Position-dependent NOR activity in barley

Two types of intraspecific nucleolar dominance/suppression are described for barley,Hordeum vulgare L. When the nucleolus organizing regions (NORs) originally belonging to chromosomes 6 and 7 are combined by translocation in one chromosome, NOR 6 is dominant over NOR 7. Neither significant loss of rDNA nor its hypermethylation is the reason for the reduced nucleolus forming activity of NOR 7. Intrachromosomal NOR suppression probably does not occur in isochromosome 6s, which has two NORs 6 in one chromosome. Meiotic and somatic pairing of the homologous arms might be the reason for early fusion of their nucleoli and thus for the lower than expected maximum number of interphase nucleoli. Variable suppression of a partial NOR (6³) is described for descendants of crosses between translocation lines with split NORs 6 and 7. In these cases also, the reduced activity of the partial NOR 6³ is not due to deletion of rDNA as shown by in situ hybridization. Unstable methylation of NOR 6³ in heterozygous F₁ individuals is probably the cause of this phenomenon.     

Distribution of silver-stained nucleolus-organizing regions in the chromosomes of the Equidae

The distribution of silver-stained nucleolus-organizing regions (NORs) in fibroblast chromosomes from all seven extant species of Equidae are described. There are variations in the number and locations of silver-stained NORs in different species but most of the cells in an individual of any speies had only 2 to 4 silver-stained NORs. In Equus przewalskii and E. caballus NORs were detected on up to three different chromosomes. In E. asinus 11 different chromosomes were observed to possess NOR sites. E. hemionus kulan and E. hemionus onager had NORs on 2 large metacentric pairs and a small acrocentric pair. In E. grevyi and E. burchelli NORs were located on 3 to 4 different pairs. E. zebra hartmannae had silver-staining over the telomere regions of the short arms of the chromosomes 1, 3 and 4. A comparison of G-banded chromosomes and silver-stained NORs has revealed one autosome with conserved G-band patterns and possessing a silver-staining NOR in all the species except E. asinus. Variations in the number and multichromosomal locations of NORs in various species could have evolved by pairing and exchanges between non-homologous chromosomal heterochromatin having similar satellite DNA sequences.     

Number and Nuclear Localisation of Nucleoli in Mammalian Spermatocytes

In seven mammalian species, including man, the position and number of nucleoli in pachytene spermatocyte nuclei were studied from electron microscope (EM) nuclear sections or bivalent microspreads. The number and position of the nucleolar organiser regions (NORs) in mitotic and meiotic chromosomes were also analysed, using silver staining techniques and in situ hybridisation protocols. The general organisation of pachytene spermatocyte nucleoli was almost the same, with only minor morphological differences between species. The terminal NORs of Thylamys elegans (Didelphoidea, Marsupialia), Dromiciops gliroides (Microbiotheridae, Marsupialia), Phyllotys osgoodi (Rodentia, Muridae) and man, always gave rise to peripheral nucleoli in the spermatocyte nucleus. In turn, the intercalated NORs from Octodon degus, Ctenomys opimus (Rodentia, Octodontidae) and Chinchilla lanigera (Rodentia, Cavidae), gave rise to central nucleoli. In species with a single nucleolar bivalent, just one nucleolus is formed, while in those with multiple nucleolar bivalents a variable number of nucleoli are formed by association of different nucleolar bivalents or NORs that occupy the same nuclear peripheral space (Phyllotis and man). It can be concluded that the position of each nucleolus within the spermatocyte nucleus is mainly dependent upon: (1) the position of the NOR in the nucleolar bivalent synaptonemal complex (SC), (2) the nuclear pathway of the nucleolar bivalent SC, being both telomeric ends attached to the nuclear envelope, and (3) the association between nucleolar bivalents by means of their NOR-nucleolar domains that occupy the same nuclear space. Thus, the distribution of nucleoli within the nuclear space of spermatocytes is non-random and it is consistent with the existence of a species-specific meiotic nuclear architecture.     

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.     

Divergent location of ribosomal genes in chromosomes of fish thorny-headed worms, <Emphasis Type="Italic">Pomphorhynchus laevis</Emphasis> and <Emphasis Type="Italic">Pomphorhynchus tereticollis</Emphasis> (Acanthocephala)

We studied distribution of ribosomal DNA (rDNA) sequences along with chromosomal location of the nucleolar organizer regions (NORs) in males of two fish parasites, Pomphorhynchus laevis and Pomphorhynchus tereticollis (Acanthocephala). Fluorescence in situ hybridization with 18S rDNA probe identified two clusters of rDNA in each species, but revealed a remarkable difference in their location on chromosomes. In P. laevis, the rDNA-FISH signals were found in long arms of the first chromosome pair and in short arms of the second pair. Whereas in P. tereticollis, rDNA clusters were located in long arms of both the first and second chromosome pairs. The divergent location of rDNA clusters in the chromosome No. 2 supports current classification of P. tereticollis, previously considered a synonym of P. laevis, as a separate species. A possible scenario of the second chromosome rearrangement during karyotype evolution of the two species involves two successive pericentric inversions. In both species, one or two prominent nucleoli were apparent within interphase nuclei stained with either silver nitrate or a fluorescent dye YOYO-1. However, a single large nucleolus was observed in early stages of mitosis and meiosis I regardless the number of rDNA clusters. Nevertheless, two bivalents with silver-stained NORs in diakinesis and two silver-stained sites in early prophase II nuclei indicated that all NORs are active. This means that each Pomphorhynchus NOR generates a nucleolus, but the resulting nucleoli have a strong tendency to associate in a large body.     

Chromosome banding in Amphibia. XXIX. The primitive XY/XX sex chromosomes of Hyla femoralis (Anura, Hylidae)

The karyotype of the pine woods treefrog, Hyla femoralis, is characterized by primitive XY female/XX male sex chromosomes. The sole difference between the X and the Y is the presence of a nucleolus organizer region (NOR) in the X. Due to a deletion of the NOR in the Y, this chromosome is distinctly smaller than the X. Since no autosomal NORs exist in the karyotype of this species, the NOR deletion in the Y results in a sex-specific difference in the number of ribosomal RNA genes, with a female:male ratio of about 2:1. Interphase nuclei of male animals contain always one silver-stained nucleolus, whereas most nuclei of female specimens exhibit two nucleoli. This is in agreement with the absence of dosage compensation for sex-linked genes in amphibian cells. The consequences of the loss of about 50% of ribosomal RNA genes for the viability of male individuals and spermatogenesis are discussed.     

The behavior of nucleolus organizers in structurally changed karyotypes of barley

Two standard karyotype barley lines and 18 lines with karyotypes reconstructed by means of induced reciprocal translocations have been studied with respect to nucleolus formation. The standard karyotype contains two pairs of satellite chromosomes (pairs 6 and 7). Five of the structurally changed karyotypes contain, as a result of reciprocal translocations between the standard satellite chromosomes, only one satellite chromosome pair, each chromosome with two satellites and two nucleolus organizing regions. Under these circumstances, only two of the four NORs are active in nucleolus formation while the other two — probably the transposed ones — remain inactive; hence the maximum number of primary nucleoli per nucleus is two. — When NORs are translocated to chromosomes with no NOR in the standard karyotyp, the normal pattern of nucleolus formation remains unchanged. The same is true after transposition of segments from other chromosomes to the satellites of the standard SAT-chromosome pairs 6 and 7. The results obtained are discussed with respect to effects of translocations on the activity and behaviour of nucleolus organizing regions.     

Fine structure of nucleoli in micronucleated cells

The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate cells.     

Chromosomal dynamics of nucleolar organizer regions (NORs) in the house mouse: micro-evolutionary insights

Variation in the number and chromosomal location of nucleolar organizer regions (NORs) was studied in the house mouse, Mus musculus (2n=40). From an origin in Western Asia, this species colonized the Middle East, Europe and Asia. This expansion was accompanied by diversification into five subspecies. NOR diversity was revealed by fluorescence in situ hybridization using 18S and 28S probes on specimens spanning Asia to Western Europe. The results showed that the house mouse genome possessed a large number of NOR-bearing autosomes and a surprisingly high rate of polymorphism for the presence/absence of rRNA genes on all these chromosomes. All NOR sites were adjacent to the centromere except for two that were telomeric. Subspecific differentiation established from the NOR frequency data was concordant with the overall pattern of radiation proposed from molecular studies, but highlighted several discrepancies that need to be further addressed. NOR diversity in M. musculus consisted of a large number of polymorphic NORs that were common to at least two subspecies, and a smaller number of NORs that were unique to one subspecies. The most parsimonious scenario argues in favor of a subspecific differentiation by lineage sorting of ancestral NOR polymorphisms; only the unique NORs would have appeared by inter-chromosomal transposition, except for the two telomeric ones that may have originated by hybridization with another species. Such a scenario provides an alternative view from the one prevailing in most systematic and phylogenetic analyses that NORs have a high transposition rate due to concerted evolution of rRNA genes.     

Differences in the location of nucleolus organizer regions in European vespertilionid bats

The karyotypes of European vespertilionid bats are distinguished by only a few, easily detectable differences in their G-banding patterns. Most rearrangements can be identified as Robertsonian translocations. Yet, there are surprising differences in the location of active nucleolus organizer regions (NORs), as revealed by silver staining. The ancestral position of the NOR is considered to be a secondary constriction on chromosome 15, as is the case in the genera Eptesicus, Nyctalus, and Vespertilio and in three of four Pipistrellus species. The remaining genera show multiple NOR sites located on minute short arms close to the centromere. In P. pipistrellus, differences in the location of the NORs correlate with the geographical origin of the animals. Some Myotis species possess NORs on numerous chromosomes and show great interindividual variability. In addition, two sibling species, M. brandtii and M. mystacinus, show completely different NOR locations.     

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH)

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA₃-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n=50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA₃-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants – (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA₃-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.     

Chromosome banding in Amphibia

The structure of the nucleolus organizer regions (NORs) in mitotic chromosomes, diploid nuclei and spermatogenesis was studied in 260 individual animals from 23 genera of the Anura. The analyses were performed with conventional cytogenetic methods as well as with Ag-staining, GC- and AT-specific fluorochromes (mithramycin, chromomycin A₃, quinacrine) and C-banding. Most of the species have only one pair of NORs in their karyotypes. The majority of individuals of all species exhibited considerable differences in the sizes of their homologous NORs. Most of these heteromorphisms are due to tandem duplications or triplications in one of the two NORs. However, duplicated or triplicated NORs never occur in a homozygous form, but are instead always in combination with a normal-structured NOR in the homologous chromosome. In three animals, a complete deletion of one NOR and its closely associated constitutive heterochromatin was determined. The cytochemistry of the specific NOR-stainings are discussed. The size differences of the Ag-, mithramycin- and chromomycin A₃-stained NORs can be traced to differences in the rDNA content in these NORs.     

Chromosome complement and nucleolar organization in Podophyllum hexandrum Royle

The karyomorphology of Podophyllum hexandrum Royle (2n=2x=12) is described for the first time. The haploid set comprises one metacentric, three sub-metacentric and two acrocentric chromosomes. Metacentrics are the longest and acrocentrics the smallest in the complement. Secondary constrictions are located in the short arm of two meta-/submetacentrics and in the long arm of the two acrocentric chromosomes. The number of NORs is in agreement with the number of nucleoli organized per root tip nucleus.     

A supernumerary chromosome segment in Locusta migratoria.

A single female of Locusta migratoria was found to be heterozygous for a supernumerary heterochromatic segment distally located on the M6 autosome close to its nucleolus organiser region (NOR). Reactions to several chromosome banding techniques revealed its heterochromatic nature and its composition of GC-rich DNA sequences and likewise the NORs in this species. This suggests an origin for the extra segment by amplification of GC-rich DNA sequences contained in the distal NOR of the M6 chromosome, which is reinforced by the observation that the NOR of segmented M6 chromosomes produced the larger nucleolus in embryo prophase cells, such as would be expected from the presence of rRNA genes in the extra segment. No accumulation mechanism was detected in this female after analyzing the 213 embryo offspring produced, but an increase in the number of nucleoli per interphase nucleus was noted in heterozygous embryos in respect to standard homozygous ones.     

Chromosome banding studies in an Indian mullet: evidence of structural rearrangements from NOR locations

C-, G- and NOR bands have been studied in the female sex of Rhinomugil corsula. (Mugilidae, Pisces) by deploying the conventional methodologies with suitable modifications of minor nature. The diploid metaphase complements contained 48 acrocentric chromosomes. The localization of C-band heterochromatin was found to be mostly at or near the centromeric regions of the acrocentric chromosomes. The G-type bands were not so well defined, but some of the G-banded chromosomes also contained C-bands. Interestingly, silver-positive NORs were found at the telomeric ends of five acrocentric chromosomes, including one homologous pair having NORs in both chromatids, while one chromosome showed NORs in both of its chromatids and the other two had only one NOR localized at one of its chromatids. This would suggest that one homologue of the second pair of NOR-bearing chromosomes possibly underwent a chromatid exchange with a non-NOR bearing chromosome. This is quite a unique situation not reported earlier in any species of fish., though some other form of NOR-polymorphism/heteromorphism has rarely been reported. Therefore, further exploration in natural populations of this species to examine the other sex and to verify if there also exists other chromosomally polymorphic races (in respect of NOR-polymorphism) of this species, would be rewarding.     

Population differences in the expression of nucleolus organizer regions in the grasshopperEyprepocnemis plorans

Summary Fluorescence in situ hybridization revealed the presence of ribosomal RNA genes in paracentromeric regions of all A chromosomes and in the distal half of B chromosomes in embryonic cells from Moroccan specimens of the grasshopperEyprepocnemis plorans. The expression of these genes was monitored by the presence of nucleoli attached to each chromosome bivalent in diplotene cells from males collected from two different Moroccan populations and was compared to previous data of Spanish populations. Whereas only the nucleolus organizer regions (NORs) on S₉–S₁₁ and X chromosomes were active in the Spanish specimens. Moroccan individuals showed NOR activity in all chromosomes. The rRNA genes on the B chromosome were inactive in both populations. The S₉ and S₁₀ NORs were less active in Moroccan specimens than in Spanish specimen, which might be partly explained by the negative interdependence for expression of the S₁₀ NOR with respect to those on L₂ and X chromosomes. On the other hand, the X NOR was more active in Moroccan specimens than in Spanish specimens, and this might be partly due to the positive effect that the presence of B chromosomes has on the expression of this NOR. The implications of these observations on current models of NOR activity regulation are discussed.Abbreviation NOR nucleolus organizer region     

Cytogenetic analysis of five Hypostomus species (Siluriformes, Loricariidae)

In this work, we analyzed the karyotypes of five Hypostomus species. Hypostomus cf. heraldoi, from the Mogi-Gua?u River, had 2n = 72 chromosomes, with a nucleolar organizer region (NOR) in one chromosomal pair. Hypostomus regani, from the Mogi-Gua?u River had 2n = 72 chromosomes with NORs in two chromosomal pairs. Hypostomus sp., from the Mogi-Gua?u River basin, had 2n = 68 chromosomes, with NORs in two chromosomal pairs. Hypostomus aff. agna, from Cavalo Stream, had 2n = 74 chromosomes with NORs in two chromosomal pairs. Hypostomus cf. topavae, from Carrapato Stream, had 2n = 80 chromosomes, with NORs in two chromosomal pairs. Hypostomus species showed marked diversity in the karyotypic formula, which suggested the occurrence of several Robertsonian rearrangements and pericentric inversions during the evolutionary history of this genus. This hypothesis was supported by the occurrence of a large number of uniarmed chromosomes and multiple NORs in a terminal position in most species and may be a derived condition in the Loricariidae.