Karyotype evolution in holocentric chromosomes of three related species of triatomines (Hemiptera-Reduviidae)

C-banded karyotypes, DNA content and the male meiiotic process ofTriatoma platensis andTriatoma delpontei are compared with those ofTriatoma infestans, the main vector of Chagas disease in South America. These three species present the same diploid chromosome number 2n=22 (20 autosomes+XX/XY). They also have several cytogenetic traits that differ from all other triatomines: large autosomes, C-heterochromatic blocks and meiotic heteropycnotic chromocenters formed by autosomes and sex chromosomes. In spite of these similarities, each species presents different chromosomal behavior during male meiosis, distinct DNA content and a specific amount and localization of the C-heterochromatin. The differences in DNA content are mainly due to the variation in C-heterochromatin amount, which may be interpreted as loss and/or gain of C-regions. This interpretation is supported by the presence of meiotic and mitotic chromocenters that facilitate the transference of C-positive material. The cytogenetic data presented in this work suggest thatT. infestans andT. platensis are more closely related to each other than toT. delpontei. It can also be inferred that the differences in distribution and amount of heterochromatin do not play a direct role in speciation in this group.     

Robertsonian polymorphism, B chromosomes variation and sex chromosomes heteromorphism in the African water rat Dasymys (Rodentia, Muridae)

Chromosome banding analysis (G- and C-bands) of Dasymys rufulus from Senegal, Mali and the Ivory Coast, and D. cf. incomtus from Eastern and South-western Ethiopia was carried out. The diploid numbers (2N) in the former species range between 36 and 39 due to the presence of 0–3 small biarmed heterochromatic B chromosomes, resulting in a corresponding variation of the number of autosomal arms (NFa) between 44 and 50. The basic autosomal set was, however, constant and identical in these specimens. The karyotypes of D. cf. incomtus from Eastern and Western Ethiopia were found to be different (2N=40 and 38, respectively). Comparison of G-banding patterns of the species studied revealed that they differ from each others by 1–2 Rb fusions/fissions, one paracentric inversion and heteromorphous sex chromosomes resulting from addition/deletion of heterochromatic blocks (X) and pericentric inversion (Y). In the light of the available chromosome banding data, the significance of intraspecies karyotypic variability within D. cf. incomtus and its relevance to the systematics of the genus are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distribution of L1-retroposons on the giant sex chromosomes of Microtus cabrerae (Arvicolidae, Rodentia): functional and evolutionary implications

Long interspersed nuclear elements (L1 or LINE-1) are the most abundant and active retroposons in the mammalian genome. Traditionally, the bulk of L1 sequences have been explained by the ‘selfish DNA’ hypothesis; however, recently it has been also argued that L1s could play an important role in genome and gene organizations. The non-random chromosomal distribution of these retroelements is a striking feature considered to reflect this functionality. In the present study we have cloned and analyzed three different L1 fragments from the genome of the rodent Microtus cabrerae. In addition, we have examined the chromosomal distribution of this L1 in several species of Microtus, a very interesting group owing to the presence in some species of enlarged (‘giant’) sex chromosomes. Interestingly, in all species analyzed, L1-retroposons have preferentially accumulated on both the giant- and the normal-sized sex chromosomes compared with the autosomes. Also we have demonstrated that L1-retroposons are not similarly distributed among the heterochromatic blocks of the giant sex chromosomes in M. cabrerae and M. agrestis, which suggest that L1 retroposition and amplification over the sex heterochromatin have been different and independent processes in each species. Finally, we proposed that the main factors responsible for the L1 distribution on the mammalian sex chromosomes are the heterochromatic nature of the Y chromosome and the possible role of L1 sequences during the X-inactivation process.     

Heteromorphic sex chromosomes inEupsophus insularis (Amphibia: Anura: Leptodactylidae)

The chromosomes of the Chilean frogEupsophus insularis are described for the first time. This species has a chromosome number of 2n=30, and based on the karyotype it is concluded thatE. insularis is closely related toE. migueli. E. insularis has an XX/XY system of sex determination, and pericentromeric constitutive heterochromatin is present in all chromosomes except in the Y chromosome. It is postulated that the Y chromosome is derived from a small ancestral metacentric chromosome that lost its heterochromatic segment.accepted for publication by M. Schmid     

Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus)

Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations.     

Dosage compensation, the origin and the afterlife of sex chromosomes

Over the past 100 years Drosophila has been developed into an outstanding model system for the study of evolutionary processes. A fascinating aspect of evolution is the differentiation of sex chromosomes. Organisms with highly differentiated sex chromosomes, such as the mammalian X and Y, must compensate for the imbalance in gene dosage that this creates. The need to adjust the expression of sex-linked genes is a potent force driving the rise of regulatory mechanisms that act on an entire chromosome. This review will contrast the process of dosage compensation in Drosophila with the divergent strategies adopted by other model organisms. While the machinery of sex chromosome compensation is different in each instance, all share the ability to direct chromatin modifications to an entire chromosome. This review will also explore the idea that chromosome-targeting systems are sometimes adapted for other purposes. This appears the likely source of a chromosome-wide targeting system displayed by the Drosophila fourth chromosome.     

Cytogenetic studies of Hynobiidae (Urodela) XIX. Morphological variation of sex chromosomes pairing behavior of sex lampbrush chromosomes in <Emphasis Type="Italic">Hynobius quelpaertensis</Emphasis> (Mori) from Cheju Island,South Korea

Using Giemsa staining, C-banding and Ag-NOR staining techniques, we analyzed chromosomes in adult male and female Hynobius quelpaertensis and in embryos of this species in egg sacs collected from eight localities of Cheju Island, South Korea. Chromosome pair 21 was consistently homomorphic in male specimens, while it was heteromorphic in female specimens, suggesting the occurrence of ZZ/ZW sex chromosome constitution in this species. The W chromosome, being much larger than the Z chromosome, was of three morphologically distinct types: W^A, W^B and W^C. Lampbrush chromosomes examined in the oocytes of one female specimen having the W^A chromosome showed that the short arm of the W^A chromosome and the long arm of the Z chromosome paired closely and hence are genetically homologous. We also tried to analyze the structural relationship among the three types of W chromosomes based on their C-banding and Ag-NOR patterns.     

Localization of uridine monophosphate synthase (UMPS) gene to river buffalo chromosomes by FISH

Uridine monophosphate synthase plays an important role in pyrimidine synthesis, converting orotic acid to uridine 5 monophosphate. In cattle,UMPS deficiency is inherited as a monogenic autosomal recessive trait. While heterozygous carriers are phenotypically normal, homozygotes are lethalin utero (Shankset al. 1992).UMPS has been mapped to human chromosome 3q13 (Qumsiyehet al. 1989), sheep chromosome 1q (Burkinet al. 1993) and cattle chromosome 1q31 (Ryanet al. 1994). In the present study we used a cattle genomic probe to localizeUMPS to river buffalo chromosomes by fluorescencein situ hybridization.     

Organisation of the nervous system in cysts of the freshwater tardigrade Thulinius ruffoi (Parachela,Isohypsibioidea: Doryphoribiidae)

Encystment is a natural process that involves cyst formation, and at least some species of tardigrades can form cysts. However, the encystment process and cyst structure among tardigrades are still poorly understood. Despite some aspects of the encysted animals' systems organisation being examined in the past, the morphology and structure of the nervous system have never been thoroughly investigated. This study covers anatomical, histological and morphological details and proposes physiological aspects of the nervous system in encysted Thulinius ruffoi up to 11 months duration in encystment. This is the first record of the nervous system organisation in a species belonging to the family Doryphoribiidae. The cyst formation results in morphological changes in the nervous system. It comprises central and peripheral elements, which may be observable even after many months since the cyst formation. Based on the nervous system's organisation in cysts, there is no sign that histolysis is a part of encystment.     

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH)

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin — perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype.This revised version was published online in November 2005 with corrections to the Cover Date.     

Localization of a vertebrate telomeric sequence in the chromosomes of two marine worms (phylum Annelida: class polychaeta)

Using the fluorescencein situ hybridization (FISH) technique, the presence of the vertebrate telomeric sequence (TTAGGG) _n was found in the chromosomes of two marine polychaetes belonging to two separate orders: one errant,Platynereis dumerilii (family Nereidae), and the other sessile,Pomatoceros lamarckii (family Serpulidae). This sequence was exclusively present at the ends of the chromosomes in both species.     

Highly conserved chromosomes in an Asian squirrel (Menetes berdmorei, Rodentia: Sciuridae) as demonstrated by ZOO-FISH with human probes

The chromosomes of Menetes berdmorei (Rodentia, Sciuridae, Sciurinae) were studied by ZOO-FISH using whole human chromosome probes. All homoeologies between M. berdmorei and human chromosomes were determined, except for two small chromosome segments. Twelve human chromosomes are conserved in a unique block of synteny; ten are split into two and one into three blocks. Thus, a small number of interchromosomal rearrangements, about twenty, separates human from this squirrel karyotype. Homoeologies between human and the presumed ancestral chromosomes of Sciurinae could also be deduced, as well as those with the presumed ancestral chromosomes of eutherian mammals. Sciurinae chromosomes appear to be much closer to those of non-rodent mammals than those of Muridae and Cricetidae species studied so far. Thus, they provide an interesting tool to link the rodent genome to those of other mammals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Karyotyping mouse chromosomes by multiplex-FISH (M-FISH)

Karyotyping of mouse chromosomes is a skillful art, which is laborious work even for experienced cytogeneticists. With the growing number of mouse models for human diseases, there is an increasing demand for automated mouse karyotyping systems. Here, such a karyotyping system for mouse chromosomes based on the multiplex-fluorescence in-situ hybridization (M-FISH) technology is shown. The system was tested on a number of individual mice with numerical and structural aberrations and its reproducibility and robustness verified. Mouse M-FISH should be a valuable tool for the analysis of chromosomal rearrangements in mice.     

Characterization of the atypical karyotype of the black-winged kite Elanus caeruleus (Falconiformes: Accipitridae) by means of classical and molecular cytogenetic techniques

The karyotype of the black-winged kite (Elanus caeruleus), a small diurnal raptor living in Africa, Asia and southern Europe, was studied with classical (G-, C-, R-banding, and Ag-NOR staining) and molecular cytogenetic methods, including primed in-situ labelling (PRINS) and fluorescence in-situ hybridization (FISH) with telomeric (TTAGGG) and centromeric DNA repeats. The study revealed that the genome size, measured by flow cytometry (3.1pg), is in the normal avian range. However, the black-winged kite karyotype is particularly unusual among birds in having a moderate diploid number of 68 chromosomes, and containing only one pair of dot-shaped microchromosomes. Moreover, the macrochromosomes are medium-sized, with the Z and W gonosomes being clearly the largest in the set. C-banding shows that constitutive heterochromatin is located at the centromeric regions of all chromosomes, and that two pairs of small acrocentrics and the pair of microchromosomes are almost entirely heterochromatic and G-band negative. The distribution pattern of a centromeric repeated DNA sequence, as demonstrated by PRINS, follows that of C-heterochromatin. The localization of telomeric sequences by FISH and PRINS reveals many strong telomeric signals but no extratelomeric signal was observed. The atypical organization of the karyotype of the black-winged kite is considered in the context of the modes of karyotypic evolution in birds.     

Cytogenetics of the bleak (Alburnus alburnus), with special emphasis on the B chromosomes

Some of the largest B chromosomes so far discovered in vertebrates are present in the cyprinid fish Alburnus alburnus. Previous cytogenetic analyses revealed a diploid chromosome number of 2n = 50. In addition, in some individuals one or two unusually large B chromosomes are present. Two morphologically different types of B chromosomes were observed. The frequency of animals bearing a supernumerary chromosome was found to vary considerably between different populations. A more detailed analysis of the A and B chromosomes of A. alburnus by conventional banding techniques, as well as fluorescence in-situ hybridization (FISH) with the telomeric DNA repeats (GGGTTA)₇/(TAACCC)₇, 18S + 28S rDNA and 5S rDNA were performed in the present study. Furthermore, a B chromosome-specific DNA probe obtained by amplified length polymorphism (AFLP) was hybridized on metaphases of A. alburnus carrying supernumerary B chromosomes. The banding analyses showed that the B chromosomes are completely heterochromatic, consist of GC-rich DNA sequences, replicate their DNA in the very late S-phase of the cell cycle and are composed mainly of a specific retrotransposable DNA element. Finally, blood probes from A. alburnus were collected for DNA-flow cytometric measurements. It could be shown that the huge supernumerary chromosomes represent nearly 10% of the total genome size of A. alburnus.     

Pericentromeric euchromatin is conserved in minute human supernumerary chromosomes: a study using cross-species colour segmenting (RxFISH)

Investigation of marker chromosomes is one of the most challenging areas of clinical cytogenetics, especially in the prenatal scenario. A range of techniques including microdissection/reverse painting, SKY and M-FISH are available for the investigation of larger markers (>3 Mb). All these techniques rely on hybridization of unique, homologous sequences with simultaneous suppression of repeat sequences. In contrast, RxFISH is based on hybridization of cross-species syntenic sequences; repeat sequences do not hybridize due to species divergence. We have used RxFISH to analyse a group of the smallest, i.e. minute, supernumerary marker chromosomes. Our results suggest that even the smallest marker chromosomes often contain conserved pericentric euchromatin. More detailed characterization of pericentric genetic content is needed to assess the clinical significance of minute supernumerary markers.     

Distribution of 5-methylcytosine-rich regions in the metaphase chromosomes ofVicia faba

The DNA methylation pattern ofVicia faba metaphase chromosomes was examined with a specific monoclonal antibody. 5-methylcytosine (5-mC) residues are present in different chromosomal sites, and are particularly abundant in telomeric and/or subtelomeric regions and in certain intercalary bands. Chromosomal localization of methylated regions enables a better knowledge of the lengthwise differentiation of this chromosome complement. Our results also indicate that there may be differences in monoclonal antibody binding between corresponding regions of homologous chromosomes inV. faba. This behaviour is detectable in specific regions with different frequencies. The data support results previously obtained forAllium cepa metaphase chromosomes using the same monoclonal antibody.accepted for publication by J. S. (Pat) Heslop-Harrison     

Segregation of holocentric chromosomes at meiosis in the nematode,Caenorhabditis elegans

The meiotic segregation of the holocentric chromosomes ofCaenorhabditis elegans in both spermatogenesis and oogenesis is described. The extended kinetochore typical of the mitotic chromosome could not be differentiated on meiotic bivalents; instead microtubules appeared to project into the chromatin. The meiotic spindles formed during spermatogenesis contain centrioles and asters, while in oogenesis the spindles are acentriolar and barrel shaped. The formation of the acentriolar spindle was studied in fixed specimens by anti-tubulin immunofluorescence. Microtubule arrays were seen first to accumulate in the vicinity of the meiotic chromosomes prior to congression. At later stages, elongated spindle structures up to 13 in length were observed parallel to the surface of the embryo. Further development of the spindle appeared to involve its shortening into a barrel shape and rotation so that one spindle pole was opposed to the membrane. By anaphase the pole-to-pole spindle length reached a minimum of 3–4 . One end of each chromatid in the meiotic bivalent was labelled byin situ hybridization of a probe DNA to show that in oogenesis the chromatids were associated end-to-end in the bivalent. Furthermore, either the right or the left ends of the homologues could be held in association. At metaphase I the bivalents were oriented axially, such that kinetic activity was restricted to one end of each pair of sister chromatids. At metaphase II the chromosomes were also aligned axially.     

Differential replication of heterochromatin in polytene chromosomes of the Old World screw-worm fly,Chrysomya bezziana (Diptera: Calliphoridae), analysed by fluorescence staining

The distribution and replication of heterochromatin in polytene trichogen chromosomes of the Old World screw-worm fly,Chrysomya bezziana, were studied using fluorescent staining techniques. Quinacrine and distamycin-DAPI, which selectively stain AT-rich DNA, and chromomycin, specific for GC-rich sequences, were used. Bright quinacrine and DA-DAPI fluorescence was found in the sex chromosome body and in all autosomal centromere regions. Chromomycin (CMA) staining results in very little bright fluorescence of the sex chromosome body and autosomal centromeric regions, but many bright bands of varying morphology are distributed in autosomal arms. The expected negative CMA staining of quinacrine and DA-DAPI bright regions was not found. The lack of reciprocal staining patterns may result from changes in the higher order chromatin structure of polytene chromosomes, or intercalation of divergent heterochromatic sequences. Comparison of the different staining techniques in mitotic and polytene cells shows that heterochromatin is differentially under-replicated, so that the proportions of the distinct fluorescent-specific chromatin changes during polytenization. CMA staining within autosomal arms suggests that repeated sequences intercalated in euchromatin are co-replicated during polytenization. The numerous fluorescent markers described also provide further morphological features for use in comparative cytological analysis ofC. bezziana.