AT-rich Heterochromatin Research Articles

Heterochromatin patterns in four diploid Zephyranthes species with different basic chromosome number (Amaryllidaceae)

Diverse species-specific karyotypes have been reported within Zephyranthes Herb., as well as different basic chromosome numbers (x = 5, 6, 7, and 9), with x = 6 being the most common. The aim of this work was to analyze the constitutive heterochromatin (C-Het) pattern of karyotypes in four diploid Zephyranthes species. For this purpose, a cytomolecular analysis of the karyotypes of Z. andalgalensis (2n = 2x = 12 = 4 m + 2 sm), Z. chacoensis (2n = 2x = 12 = 3 m + 2 sm + 1 st), Z. robusta (2n = 2x = 12 = 3 m + 2 sm + 1 st), and Z. pedunculosa (2n = 2x = 14 = 1 m + 3 sm + 3 st) was performed for the first time. All the species analyzed showed CMA+ terminal bands that could be distinguished by their heterochromatin pattern. Interestingly, DAPI+ interstitial bands were also found in the karyotype of Z. robusta. The four Zephyranthes C-Het patterns analyzed show that diploid species have little GC-rich heterochromatin, which is generally associated with terminal satellites, and that AT-rich heterochromatin is rare. The dysploidy evolutionary change between two basic chromosome numbers (x = 6 and 7) is discussed in the present study.

Dynamic development of AT‐rich heterochromatin has followed diversification and genome expansion of psammophilous Mycetophylax (Formicidae: Attini: Attina)

Heterochromatin is an important genome constituent comprised by a high density of repetitive DNA sequences that mediate chromosome structure and function. The species Mycetophylax morschi currently harbours three cytotypes: 2n=26, 2n=28 and 2n=30 chromosomes. However, Mycetophylax conformis and Mycetophylax simplex harbour 2n=30 and 2n=36 chromosomes, respectively. None of the cytotypes of M. morschi showed any AT-positive blocks, whereas the karyotypes of M. conformis and M. simplex revealed AT-rich blocks around the pericentromeric region and on the short arm of several chromosomes. This AT-rich pattern is coincident with the known heterochromatin distribution of psammophilous Mycetophylax, confirming that heterochromatin is AT-rich, in line with the genome size and AT%. Our results demonstrated that genome size among psammophilous Mycetophylax is correlated with the proportion of base pairs, biased to adenine and thymine. Thus, genome size and the proportion of adenine and thymine in the species studied here suggest that the genome changes in psammophilous Mycetophylax are related to the expansion of repetitive DNA in AT-rich heterochromatin. Considering the phylogenetic relationship of psammophilous Mycetophylax, the dynamic development of AT-rich heterochromatin and karyotype repatterning encompasses the diversification of such ants.

Comparative molecular cytogenetic characterization of five wild Vigna species (Fabaceae).

To extend our knowledge on karyotype variation of the genus Vigna Savi, 1824, the chromosomal organization of rRNA genes and fluorochrome banding patterns of five wild Vigna species were studied. Sequential combined PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindole) (CPD) staining and fluorescence in situ hybridization (FISH) with 5S and 45S rDNA probes were used to analyze the karyotypes of V. luteola (Jacquin, 1771) Bentham, 1959, V. vexillata (Linnaeus, 1753) A. Richard, 1845, V. minima (Roxburgh, 1832) Ohwi & H. Ohashi, 1969, V. trilobata (Linnaeus, 1753) Verdcourt, 1968, and V. caracalla (Linnaeus, 1753) Verdcourt,1970. For further phylogenetic analysis, genomic in situ hybridization (GISH) with the genomic DNA of V. umbellata (Thunberg, 1794) Ohwi & H.Ohashi, 1969 onto the chromosomes of five wild Vigna species was also performed. Detailed karyotypes were established for the first time using chromosome measurements, fluorochrome bands, and rDNA-FISH signals. All species had chromosome number 2n = 2x = 22, and symmetrical karyotypes that composed of only metacentric or metacentric and submetacentric chromosomes. CPD staining revealed all 45S rDNA sites in the five species analyzed, (peri)centromeric GC-rich heterochromatin in V. luteola, V. trilobata and V. caracalla, interstitial GC-rich and pericentromeric AT-rich heterochromatin in V. caracalla. rDNA-FISH revealed two 5S loci in V. caracalla and one 5S locus in the other four species; one 45S locus in V. luteola and V. caracalla, two 45S loci in V. vexillata and V. trilobata, and five 45S loci in V. minima. The karyotypes of the studied species could be clearly distinguished by the karyotypic parameters, and the patterns of the fluorochrome bands and the rDNA sites, which revealed high interspecific variation among the five species. The V. umbellata genomic DNA probe produced weak signals in all proximal regions of V. luteola and all (peri)centromeric regions of V. trilobata. The combined data demonstrate that distinct genome differentiation has occurred among the five species during evolution. The phylogenetic relationships between the five wild species and related cultivated species of Vigna are discussed based on our present and previous molecular cytogenetic data.

Advancing Eucalyptus Genomics: Cytogenomics Reveals Conservation of Eucalyptus Genomes.

The genus Eucalyptus encloses several species with high ecological and economic value, being the subgenus Symphyomyrtus one of the most important. Species such as E. grandis and E. globulus are well characterized at the molecular level but knowledge regarding genome and chromosome organization is very scarce. Here we characterized and compared the karyotypes of three economically important species, E. grandis, E. globulus, and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta, and E. occidentalis, through an integrative approach including genome size estimation, fluorochrome banding, rDNA FISH, and BAC landing comprising genes involved in lignin biosynthesis. All karyotypes show a high degree of conservation with pericentromeric 35S and 5S rDNA loci in the first and third pairs, respectively. GC-rich heterochromatin was restricted to the 35S rDNA locus while the AT-rich heterochromatin pattern was species-specific. The slight differences in karyotype formulas and distribution of AT-rich heterochromatin, along with genome sizes estimations, support the idea of Eucalyptus genome evolution by local expansions of heterochromatin clusters. The unusual co-localization of both rDNA with AT-rich heterochromatin was attributed mainly to the presence of silent transposable elements in those loci. The cinnamoyl CoA reductase gene (CCR1) previously assessed to linkage group 10 (LG10) was clearly localized distally at the long arm of chromosome 9 establishing an unexpected correlation between the cytogenetic chromosome 9 and the LG10. Our work is novel and contributes to the understanding of Eucalyptus genome organization which is essential to develop successful advanced breeding strategies for this genus.

Differential heterochromatin distribution, flow cytometric genome size and meiotic behavior of chromosomes in three Cucurbitaceae species

Basic cytogenetic investigation of economically important members of Cucurbitaceae is a prerequisite to understand their genetic architecture. The study reports detailed chromosome morphometric analyses with fluorescence banding technique, differential distribution of heterochromatin, flow cytometric nuclear DNA contents and meiotic behavior of the native populations of Benincasa hispida, Luffa cylindrica and Trichosanthes dioica in India. In spite of frequent vegetative propagation, different populations of these cultivated cucurbits show regular genetic processes evident from stable chromosome counts, nuclear DNA sizes, ploidy levels and regular meiotic behavior. Distal CMA+ve signals have been scored in two pairs of chromosomes in B. hispida and four pairs of chromosomes in L. cylindrica, representing the GC rich heterochromatic portions in genomes. The presence of terminal DAPI+ve bands in the chromosomes of male and female populations of T. dioica indicates the predominance of AT rich heterochromatin. In addition to DAPI bands, terminal CMA+ve bands in the female plants of T. dioica depict the chromosomal signs of sexual differentiation in this species. The existence of rod bivalent in meiosis of male plants facilitates further approach to characterize the genetics of chromosomal evolution since gross similarity in genome size and karyotypic features of male and female plants advocates genic control of sexual dimorphism in T. dioica.

Breakdown of the Balanced Lethals in Rhoeo: The Structure of the Alethal Renner Complex of the Homozygotic Stock of Rhoeo

Fluorescence in situ hybridization, base-specific fluorescence, C-banding and silver-staining were performed to reveal the cyto-molecular constitution of the karyotype in the bivalent-forming Rhoeo spathacea concolor. It was shown that the genome of this form is almost identical to the β-complex of the ring-forming rhoeos. In spite of some modifications in the arrangement of a few distal rDNA sites and in the amount of pericentromeric AT-rich heterochromatin, the alethal genome of the bivalent-forming Rhoeo seems segmentally unaltered. Thus, the breakdown of balanced lethals in Rhoeo is most likely uncoupled from creating new chromosome arm combinations.

Cytogenetics of Amaryllidaceae species: heterochromatin evolution in different ploidy levels

Species belonging to the Amaryllidaceae (Zephyranthes and Habranthus) were analyzed by banding with chromomycin A3 (CMA)/4,6-diamidino-2-phenylindole (DAPI) fluorochromes. The patterns of bands were studied in seven species of Zephyranthes Herb. and one of Habranthus Herb. Subterminal and interstitial DAPI+ bands were observed in Z. robusta 2n = 12 and Z. brachyandra 2n = 24. Other species showed no AT-rich heterochromatin. In species with 2n = 12, CMA+ bands were observed on one chromosome pair of Z. robusta and Zephyranthes sp., while in Z. sylvatica an additional small terminal band in the fifth chromosome pair was observed. Z. rosea and Z. grandiflora presented with 2n = 24 and had four CMA+ bands, while in Z. brachyandra, with 2n = 24 + 1B, there were eight interstitial dot bands and a larger terminal band in the short arm of the B chromosome. Z. candida with 2n = 38 presented CMA+ heterochromatin blocks on the long arms of five metacentric pairs and in the short arm of one of the submetacentric pairs; in addition a terminal band was observed on the long arm of one of the homologues of a larger submetacentric pair. H. itaobinus showed a heterozygous pair revealing a strong CMA+ band in only one of the homologues, likely a nucleolus organizing region. Taxonomic implications and karyotype evolution of this group are discussed and correlated with previous data from the literature.

Karyotype Conservation in 2 Populations of the Parthenogenetic Scorpion Tityus serrulatus (Buthidae): rDNA and Its Associated Heterochromatin Are Concentrated on Only One Chromosome

Within the order Scorpiones, the parthenogenetic mode of reproduction has been described for 11 species, 6 of which belong to the genus Tityus. In this work, an investigation of the chromosome characteristics of 2 populations of Tityus serrulatus, the first scorpion species known to be thelytokously parthenogenetic, is described. An analysis of 40 individuals revealed holocentric chromosomes of large, medium, and small sizes and an invariable diploid number of 2n = 12. In addition to the conserved macrokaryotype structure, specific chromosome regions also appeared unchanged within and between the samples studied; that is, each sample displayed only one chromosome carrier of the active nucleolar organizer region containing ribosomal genes (5.8S, 18S, and 28S) and AT-rich heterochromatin associated with the ribosomal DNA. The high conservation of the chromosomal features observed in T. serrulatus differed from that verified in certain species of other groups of animals that possess both holocentric chromosomes and parthenogenetic reproduction. Moreover, the cytogenetic results obtained herein permit us to suggest how the eggs of T. serrulatus develop, whether by apomixis or automixis.

The meaning of DAPI bands observed after C-banding and FISH procedures

Under specific technical conditions chromosome staining with 4′,6-diamidino-2-phenylindole (DAPI) permits characterization of heterochromatic regions as AT-rich (DAPI+) or AT-poor (DAPI−), especially when the chromosomes are counterstained with chromomycin A3 (CMA), which preferentially binds to GC-rich DNA. DAPI+ bands also often have been observed after C-banding or FISH. In these cases, however, it is not clear whether only AT-rich regions stain positively with DAPI or other heterochromatins with different base compositions also are stained. We evaluated the meaning of DAPI bands observed after C-banding and FISH using three plant species bearing different types of heterochromatin: DAPI+/CMA−, DAP−/CMA+ and DAPI0/CMA0 (neutral bands). Additional tests were performed using propidium iodide, a fluorochrome without preferential affinity for AT or GC. Our results indicate that AT-rich heterochromatin stains as DAPI+ bands after C-banding or FISH, but other kinds of heterochromatin also may be stained by DAPI.

Cytogenetic studies inHerbertiaSw. (Iridaceae)

Fil: Moreno, Natalia Cecilia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto Multidisciplinario de Biologia Vegetal. Universidad Nacional de Cordoba. Facultad de Ciencias Exactas Fisicas y Naturales. Instituto Multidisciplinario de Biologia Vegetal; Argentina

The meaning of DAPI bands observed after C-banding and FISH procedures

Under specific technical conditions chromosome staining with 4',6-diamidino-2-phenylindole (DAPI) permits characterization of heterochromatic regions as AT-rich (DAPI(+)) or AT-poor (DAPI(-)), especially when the chromosomes are counterstained with chromomycin A(3) (CMA), which preferentially binds to GC-rich DNA. DAPI(+) bands also often have been observed after C-banding or FISH. In these cases, however, it is not clear whether only AT-rich regions stain positively with DAPI or other heterochromatins with different base compositions also are stained. We evaluated the meaning of DAPI bands observed after C-banding and FISH using three plant species bearing different types of heterochromatin: DAPI(+)/CMA(-), DAP(-)/CMA(+) and DAPI(0)/CMA(0) (neutral bands). Additional tests were performed using propidium iodide, a fluorochrome without preferential affinity for AT or GC. Our results indicate that AT-rich heterochromatin stains as DAPI(+) bands after C-banding or FISH, but other kinds of heterochromatin also may be stained by DAPI.

Molecular and cytogenetic characterization of an AT-rich satellite DNA family in Urvillea chacoensis Hunz. (Paullinieae, Sapindaceae)

Urvillea chacoensis is a climber with 2n = 22 and some terminal AT-rich heterochromatin blocks that differentiate it from other species of the genus. The AT-rich highly repeated satellite DNA was isolated from U. chacoensis by the digestion of total nuclear DNA with HindIII and XbaI and cloned in Escherichia coli. Satellite DNA structure and chromosomal distribution were investigated. DNA sequencing revealed that the repeat length of satDNA ranges between 721 and 728 bp, the percentage of AT-base pairs was about 72-73% and the studied clones showed an identity of 92.5-95.9%. Although this monomer has a tetranucleosomal size, direct imperfect repetitions of ~180 bp subdividing it in four nucleosomal subregions were observed. The results obtained with FISH indicate that this monomer usually appears distributed in the terminal regions of most chromosomes and is associated to heterochromatin blocks observed after DAPI staining. These observations are discussed in relation to the satellite DNA evolution and compared with other features observed in several plant groups.

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). III: Extensive karyotypic variability associated with low mitochondrial haplotype differentiation in C. bivittatum

We investigated chromosomal evolution in the African killifish species Chromaphyosemion bivittatum using a combination of cytogenetic and phylogenetic methods. Specimens from five populations were examined by conventional Giemsa staining as well as sequential chromosome banding with 4′,6-diamidino-2-phenylindole (DAPI), chromomycin A₃ (CMA₃), AgNO₃-staining and C-banding. The cytogenetic analysis revealed variability in 2n ranging from 2n = 29 to 2n = 36 and in NF ranging from NF = 38 to NF = 44. Two populations showed an extensive chromosomal polymorphism (2n = 29–34, NF = 44 and 2n = 32–34, NF = 38–42, respectively). Karyotypic variability within and among populations was mainly due to Robertsonian translocations and heterochromatin additions, and chromosome banding patterns suggested that both types of chromosomal rearrangements were related to the presence of AT-rich heterochromatin. A phylogenetic analysis of the partial mitochondrial (mt) cytochrome b gene, using specimens from eleven populations, revealed a low degree of haplotype differentiation, which suggested a relatively recent divergence of the populations examined. This finding conformed to the low degree of morphological differentiation observed among C. bivittatum populations and might indicate fast chromosomal evolution. The high karyotypic variability may be caused by an elevated chromosomal mutation rate as well as certain aspects of the mating system and population dynamics of C. bivittatum facilitating the fixation of new chromosomal variants.

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

Karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S-5.8S-26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2n = 2x = 16, common to all investigated populations consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 m ; ; m. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S-5.8S-26S rDNA loci on satellite and secondary constriction (SC) of acrocentric chromosome pair 2 and terminally on accrocentric chromosome pair 3, and two 5S rDNA loci in pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity to 18S-5.8S-26S rDNA locus on chromosome pair 2. The only GC rich heterochromatin, as revealed by fluorochrome Chromomycin A3 (CMA) staining, was that associated with nucleolar organizer regions (NORs), while AT rich heterochromatin, stained with 4, 6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis-type of telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI stained heterochromatin. Heteromorphism of chromosome pair 4 which refers to terminal DAPI bands and FISH signals was observed in populations of A. hortensis. Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.

Molecular cytogenetics of Oligosarcus hepsetus (Teleostei, Characiformes) from two Brazilian locations

Karyotype and cytogenetic markers of Oligosarcus hepsetus from two Brazilian locations in the Paraiba do Sul River Basin (Brazil) were investigated using differential staining techniques (C-banding, silver (Ag)- and chromomycin A3 (CMA3)-staining) and fluorescent in situ hybridization (FISH) using 18 S rDNA and 5S rDNA probes. The diploid chromosome number was invariably 2n = 50 with 3 pairs of metacentric, 5 pairs of submetacentric, 8 pairs of subtelocentric and 9 pairs of acrocentric chromosomes. No heteromorphic sex chromosomes were observed. The nucleolar organizer regions (NORs) were detected in the short arms of the largest acrocentric pair using Ag-, CMA3- stainings and FISH with 18S rDNA probe, the latter showing also positive labeling in the short arms of a small acrocentric pair, not visualized by the former methods. FISH with 5S rDNA probe showed positive labeling in the two chromosome pairs. While the CMA3-staining exhibited GC-rich heterochromatin segments in two pairs of chromosomes, including those coincided with Ag-NORs, the DAPI staining did not reveal any signal, indicating the absence of AT-rich heterochromatin. FISH with an As-51 satellite DNA probe derived from the closely related Astyanax scabripinnis did not reveal any positive signal, demonstrating the absence of this class of DNA in the genome of the specimens under study.

Three-dimensional localization and dynamics of centromeres in mouse oocytes during folliculogenesis

Very little is known about oocyte nuclear architecture during folliculogenesis. Using antibodies to reveal centromeres, Hoechst-staining to detect the AT-rich pericentromeric heterochromatin (chromocenters), combined with confocal microscopy for the three-dimensional analysis of the nucleus, we demonstrate that during mouse folliculogenesis the oocyte nuclear architecture undergoes dynamic changes. In oocytes isolated from primordial and primary follicles, centromeres and chromocenters were preferentially located at the periphery of the nucleus. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus and then progressively clustered around the periphery of the nucleolus. Our results indicate that the oocyte nuclear achitecture is developmentally regulated and they contribute to a further understanding of the role of nuclear organization in the regulation of genome functioning during differentiation and development.

Chromosome Maps of Trilliaceae: II. A Study of the Genome Composition in Polyploid Species of the Genus Trillium by Fluorescence Nucleotide Base-Specific Staining of Heterochromatic Chromosome Regions

Chromosome banding with nucleotide base-specific fluorochromes chromomycin A3 (CMA) and Hoechst 33258 (H33258) was used to study the karyotypes and to construct cytological maps for diploid Trillium camschatcense (2n = 10), tetraploid T. tschonoskii (2n = 20), hexaploid T. rhombifolium (2n = 30), and a triploid T. camschatcense x T. tschonoskii hybrid (T. x hagae, 2n = 15). With H33258, species- and genome-specific patterns with numerous AT-rich heterochromatin bands were obtained for each of the four forms; CMA revealed a few small, mostly telomeric GC-rich bands. In T. tschonoskii, the two subgenomes were similar to each other and differed from the T. camschatcense genome; on this evidence, the species was considered to be a segmental allotetraploid. In T. x hagae, one T. camschatcense and both T. tschonoskii subgenomes were identified. The subgenomes of T. rhombifolium only partly corresponded to the T. camschatcense and T. tschonoskii genomes, in contrast to the morphologically identical Japanese species T. hagae. This was assumed to indicate that allohexaploids T. rhombifolium and T. hagae originated independently at different times; i.e., their origin is polyphyletic. Based on the chromosome maps, a new nomenclature was proposed for the Trillium genomes examined: K1K1 for T. camschatcense, T1T1T2T2 for T. tschonoskii, T1T1T2T2 for T. x hagae, and K1RK1RT1RT1RT2RT2R for T. rhombifolium.

Genome size, heterochromatin organisation, and ribosomal gene mapping in four species of Ribes

Four wild Ribes species (Ribes alpinum L., Ribes petraeum Wulf., Ribes rubrum L., and Ribes uva- crispa L.; all 2n = 2x = 16) were surveyed for their chromosome and genome organisation. Their genome size was assessed using flow cytometry. Ribes alpinum had 5.3% more nuclear DNA than did the three other species, whose average was 2C = 1.91 pg with 40.4% GC. In addition, GC- and AT-rich heterochromatin and rDNA (18S–5.8S–26S and 5S) patterns were studied using fluorochrome banding and double-target fluorescence in situ hybridization (FISH), respectively. Only GC-rich heterochromatin was detected, co-localizing with 18S–26S rDNA. Fluorochrome banding and FISH patterns revealed marked differences between species. Ribes alpinum and R. uva-crispa differed from R. rubrum and R. petraeum by the number of 18S–26S sites and the localization of 5S rDNA. Ribes alpinum and R. uva-crispa were differentiated by the number of 5S sites. Ribes rubrum and R. petraeum also differed by the number of 5S sites and by the size of the GC-rich band on the satellite chromosome pair. These results should contribute to a better understanding of phylogenetic relationships among these species.Key words: Ribes, flow cytometry, fluorochrome banding, FISH, rDNA, NORs.

Modification of position-effect variegation by competition for binding to Drosophila satellites.

White-mottled (w(m4)) position-effect variegation (PEV) arises by translocation of the white gene near the pericentric AT-rich 1.688 g/cm3 satellite III (SATIII) repeats of the X chromosome of Drosophila. The natural and artificial A*T-hook proteins D1 and MATH20 modify w(m4) PEV in opposite ways. D1 binds SATIII repeats and enhances PEV, presumably via a recruitment of protein partners, whereas MATH20 suppresses it. We show that D1 and MATH20 compete for binding to identical sites of SATIII repeats in vitro and that conditional MATH20 expression results in a displacement of D1 from pericentric heterochromatin in vivo. In the presence of intermediate levels of MATH20, we show that this displacement becomes selective for SATIII repeats. These results strongly suggest that the suppression of w(m4) PEV by MATH20 is due to a displacement of D1 from its preferred binding sites and provide additional support for a direct role of D1 in the assembly of AT-rich heterochromatin.

Dynamic of nucleolus organizer regions and karyotype evolution in Indian pygmy field mice

Ag-NOR staining and fluorescence in situ hybridization with rDNA probes showed an unusually high number of NORs in the Indian pygmy field mice, Mus booduga and the M. terricolor complex. The chromosomal location of the NORs was also altered in terricolor, they were shifted from the proximal regions of the long arms to the tips of the perceptible heterochromatic short arms of the acrocentric autosomes. The results suggested dispersion of the NORs in the booduga-terricolor lineage probably by transposition, and relocalization of the NORs in the terricolor complex by centric reorganization during the process of replacement of the Mus musculus-related AT-rich heterochromatin with the terricolor-specific heterochromatin.