Additional Heterochromatin Research Articles

Increased genome size is caused by heterochromatin addition in two non-related bat species, Hesperoptenus doriae and Philetor brachypterus (Vespertilionidae, Chiroptera, Mammalia).

The average genome size (GS) of bats, which are the only mammals capable of powered flight, is approximately 18% smaller than that of closely related mammalian orders. The low nuclear DNA content of Chiroptera is comparable to that of birds, which are also characterized by a high metabolic rate. Only a few chiropteran taxa possess notable amounts of constitutive heterochromatin. Here, we studied the karyotypes of two non-related vesper bat species with unusually high amounts of constitutive heterochromatin: Hesperoptenus doriae and Philetor brachypterus. Conventional staining methods and whole-chromosome painting with probes derived from Myotismyotis (2n = 44), showing a karyotype close to that of the presumed ancestor of Vespertilionidae, revealed Robertsonian fusions as the main type of rearrangement leading to the exceptionally reduced diploid chromosome number of 2n = 26 in both species. Moreover, both karyotypes are characterized by large blocks of pericentromeric heterochromatin composed of CMA-positive and DA-DAPI-positive segments. In H. doriae, the heterochromatin accumulation has resulted in a genome size of 3.22pg (1C), which is 40% greater than the mean genome size for the family. For P. brachypterus, a genome size of 2.94pg was determined, representing an increase of about 28%. Most notably, in H. doriae, the presence of additional constitutive heterochromatin correlates with an extended mitotic cell cycle duration in vitro. A reduction in diploid chromosome number to 30 or lower is discussed as a possible cause of the accumulation of pericentromeric heterochromatin in Vespertilionidae.

The kariotype variability in children with Down syndrome from the Odesa region

The aim of the work is to analyze the frequency of cytogenetic variants of Down syndrome among patients in Odesa and the region, as well as to identify combined karyotype anomalies. Materials and methods. Studies were conducted between 2013–2018 years in Odesa Specialized Medical Genetic Center. The experimental group was formed of patients with cytogenetically confirmed Down syndrome. Chromosomes were painted according to GTG method and identified according to ISCN 2013. Results. Among patients with Down syndrome, in 93.9 % of cases complete trisomy 21 was observed, the translocation form was in 3.7 %, and the mosaic form was in 2.4 %. Similar results were revealed in the analysis of populations belonging to different ethnic and racial groups. Complete trisomy 21 was accompanied by chromosome rearrangements of other chromosomes or additional modifications of chromosome 21. Changes in the heterochromatin in chromosome 9 were more frequently observed. In total, 5.5 % of examined karyotypes were found with additional heterochromatin in both arms of chromosome 1 and in the long arm of chromosome 21. An increase in the size of satellites in chromosomes 14, 15 and more often 21, as well as the appearance of additional satellites in chromosome 2 represented 3.6 % of the total examined karyotypes. A deletion on chromosome 6 involved in translocation with chromosome 13 also was found. Translocation forms included Robertsonian translocations involving chromosomes 21 and 21, 14 and 21, as well as translocations involving chromosomes 21 and 21, 21 and 22. Patients with a mosaic form of the disease had two cell lines: with a normal karyotype 3 (15–67 % of the studied cells) and with complete trisomy 21 without additional chromosomal abnormalities (33–85 % of the studied cells). Conclusions. Among patients with cytogenetically confirmed diagnosis of Down syndrome, the ratio of the main variants was similar to many populations studied. At the same time, additional changes in the karyotype were identified which can either be a variant of the norm or aggravate the course of the disease. This requires further studies of the disease course in such patients.

Karyotypes of Dremomys pernyi and D. pyrrhomerus (Rodentia: Sciuridae) from China

Abstract. Karyotypes of Dremomys pernyi from Kanding, Sichuan Province, China and D. pyrrhomerus from Nanling, Guangdong Province, China were examined. The karyotype of D. pernyi was of 2n = 40 and FNa = 72, while that of D. pyrrhomerus was of 2n = 38 and FNa = 70. Karyotype of D. pernyi from Kanding was different in 2n and FNa from reported karyotype of conspecific population from Taiwan, and it is suggested that the Taiwan population represents a distinct species D. owstoni from D. pernyi. The difference between karyotypes of D. pernyi and D. pyrrhomerus may involve at least a Robertsonian rearrangement and a heterochromatin addition.

Analysis of the flanking sequences of the heterochromatic JNK region in Secale vavilovii chromosomes

The JNK motif is repeated 4 000 times in an additional heterochromatin band. Flanking sequences of a JNK region, which form extra heterochromatin in 2R rye (Secale vavilovii Grossh.) chromosomes, were studied using a genome walking technique. The results clearly indicate that there were blocks of JNK sequences adjacent to the R173 family of repeated sequences. Moreover, it appears that the R173 are sequences flanking in both directions, i.e., upstream and downstream. Downstream, the R173 is adjacent to the JNKs in an anti-parallel orientation, whereas upstream it is adjacent in a parallel orientation. In order to confirm the presence of the R173 sequence, fluorescence in situ hybridization was carried out. Using both JNK and R173 molecular probes, overlapping hybridization signals in the 2RL pair of chromosomes were observed, indicating an identical location of the two sequence elements.

Similarity of heterochromatic regions in the stingless bees (Hymenoptera: Meliponini) revealed by chromosome painting

Most Meliponini share a distinctive pattern of heterochromatin distribution in relation to other bees. In general, they present one euchromatic and one heterochromatic chromosome arm, a feature explained by minimum interaction theory, which involved centric fissions followed by heterochromatin addition. In this work, two Meliponini with a distinct pattern of heterochromatin distribution, Tetragonisca fiebrigi and Melipona rufiventris, were analyzed using chromosomal microdissection of the heterochromatin region followed by FISH (fluorescent in situ hybridization). Hybridization revealed FISH signals equivalent to location of the isolated fragment that were widespread over heterochromatic portions of other chromosomes. This result showed that the heterochromatic sequences were very similar among chromosomes in the same species. Cross-hybridization of each probe in M. rufiventris and T. fiebrigi yielded no signals, revealing that both species presented differentiated and non-homologous heterochromatin seque...

Karyotypic evolution in family Hipposideridae (Chiroptera, Mammalia) revealed by comparative chromosome painting, G- and C-banding.

Comparing to its sister-family (Rhinolophidae), Hipposideridae was less studied by cytogenetic approaches. Only a few high-resolution G-banded karyotypes have been reported so far, and most of the conclusions on the karyotypic evolution in Hipposideridae were based on conventional Giemsa-staining. In this study, we applied comparative chromosome painting, a method of choice for genome-wide comparison at the molecular level, and G- and C-banding to establish comparative map between five hipposiderid species from China, using a whole set of chromosome-specific painting probes from one of them (Aselliscus stoliczkanus). G-band and C-band comparisons between homologous segments defined by chromosome painting revealed that Robertsonian translocations, paracentric inversions and heterochromatin addition could be the main mechanism of chromosome evolution in Hipposideridae. Comparative analysis of the conserved chromosomal segments among five hipposiderid species and outgroup species suggests that bi-armed chromosomes should be included into the ancestral karyotype of Hipposideridae, which was previously believed to be exclusively composed of acrocentric chromosomes.

Possible ancient origin of heterochromatic JNK sequences in chromosomes 2R ofSecale vavilovii Grossh

Employing FISH analysis as well as BLAST and CUSTAL W (1.82) programs, we investigated types of DNA nucleotide sequences building an additional heterochromatic band in 2R chromosomes of 3 lines of Secale vavilovii Grossh. The probes used in FISH analysis were designed based on the reverse transcriptase sequence of Ty1-copia and Ty3-gypsy retrotransposons and the 5S rRNA gene sequence. No hybridization signals from the reverse transcriptase probes were observed in the chromosome region where the additional band occurs. On the other hand, signals were observed after hybridization with the 5S rDNA probe, clearly suggesting the presence of that type of sequences in the analyzed heterochromatin band. Using BLAST and CUSTAL W programs, we revealed high similarity of the JNK1 sequence to the 5S rRNA gene from Hordeum chilense (HCH1016, HCH1018, 88%) and to a fragment of the 5S rRNA sequence of H. marinum (HMAR003, 97%). In addition, the same fragment of JNK1 was shown to be very similar to the part of the Angela retrotransposon (92%) as well as to the SNAC 426K20-1 transposon (89%) belonging to CACTA family, both from Triticum monococcum, and to Zingeria biebersteiniana pericentromeric sequences (78%). The similarity of JNK1 to those sequences may be accidental or the JNK1 may represent an ancient mobile genetic element that caught the 5S rRNA sequence. During the evolution those sequences might have been accumulated in the particular region on the 2R chromosome. Our results suggest that the additional heterochromatin band in chromosomes 2R of S. vavilovii is a collection of defective genes and/or mobile genetic elements.

Cytogenetic analysis of two related Deltochilum (Coleoptera, Scarabaeidae) species: Diploid number reduction, extensive heterochromatin addition and differentiation

Male mitotic and meiotic chromosomes of two species of the genus Deltochilum (Scarabaeidae) were analyzed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining (AgNO 3) and FISH (45S rDNA). The two species possessed karyotypes with 2 n = 14, neo-XY and meta-submetacentric chromosomes. The analysis of constitutive heterochromatin (CH) revealed mainly diphasic chromosomes in the two species, showing heterochromatic long arms. Silver nitrate staining labeled the blocks corresponding to CH in D. ( Deltohyboma) aff morbillosum while in D. ( Deltohyboma) calcaratum, AgNO 3 staining revealed only the CH blocks of the diphasic autosomes. The fluorochrome staining revealed in D. ( D.) calcaratum the diphasic autosomes and the sex chromosomes with CMA 3 + blocks, and in D. ( D.) aff morbillosum, the GC-rich sequences were restricted to the terminal regions of the long arms of the pairs 1 and 2 and the X. The FISH revealed 45S rDNA sites in two autosomic pairs and in the X chromosome. The analyses performed allowed for the identification of cytogenetic markers and the discussion of possible chromosome rearrangements that have been involved in the karyotypic differentiation of these species mainly related to the repetitive genome.

Sex chromosome variability outlines the pathway to the chromosomal evolution in Microtus thomasi (Rodentia, Arvicolinae)

The underground vole Microtus thomasi, a Balkan endemic, displays remarkable variability in sex chromosome size and morphology. In the present study, we demonstrate this variability in two of its chromosomal races with 2n = 44 (i.e. ‘thomasi’ and ‘atticus’) with the use of C-banding on a sample of 189 individuals from 50 localities of Greece. In ‘thomasi’, five different, acrocentric X chromosome variants (X0–X4) are described, which differ significantly in size, due to heterochromatin addition. Also, three Y chromosome variants are described (Y0–Y2), ranging in size from very small (Y0) to large (Y2). The ‘atticus’ race displays three subtelocentric variants of the X chromosome (Xst0–Xst2), which differ in arm length ratio and heterochromatin content. In Peloponnesus, males of this race exhibit Y0 and Y1, whereas, in Attiki (south-east Sterea Ellada), males carry the small metacentric, Ym. Overall, there is a trend towards sex chromosome size increase in a south to north direction. We propose that the last glaciation must have restricted M. thomasi to a refugium in southern Peloponnesus. During post-glacial colonization, limited northward expansion of its distribution area must have been accompanied by consecutive heterochromatin addition, which is proven today by comparatively larger sex chromosomes in the northern populations of ‘thomasi’ and ‘atticus’ in Greece than in their southern populations. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 685–695.

An Investigation of Heterochromatin Domains on the Fourth Chromosome ofDrosophila melanogaster

The banded portion of Drosophila melanogaster chromosome 4 exhibits euchromatic and heterochromatic characteristics. Reminiscent of heterochromatin, it contains a high percentage of repetitive elements, does not undergo recombination, and exhibits high levels of HP1 and histone-3 lysine-9 dimethylation. However, in the distal 1.2 Mb, the gene density is typical of euchromatin, and this region is polytene in salivary gland nuclei. Using P-element reporters carrying a copy of hsp70-white, alternative chromatin packaging domains can be distinguished by the eye color phenotype. Mapping studies identified the repetitive element 1360 as a candidate for heterochromatin targeting in the fourth chromosome Hcf region. We report here two new screens using this reporter to look for additional heterochromatin target sites. We confirm that reporter elements within 10 kb of 1360 are usually packaged as heterochromatin; however, heterochromatin packaging occurs in the sv region in the absence of 1360. Analyses of the sequences adjacent to P-element reporters show no simple association between specific repeated elements and transgene expression phenotype on a whole chromosome level. The data require that heterochromatin formation as a whole depends on a more complex pattern of sequence organization rather than the presence of a single sequence element.

Chromosome number reduction accompanied by extensive heterochromatin addition in the bat Glauconycteris beatrix (Mammalia; Chiroptera, Vespertilionidae)

The presumed ancestral karyotype of the bat family Vespertilionidae consists of 44 chromosomes with a fundamental number of autosomal arms (FNa) of 50. Previously, only two of the roughly 350 vespertilionid species have been reported with 2n lower than 26. In this paper we report the 2n = 22 karyotype of the African vespertilionid Glauconycteris beatrix which shows an X-autosome translocation and extended, paracentromeric, chromomycin-A3-positive heterochromatin.

The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization.

The endosperm is a seed tissue unique to flowering plants. Due to its central role in nourishing and protecting the embryo, endosperm development is subject to parental conflicts and adaptive processes, which led to the evolution of parent-of-origin-dependent gene regulation. The role of higher-order chromatin organization in regulating the endosperm genome was long ignored due to technical hindrance. We developed a combination of approaches to analyze nuclear structure and chromatin organization in Arabidopsis thaliana endosperm. Endosperm nuclei showed a less condensed chromatin than other types of nuclei and a peculiar heterochromatin organization, with smaller chromocenters and additional heterochromatic foci interspersed in euchromatin. This is accompanied by a redistribution of the heterochromatin mark H3K9me1 from chromocenters toward euchromatin and interspersed heterochromatin. Thus, endosperm nuclei have a specific nuclear architecture and organization, which we interpret as a relaxed chromocenter-loop model. The analysis of endosperm with altered parental genome dosage indicated that the additional heterochromatin may be predominantly of maternal origin, suggesting differential regulation of maternal and paternal genomes, possibly linked to genome dosage regulation.

Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome

Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells. To investigate the relationship among centromeric, transcriptionally permissive and non-permissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by beta geo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1alpha and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.

Chromosomal diversification of reef fishes from genus Centropyge(Perciformes, Pomacanthidae)

The genus Centropyge is remarkable for species richness, composing a highly specialized fish group amongst members from family Pomacanthidae. However, cytogenetical reports are nearly absent in these animals. New data are provided from karyotypical studies carried out on Centropyge aurantonotus from the Brazilian coast of the Atlantic Ocean and C. ferrugatus from the Philippines Sea of the Indo-Pacific Ocean. Both species present 2n = 48 but karyotypes are differentiated by fundamental number. C. aurantonotus has a great number of biarmed chromosomes (4 m + 14 sm + 16 st + 4 a), while C. ferrugatus presents only acrocentric chromosomes. Single nucleolar organizer regions (NORs) are located at interstitial position of an acrocentric pair in C. ferrugatus and on short arms of a subtelocentric pair in C. aurantonotus, as confirmed by fluorescent in situ hybridization (FISH) with 18S rDNA probes. Heterochromatin is distributed over NOR and centromeric regions in both species, but additional GC-rich heterochromatic blocks on short arms of up to eight chromosomal pairs can be detected in C. aurantonotus. 5S rDNA segments were located interstitially on two chromosomal pairs in C. ferrugatus and on nine pairs in C. aurantonotus, mostly equivalent to heterochromatic blocks on short arms of biarmed chromosomes. C. ferrugatus can be considered a species in which basal chromosomal features proposed for modern Teleosteans were conserved. The derived karyotype pattern of C. aurantonotus seems to be determined by pericentric inversions and heterochromatin addition which probably determined the notorious dispersion of 5S rRNA (pseudo)genes. It is demonstrated that, even within a group generally characterized by cytogenetical homogeneity as the family Pomacanthidae, diversified karyotypes can be found.

Molecular Characterization of the Pericentric Inversion That Causes Differences Between Chimpanzee Chromosome 19 and Human Chromosome 17

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.

A reexamination of spreading of position-effect variegation in the white-roughest region of Drosophila melanogaster.

In Drosophila, heterochromatin causes mosaic silencing of euchromatic genes brought next to it by chromosomal rearrangements. Silencing has been observed to "spread": genes closer to the heterochromatic rearrangement breakpoint are silenced more frequently than genes farther away. We have examined silencing of the white and roughest genes in the variegating rearrangements In(1)w(m4), In(1)w(mMc), and In(1)w(m51b). Eleven stocks bearing these chromosomes differ widely in the strength of silencing of white and roughest. Stock-specific differences in the relative frequencies of inactivation of white and roughest were found that map to the white-roughest region or the adjacent heterochromatin. Most stock-specific differences did not correlate with gross differences in the heterochromatic content of the rearranged chromosomes; however, two stocks, In(1)w(m51b) and In(1)w(mMc), were found to have anomalous additional heterochromatin that may act in trans to suppress variegating alleles. In comparing different stocks, the frequency of silencing of the roughest gene, which is more distant from heterochromatin, does not correlate with the frequency of silencing of the more proximal white gene on the same chromosome, in contradiction to the expectation of models of continuous linear propagation of silencing. We frequently observed rough eye tissue that is pigmented, as though an active white gene is skipped.

Geographical distribution and interpopulation variation in the karyotypes ofMicrotus (Terricola) thomasi(Rodentia, Arvicolidae) in Greece

SUMMARYThe karyological analysis of fifty-five voles collected in different localities of Greece showed that they belong to the following cytotypes of Microtus (Terricola) thomasi: «thomasi» 2n = 44; FN = 44, «atticus» 2n = 44; FN = 46, «subalpine» 2n = 42; FN = 42 and «Rb-subalpine» 2n = 40; FN = 42. A comparative study of the G-banding patterns among them showed that the first two, differing in a pericentric inversion, both represent the type species M. (T.) thomasi. Their distribution is intermingled and extends from central Greece to Peloponissos. The «subalpine» cytotypes distributed in mountainous central Greece, in Mt. South Pindos and in lowland of Ipiros, differ by a Robertsonian centric fusion. They display moreover a tandem fusion, a pericentric inversion and addition of heterochromatin which differentiate them to a greater extend from the type species and may consequently be consider to belong to another taxon of «thomasi» complex. The relatioships among these cytotypes is also descussed.

The effect of heterochromatin on synapsis of the sex chromosomes of <i>Peromyscus </i>(Rodentia, Cricetidae)

The pairing behavior of the sex chromosomes in male and female individuals representing seven species of Peromyscus was analyzed by electron microscopy of silver-stained zygotene and pachytene configurations. Six species possess submetacentric or metacentric X chromosomes with heterochromatic short arms. Sex-chromosome pairing in these species is initiated during early pachynema at an interstitial position on the X and Y axes. Homologous synapsis then progresses in a unidirectional fashion towards the telomeres of the X short arm and the corresponding arm of the heterochromatic Y chromosome. The distinctive pattern of synaptic initiation allowed a late-synapsing bivalent in fetal oocytes to be tentatively identified as that of the X chromosomes. In contrast to the other species, Peromyscus megalops possesses an acrocentric X chromosome and a very small Y chromosome. Sex-chromosome pairing in this species is initiated at the proximal telomeric region during late zygonema, and then proceeds interstitially towards the distal end of the Y chromosome. These observations suggest that the presence of X short-arm heterochromatin and corresponding Y heterochromatin interferes with late-zygotene alignment of the pairing initiation sites, thereby delaying XY synaptic initiation until early pachynema. The pairing initiation sites are conserved in the vicinity of the X and Y centromeres in Peromyscus, and consequently the addition of heterochromatin during sex-chromosome evolution essentially displaces these sites to an interstitial position.

Chromosomal evolution inCallithrix emiliae

We studied the karyotype of specimens of Callithrix emiliae (Callithricidae, Primates) from Rondonia, Brazil. Comparison with the karyotype of Callithrix jacchus showed that, even though these two species show many karyotypic similarities, they differ by a Robertsonian translocation, a paracentric inversion and large-scale addition of heterochromatin. The C. emiliae species appears to be in an active phase of chromosome evolution by the addition of constitutive heterochromatin.

Adding the heterochromatic YL arm to an X chromosome reduces reproductive fitnesses in Drosophila melanogaster: implications for the evolution of rDNA, heterochromatin, and reproductive isolation

For X-Y exchange to be of importance in the coevolution of X and Y rDNA, there must be a mechanism to maintain cytologically normal X chromosomes in the face of continual infusions of X.YL chromosomes produced by X-Y exchanges. Replicated populations were founded with different frequencies of isogenic X and X.YL chromosomes. The X.YL chromosome declined in frequency over time in all lines. Relative fitnesses, estimated from chromosome frequency trajectories, were 0.40, 1.01, and 1.0 for X.YL/X.YL, X.YL/X, and X/X females and 0.75 and 1.0 for X.YL/Y and X/Y males, respectively. The equilibrium frequency for the X.YL chromosome due to the balance between X-Y exchange and selection was predicted to be 4-16 x 10(-4). The results strengthen the evidence for the involvement of X-Y exchange in the coevolution of X and Y rDNA arrays. Conditions for the evolution of reproductive isolation by sex-chromosome translocation are much less probable than previously supposed since the X.YL translocation chromosome is at a selective disadvantage to cytologically normal X chromosomes. Additional heterochromatin was not neutral but was only deleterious beyond a threshold, as one dose of the heterochromatic XL arm did not reduce female reproductive fitness, but two doses did.