Giant sex chromosomes retained within the Portuguese lineage of the field vole (<Emphasis Type="Italic">Microtus agrestis</Emphasis>)

The field vole (Microtus agrestis) is characterised by extremely large blocks of heterochromatin on both the X and Y chromosome. Some other Microtus also have blocks of heterochromatin on their sex chromosomes but not as extensive and always of independent origin from the heterochromatic expansion found in M. agrestis. Coupled with evidence of geographic variation in large heterochromatic blocks within other species (e.g. in the western hedgehog Erinaceus europaeus), it might be expected that field voles would show substantial variation in size and disposition of the sex chromosome heterochromatin. In fact, only minor variation has been described up to now. Those studies conducted previously were largely on field voles from central and northern Europe. Here, we describe the karyotype of field voles from Portugal, of interest because recent molecular studies have shown field voles from western Iberia to be a separate evolutionary unit that might be considered a cryptic species, distinct from populations further to the east. The two Portuguese field voles (one female, one male) that we examined also had essentially the same karyotype as seen in other field voles, including the giant sex chromosomes, but with small differences in the structure of the Y chromosome from that described previously. The finding that field voles throughout Europe show relatively little variation in their giant sex chromosomes is consistent with molecular data which suggest a recent origin for this complex of species/near-species.     

Chromosomal evolution and phylogenetic analyses in <Emphasis Type="Italic">Tayassu pecari</Emphasis> and <Emphasis Type="Italic">Pecari tajacu</Emphasis> (Tayassuidae): tales from constitutive heterochromatin

The mammalian family Tayassuidae (peccaries) is confined to the New World and comprises three recognized extant species, white-lipped (Tayassu pecari), collared (Pecari tajacu) and chacoan (Catagonus wagneri) peccaries, which exhibit distinct morphological and chromosomal features. The phylogenetic relationships among the tayassuids are unclear and have instigated debate over the palaeontological, cytogenetic and molecular aspects. Constitutive heterochromatin analysis can be used in understanding the phylogenetic relationships between related species. Here we describe, for the first time, the constitutive heterochromatin (C-positive heterochromatin) of two tayassuid species, Tayassu pecari and Pecari tajacu. We demonstrate that in situ restriction endonuclease digestion with sequential C-banding could be a complementary tool in the study of constitutive heterochromatin heterogeneity in chromosomes of the Tayassuidae. Our characterization of peccary chromosomes suggests that the Pecari tajacu autosomal karyotype is more primitive and has accumulated great diversity in its constitutive heterochromatin. This idea is supported by several other studies that analysed nuclear and mitochondrial sequences of the living peccary species. Finally, the tayassuid X chromosome primitive form seems to be the one of Tayassu pecari.     

HP1/ORC complex and heterochromatin assembly

We have used the highly conserved heterochromatin component, heterochromatin protein 1 (HP1), as a molecular tag for purifying other protein components of Drosophila heterochromatin. A complex of HP1 associated with the origin recognition complex (ORC) and an HP1/ORC-associated protein (HOAP) was purified from the maternally loaded cytoplasm of early Drosophila embryo. We propose that the DNA-binding activities of ORC and HOAP function to recruit underphosphorylated isoforms of HP1 to sites of heterochromatin nucleation. The roles of highly phosphorylated HP1, other DNA-binding proteins known to interact with HP1, and histone modifying activities in heterochromatin assembly are also addressed.     

C-banding in the polytene chromosomes of species of the plumosus group (Diptera,Chironomidae) and their experimental hybrids

The localization and amount of heterochromatin in the plumosus group were studied, including the species Chironomus plumosus L., C. vancouveri and C. balatonicus. The appearance of C bands of Chironomus plumosus in several European populations is traced. The role of the C heterochromatin in the differentiation of this species is discussed. From the evolutionary point of view the Swiss populations, in which large centromere heterochromatin blocks have been discovered, are more varied as to the amount of heterochromatin. The importance of duplications for this process is pointed out. The chromosomes of the individuals from C. vancouveri and C. balatonicus have centromeric, telomeric and interstitial heterochromatin. The centromeric heterochromatin is represented by thin C-bands. The particularities in the appearance of C heterochromatin in C. vancouveri and C. balatonicus reflect the structural peculiarities of their chromosomes. The change in the euchromatin regions in these forms is discussed in the light of transformation of euchromatin to heterochromatin in the process of evolution.The appearance of heterochromatin in hybrids (between populations and between species) created experimentally is traced. A change has been discovered in the appearance of heterochromatin in the hybrids compared to the initial parent forms. This difference is expressed more strongly in inter-species hybrids than in interpopulation hybrids of C. plumosus.     

Structural insights into Rhino‐Deadlock complex for germline piRNA cluster specification

PIWI‐interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species‐specific manner and so defines the piRNA‐producing loci in the Drosophila genome. Here, we determine the crystal structures of Rhino‐Deadlock complex in Drosophila melanogaster and simulans. In both species, one Rhino binds the N‐terminal helix–hairpin–helix motif of one Deadlock protein through a novel interface formed by the beta‐sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. Our structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross‐species incompatibility between the sibling species. By determining the molecular architecture of this piRNA‐producing machinery, we discover a novel HP1‐partner interacting mode that is crucial to piRNA biogenesis and transposon silencing. We thus explain the cross‐species incompatibility of two sibling species at the molecular level.     

In situ digestion of satellite DNA of Scilla siberica

Restriction endonucleases have been used to digest DNA in fixed metaphase chromosomes of animal species. However, constitutive C-heterochromatin of plant species is resistant to these enzymes suggesting that the special structural organization of plant C-bands is an impediment to the activity of restriction endonucleases. In order to test this hypothesis, we have chosen the species Scilla siberica, whose purified satellite DNA, localised at the heterochromatic regions, is extensively digested by HaeIII. In situ treatment with HaeIII alone does not produce significant digestion of heterochromatin, but subsequent treatment with proteinase K results in extensive digestion of heterochromatic regions producing unstained gaps. These results indicate that HaeIII is able to access and cut chromosomal DNA from C-bands, but the DNA fragments remain attached to chromosomal proteins that characterize the complex structure of heterochromatin in this species. Although there are no reasons to suppose that accessibility of chromosomal DNA of S. siberica to restriction enzymes can be impeded, it would be reasonable to think from our results that some special features of heterochromatin organization in plants contribute to the formation of a complex structure that makes chromosomal DNA extraction impossible.by D. Schweizer     

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR‐, C‐ and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the ‘Paranense Sea’, probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.     

Analysis of the heterochromatin of <Emphasis Type="Italic">Cebus</Emphasis> (Primates,Platyrrhini) by micro-FISH and banding pattern comparisons

The karyotype of the neotropical primate genus Cebus (Platyrrhini: Cebidae), considered the most ancestral one, shows the greatest amount of heterochromatin described among Platyrrhini genera. Banding techniques and restriction enzyme digestion have previously revealed great variability of quantity and composition of heterochromatin in this genus. In this context, we use fluorescence in situ hybridization (FISH) to analyse this genomic region and discuss its possible role in the diversification of Cebus. We used a heterochromatin probe for chromosome 11 of Cebus libidinosus (11qHe+ CLI probe), obtained by chromosome microdissection. Twenty-six specimens belonging to the families Atelidae, Cebidae, Callitrichidae and Pithecidae (Platyrrhini) were studied. Fourteen out of 26 specimens were Cebus (Cebidae) individuals of C. libidinosus, C. xanthosternos, C. apella, C. nigritus, C. albifrons, C. kaapori and C. olivaceus. In Cebus specimens, we found 6 to 22 positive signals located in interstitial and telomeric positions along the different species. No hybridization signal was observed among the remaining Ceboidea species, thus reinforcing the idea of a Cebus-specific heterochromatin composed of a complex system of repetitive sequences.     

Chromosome plasticity in Ctenomys (Rodentia Octodontidae): chromosome 1 evolution and heterochromatin variation

A chromosome 1 (Cr1) pericentric inversion is described in six of seven species in the genus Ctenomys (tuco-tucos) from Uruguay. The inversion was inferred from G-band analyses of subtelocentric Cr1 hypothesised to be derived from the ancestral metacentric condition. Cr1 varies across species in heterochromatin amount and localisation including a metacentric chromosome without positive C-bands in C. torquatus, a subtelocentric chromosome with heterochromatic short arms in C. rionegrensis, and a subtelocentric chromosome negative after C-banding in five of the species analysed here. Pachytene chromosomes from C. rionegrensis, a species with the highest heterochromatin content, and C. torquatus, one of the species with the lowest heterochromatin content, were analysed in order to assess possible mechanisms of heterochromatin evolution. This analysis revealed the presence of three heterochromatic chromocenters in C. rionegrensis where bivalents converge, while in C. torquatus only one chromocenter was observed. In both species, highly repetitive DNA was observed, localised in chromocenters after “in situ” hybridisation. Heterochromatin associated protein M31 was localised in chromocenters of both species after immuno-detection. The spread of heterochromatin in Ctenomys chromosomes could be produced by chromatin exchanges at the chromocenter level. We propose the exchange of this DNA associated proteins between non-homologous chromosomes in pachytene to be the responsible for the spread of heterochromatin through the karyotypes of species like C. rionegrensis     

Characterization and Comparative Analysis of DNA from the Pericentric Heterochromatin of Chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The library containing DNA sequences from the diffuse pericentric heterochromatin from the right arm ofAnopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the library fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the A. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of A. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific library. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

A comparative study of the heterochromatin of Apodemus sylvaticus and Apodemus flavicollis

The two closely related species Apodemus sylvaticus and Apodemus flavicollis (Muridae) differ in the distribution of their heterochromatin. Two major repetitive sequences known to occur in both species were isolated from A. flavicollis after digestion of total nuclear DNA with the restriction enzymes HindIII and EcoRI respectively and characterized in both species by filter hybridisation and in situ hybridisation to metaphase chromosomes. The EcoRI clone detects a dispersed repetitive sequence family in the genome of both species. Southern blot hybridisation with the HindIII satellite DNA probe reveals major similarities and minor differences in the two species. In situ hybridisation with the HindIII probe labels all chromosomes of A. flavicollis exclusively in the centromeric heterochromatin, whereas in A. sylvaticus several autosomes are also labelled distally. The labelling patterns correspond to the distribution of heterochromatin in the two species. It is concluded that the additional distal heterochromatin of A. sylvaticus contains similar sequences to those of the centromeric heterochromatin of both species. The distal heterochromatin in A. sylvaticus most likely evolved by transposition and amplification of centromeric satellite DNA elements, after the separation of the two species.     

Cold-sensitive chromosome regions and heterochromatin inCestrum (Solanaceae):C. strigillatum,C. fasciculatum,andC. elegans

Cold-induced mitotic under-condensation of certain chromosome segments is a rare phenomenon in plants. There are about 11 genera of monocotyledons and only 3 of dicotyledons, where species are known to have such cold-sensitive regions (CSRs). The molecular causes of cold-induced undercondensation are not clear, and no consistent cytochemical characteristics of CSRs are known. Recently we have presented a chromosome banding analysis on CSRs and their relation to constitutive heterochromatin inCestrum parqui (Solanaceae), a species of sect.Cestrum. The present study is concerned with a similar analysis inC. strigillatum of sect.Cestrum, and inC. fasciculatum andC. elegans of sect.Habrothamnus. Chromomycin/DAPI fluorescent double staining, sequential C-banding, and sequential silver impregnation were applied. The species differ in detail but are similar qualitatively. Four classes of heterochromatin can be discriminated. (1) CSRs, with banding properties indicating AT-rich constitutive heterochromatin. After cold-treatment CSR heterochromatin can be silver-impregnated from interphase, as chromocentres, to metaphase, as undercondensed segments. CSRs are subject to frequent heteromorphy. (2) Nucleolar organizers. Two pairs were identified in the karyotypes. Banding properties indicate GC-rich heterochromatin. The nucleolar organizing regions are less evident and their silver-reducing capability reduces during metaphase. (3) Non-nucleolar CMA-positively fluorescing bands. These are minute, polymorphic, positively C-stained, and restricted to one or a few sites in the karyotypes. (4) Indifferently fluorescing, positively C-stained bands. They occur on centromeres, some chromosome ends, and clustered over the chromosome arms. They are mostly very delicate and do not resist harsh banding treatments. — The species investigated here andC. parqui resemble each other qualitatively in heterochromatin classes (1), (2), and (3), but differ much in banding properties of class (4). Therefore, heterochromatin characteristics in the genus are not so uniform as the present results inC. strigillatum, C. fasciculatum, andC. elegans appear to show.     

THE DISTRIBUTION OF HETEROCHROMATIN IN THE GENUS NICOTIANA (SOLANACEAE)

A survey of the species of the genus Nicotiana was carried out to determine the distribution and the cytological characteristics of heterochromatin in this genus. All examined species of the genus possess knob-type heterochromatin, which is defined as spherical, densely staining regions of the pachytene chromosomes. These knobs are most frequently located near the centromere, the nucleolar organizer, and the ends of the chromosomes. Block-type heterochromatin, defined as any longer-than-broad heterochromatic segment seen at pachytene, was found in three species of the section Paniculatae, three species of the section Tomentosae, and two species of the section Noctiflorae. Three categories of the block-type heterochromatin, corresponding to the three subgenera, were found to differ with respect to overall size, staining properties, and location of the blocks. The distribution of these three types of block heterochromatin is discussed in the light of the latest taxonomic treatments of the genus.     

Hidden heterochromatin: Characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

Rapid,localized amplification of a unique satellite DNA family in the rodent Microtus chrotorrhinus

A novel satellite DNA family (called MSAT-2570) was isolated and characterized from the rodent Microtus chrotorrhinus. With a length of 2,570 bp the repeat unit is among the largest yet reported in mammals and comprises a series of short direct and inverted repeats. These repeat motifs may prevent nucleosome formation or represent an endless source of genetic variation. Restriction enzyme digestion using the two pairs of isoschizomers HpaII/MspI and MboI/Sau3AI demonstrated tissue specific differences in satellite DNA methylation that may reflect variable chromatin conformation or differences in patterns of gene expression. The sex chromosomes of M. chrotorrhinus are unusually large in size among mammals, comprising 15%–20% of the karyotype and containing large blocks of heterochromatin. In situ hybridization of the satellite DNa revealed chromosomal localization predominantly to sex chromosome heterochromatin. A survey of related rodents including three congeneric species also with giant sized sex chromosomes demonstrated that MSAT-2570 is present only in the genome of M. chrotorrhinus. However, another previously reported satellite DNA also isolated from M. chrotorrhinus has been shown to reside on sex chromosome heterochromatin in one of the other three species, indicating that these giant blocks of heterochromatin are complex in structure and comprise multiple, unrelatined satellite DNA families.     

Heterogeneity of heterochromatin in six species ofCtenomys (Rodentia: Octodontoidea: Ctenomyidae) from Argentina revealed by a combined analysis of C- and RE-banding

Exceptional chromosomal variability makesCtenomys an excellent model for evolutionary cytogenetic analysis. Six species belonging to three evolutionary lineages were studied by means of restriction endonuclease and C-chromosome banding. The resulting banding patterns were used for comparative analysis of heterochromatin distribution on chromosomes. This combined analysis allowed intra- and inter-specific heterochromatin variability to be detected, groups of species belonging to different lineages to be characterized, and phylogenetic relationships hypothesized from other data to be supported. The “ancestral group”,Ctenomys pundti andC. talarum, share three types of heterochromatin, the most abundant of which was also found in C. aff.C. opimus, suggesting that the latter species also belongs to the “ancestral group”. Additionally, within the subspeciesC. t. talarum, putative chromosomal rearrangements distinguishing two of the three chromosomal races were identified. Two species belong to an “eastern lineage”,C. osvaldoreigi andC. rosendopascuali, and share only one type of heterochromatin homogeneously distributed across their karyotypes.C. latro, the only analyzed species from the “chacoan” lineage, showed three types of heterochromatin, one of them being that which characterizes the “eastern lineage”.C. aff.C. opimus, because of its low heterochromatin content, is the most primitive karyotype of the genus yet described. The heterochromatin variability showed by these species, reflecting the evolutionary divergence toward different heterochromatin types, may have diverged since the origin of the genus. Heterochromatin amplification is proposed as a trend withinCtenomys, occurring independently of chromosomal change in diploid numbers.     

Drosophila Telomere Transposons: Genetically Active Elements in Heterochromatin

In Drosophilatwo non-LTR retrotransposons, HeT-Aand TART, offer a novel experimental system for the study of heterochromatin. These elements, found only in heterochromatin, form Drosophilatelomeres by repeated transposition onto chromosome ends. Their transposition yields arrays of repeats larger and more irregular than the repeats produced by telomeras; nevertheless, the transpositions are, in principle, equivalent to the telomere-building action of telomerase. The identification of the HeT-Apromoter has given the first view of the molecular structure of a promoter active in heterochromatin. These telomere-specific elements are unusual in having a large amount of non-coding sequence. Like many other heterochromatic sequences, the HeT-Anon-coding sequence has a repetitive organization strongly conserved within the species, although the sequence itself can undergo significant change between species (atypical example of concerted evolution). Such heterochromatic sequences could be important for the cell, perhaps as docking stations for essential proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Karyomorphological parameters and C-band distribution suggest phyletic relationships within the subtribeLimodorinae (Orchidaceae)

Karyotype attributes and heterochromatin distribution were used to characterize fourteen taxa of the subtribeLimodorinae (Orchidaceae). The karyotypes were established using morphometrical parameters following Feulgen staining and C-banding. No significant differences in heterochromatin content were found between specimens collected from various sites. Four species of theEpipactis helleborine group possess some chromosome pairs with quite similar heterochromatin patterns; some differences were found inE. distans with respect to other species of this group.Epipactis palustris differed significantly from otherEpipactis species in its different karyotype and its numerous terminal C-bands. The largest differences from the other genera were shown inLimodorum as far as karyomorphology and heterochromatin patterns were concerned. C-band distribution indicated similarity among non-homologous chromosomes, supporting a possible palaeo-polyploid origin for theCephalanthera andEpipactis karyotypes.     

Constitutive heterochromatin: a surprising variety of expressed sequences

The organization of chromosomes into euchromatin and heterochromatin is amongst the most important and enigmatic aspects of genome evolution. Constitutive heterochromatin is a basic yet still poorly understood component of eukaryotic chromosomes, and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Although recent evidence indicates that the presence of transcribed genes in constitutive heterochromatin is a conserved trait that accompanies the evolution of eukaryotic genomes, the term heterochromatin is still considered by many as synonymous of gene silencing. In this paper, we comprehensively review data that provide a clearer picture of transcribed sequences within constitutive heterochromatin, with a special emphasis on Drosophila and humans.