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.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

HETEROCHROMATIN BANDING IN BOYKINIA,HEUCHERA, MITELLA,SULLIVANTIA, TIARELLA,AND TOLMIEA (SAXIFRAGACEAE)

Karyotypic differences were sought among species of Boykinia, Heuchera, Mitella, Sullivantia, Tiarella, and Tolmiea utilizing a modification of the Hy-banding technique. Prominent centromeric and some telomeric heterochromatin banding was observed. Boykinia aconitifolia and species of Sullivantia possess an identical banded karyotype, while four species of Heuchera, Mitella diphylla, Tiarella cordifolia, and Tolmiea menziesii (the latter at the tetraploid level) are characterized by a second, slightly different banded karyotype. In Sullivantia, Giemsa C-banding stains the same chromosomal regions revealed by Hy-banding. Larger amounts of heterochromatin are present in chromosomes of species of Heuchera, Mitella, Tiarella, and Tolmiea than in chromosomes of Sullivantia species and Boykinia aconitifolia. These karyological observations confirm generic relationships and demonstrate the systematic applicability of chromosome banding techniques to plants with very small chromosomes.     

玉米花粉单倍体植株染色体上异染色质的变异

我们用Giemsa BSG C-带技术检查了玉米花药培养获得的花粉单倍体植株根尖细胞染色体上异染色质的变异,观察结果表明,有的植株所显示的C-带数目是与供体植株的相一致,有的植株所显示的C-带数目则发生了显著变化,其中有的增加,有的减少。并讨论了异染色质发生变异的可能原因。还相应地观察到间期核中染色中心的变化是与中期染色体上C-带数目的变化相一致。     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

The constitutive heterochromatin in chromosomes of Fritillaria Sp., as revealed by Giemsa banding.

The incidence of C-bands (constitutive heterochromatin), as determined by differential Giemsa staining, was studied in the chromosomes of 56 species, varietal forms and subgenera of Fritillaria and 30 of them are illustrated. With the exception of the subgenera Korolkowi, a supposed link between lilies and fritillaries, and chromsome complements of all plants contained bands. There were wide differences in the size and number of these bands among species both within and between groups. In those with the largest and most abundant bands, there was a pronounced tendency for centromeric localization, both in Old and New World species. The Giemsa positive centromeres were masked when this occurred. Heteromorphy in respect of banding occurred in most species. The relation of repetitive DNA sequences with heterochromatin is discussed, as is also the problem of evolution in Fritillaria.     

The karyotypes of the thorny catfishes <Emphasis Type="Italic">Wertheimeria maculata</Emphasis> Steindachner, 1877 and <Emphasis Type="Italic">Hassar wilderi</Emphasis> Kindle, 1895 (Siluriformes: Doradidae) and their relevance in doradids chromosomal evolution

We studied the karyotypes of two doradids, the rare and endangered Wertheimeria maculata and a derived Amazonian species, Hassar wilderi. Cytogenetic characterization was assessed using conventional staining (Giemsa), C-banding, and NOR banding. Both species had 2n = 58 chromosomes but differed in their chromosome formulae, 24 m + 14sm + 8st + 12a for W. maculata and 32 m + 16sm + 10st for H. wilderi. In W. maculata heterochromatin was mainly telomeric, and three chromosomes had a fully heterochromatic arm; in H. wilderi heterochromatin was also predominantly telomeric and evident in many more chromosomes. Hassar wilderi also presented one pair of homologues with a fully heterochromatic arm. In both species, nucleolar organizer regions were restricted to one pair of subtelocentric chromosomes. Assuming a basal position for W. maculata, we hypothesized that underlying conserved diploid and NOR-bearing chromosome numbers, chromosomal evolution in doradids has involved pericentric inversions and an increase of heterochromatic blocks.     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.     

Analysis of different banding patterns and late replicating regions in chromosomes of Allium cepa,A. sativum and A. nigrum

Different banding procedures and preferential Giemsa staining of late replicating DNA-rich regions were carried out in metaphase chromosomes of three species belonging to different sections of the genus Allium (A. cepa, A. sativum and A. nigrum). The banding, as well as the late replicating patterns were species-specific. The late replicating pattern proved to be, in all cases, the more detailed, and represented the highest percentage of the karyotype differentially stained. Lower percents of the karyotype positively stained were accounted for by C-banding, by modified C-banding and by N-banding. In A. cepa interphase nuclei the pattern of constitutive heterochromatin fitted well with that of late replicating DNA-rich regions, but the coincidence with that revealed by C-banding was only partial. This supports the suggestion that late replicating regions may be considered to be a special category of heterochromatin. On the other hand, it seems that not all C-banded material replicates at the end of the S phase. By the modified C-banding, stained centromere dots or small bands, as well as bands at the NORs are observed.     

Cytochemical heterochromatin differentiation inSinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads

The fluorochrome and Giemsa chromosome banding patterns and the Ag-NOR histochemical staining ofSinapis alba are described. Two major types of heterochromatin can be distinguished, one of which contains GC-rich DNA. The observations are discussed as they relate to the known satellite DNAs ofS. alba. — A simple air-drying technique for producing spreads of plant mitotic chromosomes is presented. Different materials and staining techniques were tested showing that the method has wide applications.Dedicated in gratitude to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday.     

R-banding produced by DNase I digestion of chromomycin-stained chromosomes

A distinct reverse (R-) banding pattern was produced on human chromosomes by digesting chromosome spreads with pancreatic deoxyribonuclease I (DNase I) in the presence of an excess of chromomycin A3 (CMA), followed by staining with Giemsa. The banding pattern corresponds with that obtained by chromomycin A3 fluorescence, and bands which fluorescence brightly with chromomycin appear darkly with Giemsa. The same relationship was observed in two plants, Scilla siberica and Ornithogalum caudatum, which have contrasting types of heterochromatin. Chromomycin bright C-bands stained darkly with the CMA/DNase I technique, whereas chromomycin negative C-bands appeared lightly stained. The digestion patterns are thought to reflect the variation in chromomycin binding capacity along the chromosome with R-bands and dark C-bands being sites which preferentially bind the antibiotic.     

The karyotype ofFestucopsis serpentini (Poaceae Triticeae) from Albania studied by banding techniques and in situ hybridization

The karyotypes of two populations ofFestucopsis serpentini (2n = 2x = 14) endemic to Albania were investigated in detail by Giemsa C- and N-banding, AgNO₃ staining, and in situ hybridization with an rDNA probe. The complements consisted of 14 large chromosomes, 10 metacentric and 4 SAT-chromosomes, a metacentric and a submetacentric pair. SAT-chromosomes from one population carried exclusively minute satellites, whereas SAT-chromosomes from another population also carried larger polymorphic satellites, suggesting a geographical differentiation. The existence of four chromosomes with nucleolus forming activity was established through AgNO₃ staining; however, the rDNA probe additionally hybridized to intercalary positions in the short arms of two metacentric chromosomes revealing two inactive rDNA sites. C-banding patterns comprised from zero and up to four very small to larger, generally telomeric bands per chromosome giving low levels of constitutive heterochromatin. Similarities in chromosome morphology and C-banding patterns identified the homologous relationships of all chromosomes in one population, but of three pairs only in the other. Reliable identification of homologous chromosomes between plants was only possible for the SAT-chromosomes. A comparison between the C-banded karyotypes ofF. serpentini andPeridictyon sanctum supports their position in two genera.     

X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid <Emphasis Type="Italic">Aphis pomi</Emphasis>

Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA₃ staining reveals that these silver positive telomeres are the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.     

Chromosome homology and heterochromatin in goat,sheep and ox studied by banding techniques

Peripheral blood lymphocyte metaphase chromosomes of three Bovoidean species have been studied using Quinacrine fluorescence and Giemsa banding techniques to give Q-, G-, and C-banding patterns. Q- and G-banding characteristics, coupled with chromosome length, enabled all of the chromosomes in each of the chromosome complements to be clearly distinguished, although some difficulties were encountered with the very smallest chromosomes. A comparison of G-banding patterns between the species revealed a remarkable degree of homology of banding patterns. Each of the 23 different acrocentric autosomes of the domestic sheep (2n=54) was represented by an identical chromosome in the goat (2n=60) and the arms of the 3 pairs of sheep metacentric autosomes were identical matches with the remaining 6 goat acrocentrics. A similar interspecies homology was evident for all but two of the autosomes in the ox (2n=60). This homology between sheep metacentric and goat acrocentric elements confirms a previously suggested Robertsonian variation. The close homology in G-banding patterns between these related species indicates that the banding patterns are evolutionarily conservative and may be a useful guide in assessing interspecific relationships. —The centromeric heterochromatin in the autosomes of the three species was found to show little or no Q-or G-staining, in contrast to the sex chromosomes. This lack of centromeric staining with the G-technique (ASG) contrasts markedly with results obtained with other mammalian species. However, with the C-banding technique these regions show a normal intense Giemsa stain and the C-bands in the sex chromosomes are inconspicuous. The amount of centromeric heterochromatin in the sheep metacentric chromosomes is considerable less than in the acrocentric autosomes or in a newly derived metacentric element discovered in a goat. It is suggested that the pale G-staining of the centromeric heterochromatin in these species might be related to the presence of G-Crich satellite DNA.     

Restrictive distribution of asymmetric C-bands in the chromosome complement of the rhesus (Macaca mulatto)

Lymphocyte chromosomes from a cercopithecoid species, Macaca mulatta, were studied for the occurrence of lateral asymmetry in constitutive heterochromatin. The technique consisted of growing the lymphocytes for one cell cycle in BrdUrd, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 SSC and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin of the nucleolar organizer marker chromosome (no. 13 of the complement). In these chromosomes, the darkly staining region was confined at any given point to a single chromatid, while the corresponding region on the sister chromatid was lightly stained. This pattern of asymmetric staining in the constitutive heterochromatic region was not observed in any other chromosome of Macaca mulatta. The lateral asymmetry of constitutive heterochromatin in this species is presumed to reflect the strand bias in the distribution of thymine in the alphoid DNA fractions.     

Distribution of heterochromatin in a reconstructed karyotype of Vicia faba as identified by banding- and DNA-late replication patterns

1) The distribution pattern of heterochromatin characterized by Giemsa-banding, Quinacrine-banding and DNA-late replication has been studied in a reconstructed karyotype of Vicia faba with all chromosome pairs interdistinguishable. 2) By means of two Giemsa-banding methods both an interstitial and a centromeric Giemsa-banding pattern are described. The former one comprehends 14 marker and 18 additional bands of lower but characteristic visualization frequencies. The centromeric Giemsa-banding pattern consists of 7 bands, located in the centromeric and in the secondary constrictions of the metaphase chromosomes. Chromosomes with banding patterns intermediate between the interstitial and the centromeric Giemsa-banding have also been observed. 3) Quinacrine-banding revealed 10–12 brightly fluorescent bands and 1–2 regions of dim fluorescence. Most Q-bands occupy chromosomal positions also characterized by interstitial Giemsa bands. 4) The DNA-late replication pattern, analyzed both by autoradiography and by FPG-technique, revealed 9 late replicating chromosome regions; all of these correspond positionally to the sites of interstitial Giemsa bands. 5) The results are discussed with respect to (a) the relationships between the banding- and the DNA-late replication pattern; (b) banding and heterochromatin characteristics; (c) the correlations between the distribution of chromatid aberrations and special types of heterochromatin. — The patterns of heterochromatin distribution found are in basic conformity with the corresponding patterns reported for the standard karyotype of Vicia faba. The heterochromatin type characterized by both Giemsabanding and late replication is characteristic of all those chromosome regions which after mutagen treatments show up as aberration hot spots. Positional correlations between interstitial Giemsa marker bands and chemically induced isochromatid breaks are indicative of preferential aberration clustering in heterochromatin/euchromatin junctions.     

Characterization of different types of condensed chromatin inCostus (Zingiberaceae)

The chromatin structure of six diploids species ofCostus was analysed using conventional Giemsa staining, C-banding and DAPI/CMA fluorochromes. The interphase nuclei in all the species show an areticulate structure and the prophase chromosomes show large blocks of proximal condensed chromatin. After banding procedures, each chromosome exhibits only centromeric dot-like DAPI⁺/CMA^– C-bands whereas the satellites (one pair at each karyotype) are weakly stained after C-banding and show a DAPI^–/CMA⁺ fluorescence. Two chromocentres show bright fluorescence with CMA and weak staining after C-banding whereas the others chromocentres show only a small fraction of DAPI⁺ heterochromatin. These results were interpreted to mean that the greater part of the condensed chromatin has an euchromatic nature whereas two types of well localized heterochromatin occur in a small proportion. The Z-stage analysis suggests that heterochromatin and condensed euchromatin decondense at different times. The chromosome number and morphology of all species are given and the implications of the condensed euchromatin are discussed.Dedicated to Prof.Elisabeth Tschermak-Woess on the occasion of her 70th birthday.     

黄颡鱼染色体的C─带、Ag—NORs、荧光带和复制带显带的研究

采用Giemas染色、C─带、Ag—NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明,黄颡鱼只有部分的染色体呈现阳性C─带,可分为三类,其中NORs区是染色最深、染色面积最大的区域,为深染居间C─带。其Ag－NORs位于m5q末端。CMA3染色显示NORs区呈现出明亮的荧光。中复制染色体上着丝粒区、端粒区和居间区浅染。发现核仁缢痕、深染居间C─带、Ag—NORs、CMA3明亮区和中复制带浅染NORs区位置基本一致,C─带阳性区和中复制带浅染区具有对应性。     

Karyomorphology of six taxa in Chrysanthemum sensu lato (Anthemideae) in Egypt and their genetic relationships by Giemsa C‐banding

Abstract Giemsa C‐banding was applied to the chromosome complements of six diploid species belonging to six genera in Chrysanthemum sensu lato (Anthemideae) distributed in Egypt. Four types of C‐banding distribution were observed in the taxa as follows: (i) negative C‐banding in Anacyclus monanthos (L.) Thell.; (ii) all bands in terminal regions in Achillea fragrantissima (Forssk.) Sch. Bip, which showed 32 bands on 18 chromosomes; (iii) all eight bands at centromeric regions on eight chromosomes in Matricaria recutita L.; and (iv) bands at terminal and centromeric regions in Brocchia cinerea Vis. (12 terminal and six centromeric bands on 12 chromosomes), Cotula barbata DC. (four terminal, six centromeric, and eight short arm bands on 16 chromosomes), and Glebionis coronaria (L.) Cass. ex Spach. (eight terminal on the short arms and four large bands in centromeric regions on 12 chromosomes).     

Comparison of areas of quinacrine mustard fluorescence and modified Giemsa staining in human metaphase chromosomes

The methods of quinacrine mustard fluorescence and modified Giemsa staining were compared in view of the structural details revealed in human mitotic chromosomes derived from the peripheral blood of normal healthy humans. Over the chromatids both techniques produced a crossbanding pattern where larger segments of heavy staining in the latter technique and the fluorescing bands in the former occurred at similar locations. The centromeric heterochromatin, intensely stained with Giemsa was, however, negative in fluorescence, except for chromosome no. 3 and less often no. 6. The regularly occurring secondary constrictions in chromosomes 1, 9, and 16 behaved generally like areas of centromeric heterochromatin. The area of secondary constriction in the Y chromosome as also that of chromosome 9 in the ASG modification of the Giemsa technique was both non-fluorescent and non-staining.