Heterochromatic Segments Research Articles

Characterization of diploid, tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe.

Comparative karyotype analyses of five diploid, two tetraploid, and three hexaploid species of Helianthus were performed using Feulgen staining, Giemsa C and CMA3 (C-CMA) staining, and FISH with 45S rDNA probe. The karyotypes are composed by a basic number of x = 17 with a predominance of meta- and submetacentric chromosome types. A polyploid series is associated with the basic number. Giemsa C- and C-CMA banding revealed terminal or interstitial heterochromatin according to the species, suggesting the existence of a mechanism that may be acting in the dispersion of heterochromatic segments in Helianthus. The nucleolar organizer regions were located at terminal chromosome positions by FISH with 45S rDNA probe. Diploid species presented four, six, and eight rDNA sites, tetraploid species showed eight sites and hexaploid species presented 12 rDNA sites. Karyomorphological differences include variation in number, size and chromosome morphology, suggesting that rearrangements involving small heterochromatic and rDNA segments played a major role in karyotype evolution.

Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods.

Chromatin diminution is a precisely controlled, highly repeatable, genome-wide deletion of noncoding heterochromatic segments from the presomatic line. The somatic line is reduced in size and reorganized; the germ line remains unaltered. Little is understood about its mechanistic underpinnings and adaptive significance in the nematodes, copepods, and hagfish in which it occurs. Here, we propose that microcrustacean copepods, whose cytology, development, and evolutionary ecology are well understood from an adaptationist point of view, provide the vehicle to test how chromatin diminution might orchestrate certain cell cycle dynamics, with the consequence of influencing the evolution of nuclear DNA contents, organismal development rates, and body size.

Genetic variation and zoogeography of pine voles of the Microtus subterraneus/majori group in Europe and Asia Minor

AbstractKaryotypic and allozyme analyses were carried out on 55 individuals of Microtus subterraneus and M. majori from 19 populations in Europe and Anatolia. The occurrence of M. subterraneus in Asia Minor was proven. Standard karyotypes were found in M. majori (2n=54), M. subterraneus from Poland (2n=54), and central and south‐eastern Europe (2n=52). Although the karyotype of M. subterraneus populations from Anatolia (2n=54), resembled that from Poland, there were apparent differences in the X chromosomes: it was larger and submetacentric (instead of metacentric) in the Anatolian populations owing to the presence of a large block of heterochromatin. The heterochromatic segment was also found to cause the size and shape differences in the X chromosomes between M. majori and European M. subterraneus (both in the 2n=52 and 2n=54 karyotypes). However, the location of this segment differed between the latter species and M. subterraneus from Anatolia. The distribution of the 2n=52 and both the 2n=54 karyotypes of M. subterraneus suggests that the Robertsonian fusion, distinguishing these different karyotypes, originated within the range of the species (i.e. within the 54‐chromosome karyotype) and has spread subsequently towards its periphery. It is also suggested that both the fusion in European populations and amplification of heterochromatin in Anatolian populations are quite recent phenomena. The allozyme data indicate that gene flow is limited between populations of M. subterraneus and they are concordant with the isolation‐by‐distance model. There seems to be little correspondence between patterns of chromosomal and allozyme variation in M. subterraneus. Nei's genetic distance between M. subterraneus and M. majori was low (D=0.169), suggesting their recent divergence.

Non-random Distribution of the GC-rich Heterochromatin and Nucleolar rDNA Sites on Astyanax scabripinnis Chromosomes.

A close relationship has been verified between presumedly GC-rich conspicuous segments and the nucleolus organizer regions on chromosomes of cold-blooded vertebrates. C-banding, and chromomycin A3 (CMA) or mithramycin A (MM) staining as well as rDNA in situ hybridization were performed in mitotic chromosomes of a population of the characid fish Astyanax scabripinnis in order to study GC-rich heterochromatin and the pattern of nucleolar rDNA location. The results showed a close relationship between large telomeric heterochromatic segments and GC-specific fluorochrome bands. Some CMA+ (or MM+) bands presented no correspondence with nucleolar rDNA sites detected by fluorescence in situ hybridization (FISH). Although GC specific fluorochromes have been widely used to infer nucleolus organizer regions location in fish chromosomes, a detailed FISH analysis indicates that, in this population, rDNA clusters are not always located on CMA+ (or MM+) bands, and some GC-rich bands do not bear rDNA clusters. Based on some available models we discussed some possible mechanisms for the non-random nucleolar rDNA and CMA+ heterochromatin distribution in A. scabripinnis chromosomes.

INTEGRATING THE GENETIC AND PHYSICAL CHROMOSOME MAPS OF BRASSICA OLERACEA VAR. ALBOGLABRA

Linking genetic maps constructed with molecular markers to the corresponding physical maps is an important goal in the investigation of crops. The relationship between these maps is reviewed, and we illustrate our approach by the analysis of the short arm of chromosome 4 of the model dicotyledonous plant, Arabidopsis thaliana L. We show that it is possible to produce a detailed molecular cytogenetic map by placing multicopy and unique sequences in relation to euchromatic and heterochromatic segments. This map correlates well with the molecular physical map (sequence map) but, as expected, shows differences from the genetic map. For Brassica oleracea L., as a first step we have produced a partial karyotype based on the combination of chromosome size, arm ratios and the location of repeated 45S rDNA, 5S rDNA and pericentromeric heterochromatin sequences by fluorescence in situ hybridisation (FISH). We are currently using BACs, cosmids and RFLPs, with known genetic map positions, as FISH probes to identify additional linkage group associated markers for the chromosomes. The construction of a definitive karyotype, with all linkage groups assigned to our cytogenetic map is approaching completion.

Amplification of a GC-rich heterochromatin in the freshwater fish Leporinus desmotes (Characiformes, Anostomidae)

This is the first description of the karyotype of Leporinus desmotes. The diploid female number was 2n = 54 meta- and submetacentric chromosomes. The nucleolar organizing regions (NORs) were studied by silver nitrate staining and rDNA fluorescence in situ hybridization (FISH) and were found to be located in the telomeric region of the long arm of the 9th pair. C-banding revealed centromeric and telomeric heterochromatin segments in most chromosomes. Intercalar blocks of heterochromatin were observed in the long arm of six chromosome pairs. Besides a NOR-adjacent heterochromatin, all of the intercalar heterochromatic segments were brightly fluorescent by mithramycin staining. These data suggest that a unique amplification of a primordial GC-rich heterochromatin, probably NOR-associated, may have taken place in the karyotype diversification of this Leporinus species.

Cloning and characterisation of polymorphic heterochromatic segments of Brachycome dichromosomatica.

After selective enrichment and differential hybridisation of Cot-1 DNA fractions of plants with and without polymorphic heterochromatic segments, a repetitive sequence (called Bds1) specific to the polymorphic chromosome segments of Brachycome dichromosomatica (Brachyscome dichromosomatica) was isolated. A single repeat unit of Bds1 is 92 bp long and is organised in tandem arrays at three different polymorphic segment sites on the chromosomes of cytodeme A2. Although all three sites showed extensive polymorphism between plants, the karyotypes of all analysed mitotic root cells were stable within a single plant. Electron microscopy revealed heavily condensed chromatin structures at the most obvious polymorphic site. The mechanisms that generate and maintain the observed chromosome structure polymorphisms are discussed.

Molecular cytogenetic analysis of supernumerary heterochromatic segments in Rumex acetosa.

The dioecious plant Rumex acetosa shows intraspecific karyotype variation, caused by supernumerary heterochromatic segments or DAPI (4',6-diamidino-2 phenylindole)-bands at the ends of the short arms of three pairs of autosomes. A DNA sequence (RAE730) specific to the supernumerary heterochromatic segments was cloned and sequenced. RAE730 was about 730 bp and AT-rich (71% AT-content). Using fluorescence in situ hybridization (FISH), RAE730 was localized in the supernumerary DAPI-positive heterochromatic segments on several mitotic chromosomes and chromocenters in interphase nuclei, but not in the DAPI-bands of Y or B chromosomes. RAE730 was tandemly arranged in the genome, and the copy number varied between plants from 40000 to 304000 copies per 2C, corresponding to the relative amount of supernumerary heterochromatic segments per genome. These results indicate that the karyotype variation caused by the supernumerary heterochromatic segment was generated by amplification or reduction of the tandem repeats of RAE730.

Allium cepa L. Cultivars from Four Continents Compared by Flow Cytometry show Nuclear DNA Constancy

In 1965 Van't Hof estimated the nuclear DNA amount of an unidentified Allium cepa L. cultivar as 2C=33.55pg (Experimental Cell Research39: 8–58). This value has been adopted by common usage as the main calibration standard for angiosperm DNA C-value estimations. However, different cultivars have been used while assuming species DNA C-value constancy. Surprisingly this assumption has never been tested. A. cepa is an outbreeder with telomeric heterochromatic segments, so intraspecific variation in C-value, possibly correlated with environmental factors as seen in Zea mays L., might be expected. We used laser flow cytometry to compare nuclear DNA amounts in roots of six A. cepa cultivars used as calibration standards or from different environments. Tissues from one cultivar, or similar volumes of tissue from two cultivars, were run and the variance between nuclei in 2C peaks compared. Only one shoulderless 2C peak was seen for all pairs of co-chopped cultivars. Thus, no large differences in C-value between cultivars from different environments were found. Moreover, comparing cultivars run singly or as pairs showed no evidence for increased variation in 2C peaks in the latter, and hence of critical differences in DNA amounts between ‘Ailsa Craig’ and another cultivar. Such variation was insufficient to make their use as alternative calibration standards, or the practice of imputing Van't Hof's original C-value estimate to them, unacceptable for most practical purposes. Given the mechanisms known which can generate genome size variation, the degree of constancy in DNA C-value found seems remarkable.

Cytogenetics of hepatoblastoma: further characterization of 1q rearrangements by fluorescence in situ hybridization: an international collaborative study.

Hepatoblastoma (HBT) is the most common hepatic neoplasm in children. This notwithstanding, little is known about pathogenetic factors, such as genetic abnormalities, of importance for the development and progression of this tumor type. To date, only 33 cytogenetically abnormal HBT have been published, and trisomies for chromosomes 2 and 20 have been shown to be the most frequent aberrations. Recently, unbalanced translocations involving proximal 1q have been described in several HBT, suggesting that a pathogenetically important gene maps to 1q. Six primary and one recurrent HBT were cytogenetically analyzed after short-term tissue culture. In addition, fluorescence in situ hybridization (FISH) studies, using locus-specific probes, were performed on three of these pediatric HBT as well as on one previously reported adult HBT. Total or partial trisomy 8, gain of chromosome 20, and structural rearrangements of chromosome 1 were detected in three HBT, and overrepresentation of chromosome 2 material was found in two HBT. The adjacent chromosome bands 1q12 and 1q21 were involved in three translocations, t(1;2), t(1;4), and t(1;11), which were all unbalanced and resulted in gain of 1q material. The previously reported adult HBT displayed 1q deletions with breakpoints at 1q12-21. FISH analyses of the 1q rearrangements revealed that all breakpoints were within the heterochromatic region. These findings provide further support for the importance of trisomies 2, 8, and 20 and rearrangements of 1q in the development of HBT. Furthermore, the consistent localization of breakpoints within the heterochromatic segment of chromosome 1 suggests that the important pathogenetic consequence of 1q abnormalities is the resulting genomic imbalance rather than a specific gene rearrangement.

Replication timing of homologous alpha-satellite DNA in Roberts syndrome.

Roberts syndrome (RS) is associated with a characteristic constitutive heterochromatin anomaly, namely, at metaphase the centromeres and heterochromatic segments appear split. In addition to this cytogenetic phenomenon, known as the RS effect, several other cytological features, especially affecting mitotic chromosome disjunction, are also observed. Applying FISH to interphase nuclei, we investigated the replication patterns of homologous alphoid centromeric DNA of chromosomes 9, 11, 16 and 17 in three patients showing the RS effect and in four normal individuals. A tendency for homologous centromeres to replicate asynchronously was observed in RS patients. This tendency was more evident in chromosomes 9 and 16, with large heterochromatic blocks and particularly subject to RS effect. This asynchrony could reflect a more generalized alteration in repetitive DNA replication timing that, in turn, would prevent the establishment of proper cohesion between sister chromatid heterochromatin, leading to the RS effect.

A revised cytotaxonomy of the genus Tulbaghia (Alliaceae)

Abstract A revision of Tulbaghia (Alliaceae) has been carried out. The genus includes twentyone species and one variety. On the basis of the different karyotypes, the genus can be subdivided into seven distinct Groups. Some species have poly-ploid forms but no true polyploid species has been found so far. A number of species possess heterochromatic segments which may be either mostly distally or proximally located. The heterochromatic segments of Tulbaghia are cold sensitive and specific fluorochrome staining shows that they are composed mainly of highly repetitive sequences of Guanine-Cytidine nucleotides. The possible evolutionary role of heterochromatin as well as that of the B-chromosomes, present in some species, is briefly discussed. The illustrations include micrographs of the mitotic chromosomes as well as colour photographs of the inflorescence of all the species. The typical habitat of two of the species is also illustrated in colour.

Cytogenetic characterization of a supernumerary chromosome segment and of B-chromosomes in Astyanax scabripinnis (Teleostei, Characidae).

The chromosome complement of a local population of Astyanax scabripinnis in Brazil was investigated with emphasis on the study of the heterochromatin attached to the A-chromosomes and present in the macro B-chromosome. Analysis after C-banding, silver and CMA3 staining, incorporation of 5-bromo-2'-deoxyuridine and chromosome digestion with nine restriction endonucleases revealed that the heterochromatin in the B-chromosomes was different from that found in the A-chromosomes. A polymorphism due to the presence of a supernumerary heterochromatic chromosome segment was observed in the population investigated. Some aspects related to the origin of the heterochromatin polymorphism in Astyanax scabripinnis are discussed.

Differences in the meiotic pairing behavior of gonosomal heterochromatin between female and male Microtus agrestis: implications for the mechanism of heterochromatin amplification on the X and Y

It is generally thought that pairing and recombination between the X and Y chromosome in eutherian mammals is important for the occurrence of normal meiotic division and the production of functional gametes. Microtus agrestis is one of the examples whose giant and heterochromatin-rich sex chromosomes fail to establish a durable association at any stage of the first meiotic division in males. In contrast, in females, synapsis starts in the euchromatic short arm and pairing progresses unidirectionally and continues until both X chromosomes have paired completely, as can be demonstrated by the use of fluorescence in situ hybridization with a sequence confined to the non-centromeric, gonosomal heterochromatin. However, compared with euchromatin, this association is apparently ephemeral and breaks off precociously in the pachytene and metaphase I stages. We demonstrate that a middle repetitive element is localized interspersed in the noncentromeric heterochromatin of both X and Y, except the telomeric region of the Y. No differences could be detected at the molecular level between male and female DNA, indicating that at least the bulk of these elements are organized in the same manner on the X and Y. Our data imply that the loss of synapsis and recombination between the X and Y might have preceded the process of heterochromatin amplification in the course of Microtinae evolution. Since asynapsed elements are particularly susceptible to DNA strand breaks during prophase I, DNA repair of double-strand breaks involving heterochromatic segments of the X and Y could have resulted in translocations of larger segments from the X to the Y or vice versa during the course of chromosome evolution of the gonosomes, explaining the homology at the molecular level between the heterochromatin of the asynaptic X and Y in M. agrestis.

An XX/XY sex chromosome system in a fish species, Hoplias malabaricus, with a polymorphic NOR-bearing X chromosome.

Cytogenetic studies were carried out in the fish, Hoplias malabaricus, from the Parque Florestal do Rio Doce (Brazil). This population is characterized by 2n = 42 chromosomes for both males and females and an XX/XY sex chromosome system, confirmed through several banding methods. Females show 24 metacentric, 16 submetacentric and 2 subtelocentric chromosomes. Males show 24 metacentric, 17 submetacentric and 1 subtelocentric chromosomes. While the X chromosome is easily recognized (the only subtelocentric element), the Y chromosome is somewhat difficult to identify but appears to correspond to the smallest submetacentric in the male karyotype. In-situ hybridization with an 18S rDNA probe showed 10 well-labeled chromosomes, including the X chromosome. The 5S rDNA is interstitially located in a single metacentric pair independent of the 18S rDNA sites. The NOR on the X chromosome is always active and occurs adjacent to a heterochromatic distal segment on the long arm. Variations in size of the NORs and/or heterochromatic segment correspond to a polymorphic size condition observed in the X chromosome. The present results confirm the XX/XY sex chromosome system in the population analyzed as well as a new cytotype in the Hoplias malabaricus group.

Nature and distribution of constitutive heterochromatin in fishes, genus Hypostomus (Loricariidae).

Some Hypostomus species were studied concerning the features of the karyotype structure and the constitutive heterochromatin. The karyotype of Hypostomus sp. F from the São Francisco river (Minas Gerais state, Brazil) is now described for the first time. A diversity in the diploid number, ranging from 2n = 68 to 2n = 80, as well as in the karyotype formulae, is evident in this fish group. Two types of heterochromatin, GC- and AT-rich, could be identified with the use of base-specific fluorochromes. In some species heterochromatic bands are mainly located on the centromeric and telomeric chromosomal regions, while in other species they are also observed at interstitial locations. Hypotheses concerning this heterochromatic distribution in Hypostomus karyotypes are discussed. A case of supernumerary heterochromatic segment and a centric fusion appear to be related with two variant karyotypic formulae observed among specimens from the Mogi-Guaçu and São Francisco rivers, respectively. The available data permit us to characterize a divergent karyotypic evolution among the Hypostomus species already analyzed, both at the macro- and microstructural levels, that is, their general karyotype organization and particular features related to chromosomal banding or staining, respectively.

Influence of hcterochromatin on fetal loss: Clinical and counseling aspects

Morphological variations of human chromosomes due to heterochromatic DNA have been the subject of numerous speculations for a quarter of century. Chromosomal variations or heteromorphisms have been correlated to a number of clinical parameters and a barrage of conflicting views have emerged. An enlarged secondary constriction region [qh] of chromosome 9 and the heterochromatin of the Y chromosome are frequent examples that have been correlated with increased risk of abortions. Recently, we were referred two couples for cytogenetic evaluation, each having three consecutive fetal losses. Both males had very large heterochromatic segments on their Yq chromosomes, while the male of the second couple also had an enlarged qh region on chromosome 16. To the best of our knowledge, such an enlarged qh region on chromosome 16 has not been reported before in combination with a large Y chromosome. A chromosome 16 specific alphoid probe [Oncor] was used to evaluate centromeric size by the FISH technique. A normal size centromere was noted, suggesting that the morphological variation of the long arm of chromosome 16 [16q] was due to the enlarged qh region. Does the male have double trouble or is this a fortuitous finding? Utmost caution should be exercised during counseling of such couples as there are no convincing evidence suggesting heterochromatin plays a role in fetal loss.

The karyotype of Alouatta fusca clamitans from Rio de Janeiro, Brazil: Evidence for a y-autosome translocation

The chromosome complements of four males of Alouatta fusca clamitans, caught in Rio de Janeiro State, Brazil, were analyzed by G-, C-, and NOR-banding techniques. The diploid number found was 49 in all the specimens. The presence of a heteromorphic pair of submetacentric chromosomes in the analyzed specimens, not present in males and females with 2n = 50 previously reported, and its G-banding pattern, led us to assume that this pair is involved in a Y-autosome translocation. Thus, the sex determination system appears modified to X1X1X2X2 /X1X2Y. Heterochromatic segments were found in the pericentromeric region of all the chromosomes, in the telomeric region of the short arm in pair 2, in the complete length of the short arm of pairs 5 and 6 and in the intercalary region of the long arm in pair 17. The nucleolar organizer regions were situated in the intercalary region of the long arm in two small acrocentric pairs.

Heterochromatin heterogeneity and chromosome heteromorphism in Cerdocyon thous (Mammalia, Canidae)

Few cytogenetic studies have been conducted on South American canids. Cerdocyon thous is a representative of the Canidae, living in the forests, open wooded areas and savannahs of South America. Compared to other canid species, C. thous has a large proportion of metacentric and submetacentric chromosomes as well as a large amount of constitutive heterochromatin, especially along the short arm of submetacentric chromosomes. In the present study, different chromosome banding methods were used to characterize the heterogeneous nature of the large heterochromatic segments and to propose an organizational model for this segment that occupies the entire short arm of most two-armed chromosomes. Furthermore, chromosome heteromorphism related to the short arm of large submetacentric chromosome corresponding to no. 3 in male and female karyotypes is described.

C-bands and NORs on chromosomes in four species of the genusTrechusClairv. (Coleoptera, Carabidae)

SUMMARYC-banding pattern and Ag-NORs on chromosomes of Trechus latus, T. pilisensis, T. pulchellus, and T. quadristriatus are presented. T. pulchellus has a paracentromeric C-band similar in size on all chromosomes. T. latus has euchromatic the X chromosome but all autosomes have a paracentromeric C-positive band. In the karyotype of T. quadristriatus the 1st pair of autosomes is euchromatic while the autosomes have a paracentromeric C-band. T. pilisensis has a constitutive heterochromatic segment in a paracentromeric position on all chromosomes and in addition the terminal C-band on the 1st pair of autosomes. Males of the examined species have the same pattern of silver precipitation during spermatogenesis. In the mitotic stages, from the prophase to the prometaphase stage one silver dot on the end of two autosomes is visible. In prophase I (to diakinesis) one active NOR is observed.