Relationship between secondary constrictions and nucleolus organizing regions inAllium sativum chromosomes

Summary Allium sativum L. (2 n=16) had three types of clones with regard to the number of chromosomes carrying well-defined secondary constrictions: the first type had two secondary constricted chromosomes (type I), the second had three (type II) and the third had four (type III). Silver staining was applied to these three types of cells to determine the number of nucleolus organizing regions (NORs) per cell and to study the relationship between the morphological appearance of the secondary constrictions and the ability of the chromosomes to form nucleoli. Ag-positive regions appeared on two chromosomes in type I, on three in type II and on four in type III. The comparison of Giemsa and Feulgen stained chromosomes with the silver stained ones clearly indicated that the positive reaction with silver occurred exclusively on the secondary constricted regions that responded negatively to both Giemsa and Feulgen staining, indicating that the size of the achromatic secondary constrictions directly reflects the volume of the Ag-positive materials. However, all three types of clones had a maximum of four nucleoli at interphase. Of the four nucleoli, either two or one was extremely small (less than 1 m in diameter) in types I and II respectively. The size variations of the other nucleoli seemed to be positively correlated with those of the Ag-positive regions. This and the observation that the maximum number of nucleoli per cell did not coincide with the number of Ag-positive regions on the metaphase chromosome complement suggest strongly that the NORs responsible for the minute nucleoli cannot be detected on the metaphase chromosomes. The present observations indicate that not all NORs are indicated by the morphological appearance of secondary constrictions.     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.     

Morphology of the axial structures in the neo-XY sex bivalent of Pycnogaster cucullata (Orthoptera) by silver impregnation

A simple method, using silver impregnation after 2 x SSC pretreatment allowed us to demonstrate axial structures (cores) in the metaphase I bivalents of the neo-XY race of Pycnogaster cucullata under bright-field microscopy. Axial structures can also be shown in DNA-depleted metaphase I bivalents, suggesting that DNA is not essential to demonstrate these elements. Specifically differentiated regions, which coincide with kinetochores, secondary constrictions (including the NOR), and chiasmata were also found. These regions have a characteristic morphology and therefore may be utilized for cytogenetic analysis. The simultaneous visualization of all these regions allowed us to establish their spatial relationships and hence the basic structural organization of the neo-XY sex bivalent in this species.     

Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are — at least in species with large genomes — intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.     

The structure and behavior of the nucleolus organizers in mammalian cells

The regularly occurring secondary constrictions on metaphase chromosomes of mammalian cells prove to be nucleolus organizers as expected. The expression of nucleolus organizers as secondary constrictions, however, varies from cell to cell and from tissue to tissue, including cultivation in vitro. Electron micrographs of the organizer region show that the nucleolus organizer at metaphase is not a constriction. The width of the organizer area is the same as the condensed chromosomal arms; but the filaments, which are the major components of this region, show a diameter of 50–70 Å. The condensed chromosome arms consist of filaments 150–200 Å in diameter. In some mammalian species, structures similar to the nucleolus organizer are located at the end of chromosomes. These may be terminal nucleolus organizers.Supported in part by Research Grants DRG-269 from Damon Runyon Memorial Fund for Cancer Research, E-286 from American Cancer Society, and HD-2590 from National Institutes of Health.     

The surface and internal structure of metaphase chromatin

Mammalian metaphase chromatin has been isolated by an ultrasonication technique and examined by both surface scanning and high-voltage electron microscopy (H.V.E.M.). By both techniques, rod-like structures 0.5 to 0.8 wide and varying in length from 3 to 5 were seen. Evidence is submitted that these represented parts of metaphase chromosomes. — By scanning microscopy the rods showed repeated patterns of wide and constricted areas. The constrictions were spaced 3,000 to 4,000 Å apart and the entire surface of the rods was covered with smaller rounded projections. In addition, longitudinal grooves were occasionally seen. — H.V.E.M. revealed gross folded and unfolded fibres whose sizes correlated with the surface projections seen by scanning microscopy. In addition, microfibrils, 20–40 Å in diameter were seen. The possibility that these fibrils represent the DNA co-helix is discussed.     

In situ localization and characterization of different classes of chromosomal DNA: acridine orange and quinacrine mustard fluorescence

In situ denaturation of metaphase chromosomes with alkali results in a shift from green to yellow, orange, brown and red fluorescence with acridine orange, indicating increasing denaturation of chromosomal DNA. The kinetics and characteristics of denaturation are described. Mouse and Microtus agrestis chromosomes denature uniformly but human cells show sequential denaturation. With increasing concentrations of alkali, the secondary constrictions in chromosomes 1, 9 and 16 are the first, and the distal half of the Y chromosome the last, to become denatured. — Reassociation of chromosomal DNA occurs within seconds after the start of incubation in salt solution. Areas containing repetitious DNA, e.g. mouse centromeres, fluoresce much more strongly than other regions with acridine orange after prolonged reassociation. Since human and Microtus centromeric regions behave similarly, it is proposed that they, too, contain repetitious DNA. — Reassociation treatment leads to enhancement of bright quinacrine mustard fluorescence in regions already bright before treatment. Furthermore, regions containing repetitious DNA, e.g. the secondary constrictions in human chromosomes 1, 9 and 16, whose fluorescence is dull before treatment, turn bright after reassociation. — The methods of fluorescence analysis of mammalian chromosomes with acridine orange and quinacrine mustard permit the localization and study of different classes of chromosomal DNA.     

Chromosome banding in the genusPinus

Somatic chromosomes (2n=24) ofPinus luchuensis Mayr at metaphase were observed by fluorescent banding methods with chromomycin A₃ (CMA) and DAPI. CMA-bands appeared at the interstitial and/or proximal regions of nearly all chromosomes. DAPI-bands appeared at the interstitial and/or centromeric regions of nearly all chromosomes, and pairs of DAPI-dots appeared at the centromeric regions. Each homologous pair of chromosomes in the chromosome complement was identified by the CMA and DAPI fluorescent banding patterns. The interstitial CMA-bands were mostly localized at the secondary constrictions of the Feulgen-stained chromosomes. The fluorescent banding pattern ofP. luchuensis was very similar to that ofP. thunbergii, but was different from that ofP. densiflora.     

Control of conformation changes associated with homologue recognition during meiosis

During early meiosis, chromosomes pair via their telomeres and centromeres. This pairing induces a conformational change which propagates from these regions along each chromosome, making the chromatin of the partners accessible for intimate pairing. In the present study, we show by exploiting wheat–rye hybrids that the signal is initiated in both the presence and absence of either the Ph1 or Ph2 locus. However, the chromatin change only continues to propagate through rye telomeric heterochromatin when Ph1 is absent. This failure to propagate the chromatin change through the rye heterochromatin in the absence of Ph2 correlates with a subsequent lack of wheat–rye chromosome association at metaphase I.     

Dimorphic nucleolar organizer regions in the frog Rana blairi.

The nucleolar organizer-specific staining procedure, ammoniacal silver (Ag-AS), has been used to study the distribution and size of the nucleolar organizer regions (NORs) in chromosomes of the frog Rana blairi (Mecham, Littlejohn, Oldham, Brown and Brown). The somatic metaphase karyotype of this frog is similar to that of other frogs of the Rana pipiens species complex, numerically (2n=26) and morphologically. Secondary constrictions are detectable in untreated Giemsa-stained metaphase preparations as achromatic gaps in the long arms of a pair of submetacentric chromosomes (no. 10). These constrictions are the only regions which are deeply stained with the Ag-AS method and are thus identified as the nucleolar organizer regions (Ag-NORs). In each of the three individuals, the Ag-NORs as visualized on the homologues are of unequal length.     

CRUCIFORM NUCLEAR DIVISION IN WORONINA PYTHII (PLASMODIOPHOROMYCETES)

Somatic nuclear divisions in sporangiogenous plasmodia of Woronina pythii Goldie-Smith were studied with transmission electron microscopy. During metaphase, each nucleus formed a cruciform configuration as chromatin became aligned at the equatorial plate perpendicular to the persistent nucleolus. Except for polar fenestrations, the original nuclear envelope remained intact throughout the mitotic division. Intranuclear membranous vesicles appeared to bleb off the inner membrane of the original nuclear envelope, adhered to the surfaces of the separating chromatin, and eventually formed new daughter nuclear envelope within the original nuclear envelope. During the first 24 hr of vegetative plasmodial growth, each telophase nucleus exhibited an obvious constriction of the original nuclear envelope in the interzonal region. Similar constrictions were not evident in telophase nuclei found in 24–36-hr-old plasmodia. This variation in the ultrastructural morphology of cruciform division appears to be related to the age and size of each sporangiogenous plasmodium, and is the first to be documented within this group of fungal pathogens.     

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytological localization of inverted repeated DNA sequences in Vicia faba

Inverted repeated DNA sequences have been isolated from sheared Vicia faba DNA by hydroxylapatite column chromatography, treated with nuclease S₁, tritiated by the nick translation method and hybridized in situ on squashes of Vicia faba root tips. Silver grains appear grouped in a rather limited portion of interphase nuclei and form a sort of band across them. The central regions of metaphase chromosomes are preferentially labeled, labeling being excluded from telomeres, centromeres and secondary constrictions. These results are briefly discussed in relation to those obtained in other species and the functional significance of inverted repeats.     

C-Banding studies on australian hylid frogs: secondary constriction structure and the concept of euchromatin transformation

A C-banding and silver staining analysis of 12 species of Australian frogs of the genus Litoria, has shown that 6 morphologically distinct classes of secondary constrictions are present. These constrictions are distinguished by the distribution and type of C-banding chromatin and the distribution of silver staining material. Not all of these constrictions are nucleolus organizers. Groups of closely related species often share particular constrictions, although previously unencountered constrictions do occur in some species. It is argued that changes in position of nucleolar organizing constrictions is most easily explained by the amplification of latent nucleolus organizing sites. One of the more unusual features of this group of species is the shared similarity in gross chromosome morphology, contrasted to the extensive C-banding variation at secondary constriction sites. While in some of these cases chromosomal evolution has undoubtedly proceeded by the addition of heterochromatic segments, the predominant mechanism of change appears to involve the large scale transformation of euchromatin to heterochromatin.     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.     

Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination‐poor region comprises more than half of barley chromosome 3H

Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single‐copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination‐poor region is preferentially marked by a heterochromatin‐typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin‐typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.