sexual determination system. This finding is very important, as this is the first record of ZZ/ZW sex chromosomes within Cycloramphidae. We believe that the cytogenetic differences found between southeastern and northeastern Brazilian population samples of P. boiei strongly supports the existence of a species complex under the name P. boiei, and the requirement of taxonomic and systematic reviews by morphological, bioacoustical, molecular, and cytogenetic data could define this taxonomic issue in the future.     

Length and restriction site heteroplasmy in the mitochondrial DNA of american shad (alosa sapidissima)

Restriction endonuclease analysis was used to assess mitochondrial DNA (mtDNA) variation in American shad (Alosa sapidissima) collected from 14 rivers ranging from Florida to Quebec. Two types of heteroplasmy were observed, one involving a major length polymorphism and the other a single restriction site. Shad mtDNA occurred in two principal size classes, 18.3 and 19.8 kb. Of 244 shad examined, 30 were heteroplasmic and carried both size classes of mtDNA in varying proportions; the remainder were homoplasmic for the smaller size class of mtDNA. The large mtDNA variant occurred most frequently at the southern end of the range, and except for two individuals from Nova Scotia, was not detected among shad from rivers north of the Delaware. In contrast, ten shad heteroplasmic for a SalI restriction site originated from rivers ranging from South Carolina to Nova Scotia. DNA mapping and hybridization experiments indicated that the length polymorphism is in the D-loop-containing region and consists of a tandemly repeated 1.5-kb DNA sequence occurring in two and three copies, respectively, in the two major size classes of shad mtDNA. Continuous length variation up to approximately 40 bp occurs among copies of the repeat both within and among individuals. Restriction site data support the conclusion that both forms of heteroplasmy in shad mtDNA have originated more than once.     

Karyotypes of two American field crickets: Gryllus rubens and Gryllus sp. (Orthoptera: Gryllidae)

The karyotypes of Gryllus rubens (Scudder) and Gryllus sp. collected in the US were studied using conventional Giemsa staining and two differential staining methods. Both species had a chromosome complement of 2n = 28 + XX/XO, and the X chromosome was large and metacentric. In addition, nucleolus organizer regions (NOR) were detected in the short arm of one pair in the two species, and the NOR showed variation in size. The two species had species‐specific chromosome configuration and C‐banding patterns. In Gryllus sp., the chromosome configuration showed polymorphism in size and type among individuals, and the distal C‐bands of Gryllus sp. were larger than those of G. rubens.     

An assessment of the size structure, distribution and behaviour of fish populations within a temporarily closed estuary using dual frequency identification sonar (DIDSON)

The fish populations within the East Kleinemonde Estuary, South Africa, were studied using Dual frequency IDentification SONar (DIDSON) during March 2010 which allowed for direct comparisons of the abundance, distribution and behaviour of a wide size range of fishes along the length of the system. Small schooling fishes <100 mm in total length (L(T) ) were most abundant while larger size classes 100-300 and 300-500 mm were less abundant, but evenly distributed longitudinally within the estuary. Fishes <100 mm were most abundant within the mouth region which may reflect the feeding ecology of species that fall into these size classes and the effects of higher turbidities further up the estuary. Large fishes >500 mm were similarly most abundant near the mouth, and fishes of this size being mostly piscivorous, probably showed these distribution patterns due to the location of smaller prey fishes. Differential behaviour was observed among the longitudinal sections, with small fishes forming schools in areas of low turbidity and particularly in the presence of large predatory fishes. Results from this study present a snap-shot of the composition, distribution and behaviour of the ichthyofauna within a small temporarily closed estuary, and also show that abiotic factors and interactions of various size classes can influence their distribution. There is significant potential for DIDSON in future ecological research within these systems.     

玉米8个栽培亚种（类型）的核型和C—带带型的比较研究

本文首次报道了玉米8个亚种、2个亚型和2个杂交品种的核型和Giemsa C-带带型。所有材料的根尖细胞染色体数目均为2n=20。主要由中部和亚中部着丝点染色体组成。第6染色体短臂均具随体,但大小不同。所有材料均显示有亚端带和端带,在第6染色体的短臂上显示有NOR或/和随体带。C-带的分布、总数目和总长度各不相同。其总带数变异于6至18之间,C-带总长度为5.65—11.40％之间。在核型中,具中部着丝点的染色体数目及C-带总数,罕见栽培或原始的类型通常多于广泛栽培的类型。此外,有关核型和C-帝的变异和进化也进行了简略的讨论。     

Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae)

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region (NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic populations of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied, even those of distant islands. Diploid karyotypes (2 n = 16) always possessed two NOR-bearing chromosome pairs with pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluorochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands was noticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was correlated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as number and size of silver stained NORs, existed between cells and between individuals of the same population. The different size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only 5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich constitutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed between homologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 99–108.