Nature of telomere dimers and chromosome looping in human spermatozoa

Specific and well-organized chromosome architecture in human sperm cells is supported by the prominent interactions between centromeres and between telomeres. The telomere-telomere interactions result in telomere dimers that are positioned at the nuclear periphery. It is unknown whether composition of sperm telomere dimers is random or specific. We now report that telomere dimers result from specific interactions between the two ends of each chromosome. FISH using pairs of subtelomeric DNA probes that correspond to the small and long arms of seven human chromosomes demonstrates that subtelomeres of one chromosome are brought together. Statistical analysis confirmed that telomere associations could not result from the random proximity of DNA sequences. Therefore, chromosomes in human sperm nuclei adopt a looped conformation. This higher-order chromosome structure is most likely required for chromosome withdrawal/decondensation during the early fertilization events leading to zygote formation. These individuals contributed equally to the work     

Highly comprehensive karyotype analysis by a combination of spectral karyotyping (SKY), microdissection, and reverse painting (SKY-MD)

A technique disclosing most information about chromosome modifications is the technique of choice for the analysis of chromosome alterations. The newly developed method for microdissection of fluorescence-labeled chromosomes (FISH-MD) can improve upon this expectation in combination with 24-color spectral karyotyping (SKY). The highly efficient way to detect chromosome modifications by SKY and the detailed specification of aberrant chromosomes by FISH-MD prompted us to use both techniques in a combined approach called SKY-MD. First, an overview of chromosomal aberrations is obtained by spectral karyotyping and subsequently the derivative chromosomes recognized are characterized in a highly specific manner by microdissection and reverse painting. A small quantity of isolated material dissected directly from a 24-color metaphase is sufficient to obtain very detailed information about the chromosome regions and the breakpoints involved in the derivative chromosomes. Therefore, the combination of spectral karyotyping and microdissection in one procedure, and reverse painting can characterize chromosomal aberrations with a degree of specificity hitherto unknown from individual karyotyping experiments. In this article we compare the efficiency of both the SKY technique and that of classical microdissection with the efficiency obtained by SKY-MD.     

Chromosomal localization of the telomeric (TTAGGG)<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> sequence in four species of Armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra

The distribution of the vertebrate telomeric sequence (TTAGGG)_n in four species of armadillos (Dasypodidae, Xenarthra), i.e. Chaetophractus villosus (2n = 60), Chaetophractus vellerosus (2n = 62), Dasypus hybridus (2n = 64) and Zaedyus pichiy (2n = 62) was examined by FISH with a peptide nucleic acid (PNA) probe. Besides the expected telomeric hybridization, interstitial (centromeric) locations of the (TTAGGG)n sequence were observed in one chromosome pair of Chaetophractus vellerosus and Zaedyus pichiy, suggesting chromosome fusion of ancestral chromosomes occurring during the evolution of Dasypodidae. In addition, all the species analysed showed one to four apparently telocentric chromosomes, exhibiting only two telomeric signals. However, the immunodetection study of kinetochore proteins on synaptonemal complex spreads from C. villosus showed that the apparently telocentric chromosomes have a tiny short arm that can be resolved only in the more elongated pachytene bivalents. This finding suggests that none of the species of armadillos possess true telocentric chromosomes. Our present results support a reduction in the diploid number by fusion of acrocentrics with loss of chromosome material as a tendency in Dasypodidae.     

Whole-comparative genomic hybridization (W-CGH): 1. The quick overview of repetitive DNA sequences on a genome

We present a technique (W-CGH) based on Comparative Genomic Hybridization (CGH), but using whole DNA probes, which permits the identification of chromosomal polymorphisms related to highly repetitive DNA sequences that exist between the two genomes compared. The procedure employs two differently colored whole DNA probes from two different individuals that are mixed and hybridized to metaphase chromosomes. The method provides a simple way to map whole genome differences for highly repetitive DNA sequences between two individuals, since it does not require chromosome-specific probes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mapping the Distribution of the Telomeric Sequence (T2AG3) n in the 2n = 14 Ancestral Marsupial Complement and in the Macropodines (Marsupialia: Macropodidae) by fluorescence in situ hybridization

In this study we test the theory that the presence of the conserved vertebrate telomeric sequence (T(2)AG(3))(n) at the centromeres of Australian marsupial 2n = 14 complements is evidence that these karyotypes are recently derived, which is contrary to the generally held view that the 2n = 14 karyotype is ancestral for Australasian and American marsupials. Here we compare the distribution of the (T(2)AG(3))( n ) sequence and constitutive heterochromatin in the presumed ancestral 2n = 14 complement and in complements with known rearrangements. We found that where there were moderate to large amounts of constitutive heterochromatin, the distribution of the (T(2)AG(3))(n) sequence reflected its presence as a native component of satellite DNA rather than its involvement in past rearrangements. The presence of centromeric heterochromatin in all Australian 2n = 14 complements therefore suggests that centromeric sites of the (T(2)AG(3))(n) sequence do not represent evidence for recent rearrangements.     

Comparison of spontaneous background genomic aberration frequencies among cattle, pig and humans using dual-colored FISH

Spontaneous frequencies of stable chromosomal aberrations in farm animals have not been established yet. The aim of this study was to determine the spontaneous background frequencies of structural chromosomal aberrations in cattle and pig, and to compare them with the established findings in humans. Analysis was carried out on peripheral blood samples taken from 29 cows, 15 calves, 15 boars, 13 piglets, and 23 adult and 12 newborn humans. Dual-colored FISH using whole chromosome painting probes specific for human chromosomes 1 and 4, bovine chromosomes 1 and 7, and pig chromosomes 1 and 13 was performed. Chromosome aberrations were classified according to the PAINT nomenclature. The proportions of aberrant cells and the genomic frequencies of translocations, insertions and dicentrics were measured. The highest background translocation frequency was observed in humans (1.40±0.92). Data obtained in boars were similar to those obtained in humans. Cows showed much lower values of studied parameters than was expected. There was no statistical difference in any category of aberration frequencies between cows and calves. Significant differences in genomic frequencies of both total and reciprocal translocations were found when comparing boars with piglets and adult humans with newborn babies. Very low levels of spontaneous background translocation frequencies were seen among calves, piglets and newborn human babies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomes ofDamaliscus (Artiodactyla,Bovidae): Simple and complex centric fusion rearrangements

G- and C-banded karyotypes ofDamaliscus hunteri, D. lunatus andD. pygargus were compared using the standard karyotype ofBos taurus. Chromosomal complements were 2n=36 inD. lunatus jimela, 2n=38 inD. pygargus phillipsi andD. p. pygargus, and 2n=44 inD. hunteri. The fundamental number in all karyotypes was 60. Among the three species ofDamaliscus, seven autosomal pairs and the X chromosomes were conserved. Y-chromosome differences were attributed to heterochromatic additions or deletions. Banded karyotypes of the two subspecies ofD. pygargus exhibited complete homology. Chromosomal complements ofD. pygargus andD. lunatus differed by a simple centric fusion. However, karyotypes ofD. pygargus andD. lunatus differed fromD. hunteri by numerous centric fusions, several of which were related by monobrachial chain complexes. Between the karyotypes ofD. hunteri andD. pygargus orD. lunatus, there were two chain complexes, one involving five chromosomes (chain V) and the other involving 12 inpygargus (chain XII) or 13 inlunatus (chain XIII). There were also two simple centric fusions betweenD. hunteri andD. lunatus/D. pygargus; acrocentric chromosomes 13, 15, 20 and 22 inD. hunteri were fused as 13;15 and 20;22 inD. lunatus andD. pygargus.accepted for publication by D. Ward     

Three-dimensional arrangements of centromeres and telomeres in nuclei of human and murine lymphocytes

The location of centromeres and telomeres was studied in human and mouse lymphocyte nuclei (G0) employing 3D-FISH, confocal microscopy, and quantitative image analysis. In both human and murine lymphocytes, most centromeres were found in clusters at the nuclear periphery. The distribution of telomere clusters, however, differed: in mouse nuclei, most clusters were detected at the nuclear periphery, while, in human nuclei, most clusters were located in the nuclear interior. In human cell nuclei we further studied the nuclear location of individual centromeres and their respective chromosome territories (CTs) for chromosomes 1, 11, 12, 15, 17, 18, 20, and X. We found a peripheral location of both centromeres and CTs for 1, 11, 12, 18, X. A mostly interior nuclear location was observed for CTs 17 and 20 and the CTs of the NOR-bearing acrocentric 15 but the corresponding centromeres were still positioned in the nuclear periphery. Autosomal centromeres, as well as the centromere of the active X, were typically located at the periphery of the respective CTs. In contrast, in about half of the inactive X-CTs, the centromere was located in the territory interior. While the centromere of the active X often participated in the formation of centromere clusters, such a participation was never observed for the centromere of the inactive X. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cattle rob(1;29) originating from complex chromosome rearrangements as revealed by both banding and FISH-mapping techniques

Sixteen carriers of rob(1;29) (one of which was homozygous) from six different breeds (four Italian and two Portuguese), two heterozygous carriers of rob(26;29), three river buffaloes and two sheep were cytogenetically investigated in this study by using banding and FISH-mapping techniques (the latter only in cattle and river buffalo). Single- and dual- colour FISH were used with bovine probes containing both INRA143 (mapping proximally to BTA29) and bovine satellite (SAT) DNA SAT I, SAT III and SAT IV (mapping at the centromeric regions of cattle chromosomes). The combined use of these probes, the comparison of rob(1;29) with the dicentric rob(26;29) and with both river buffalo and sheep chromosomes (biarmed pairs) allowed us to hypothezise that rob(1;29) originated from complex chromosomal rearrangements through at least three sequential events: (a) centric fusion with the formation of a dicentric chromosome; (b) formation of a monocentric chromosome with loss of SAT I from both BTA1 and BTA29, most of SAT IV from BTA29 and, probably, some repeats of SAT III from BTA1; (c) double pericentric inversion or, more probably, a chromosome transposition of a small chromosome segment containing INRA143 from proximal p-arms to proximal q-arm of the translocated chromosome. This study is dedicated to Professor Ingemar Gustavsson, a teacher for many of us, recently retired.     

Transgene integration and chromosome alterations in two transgenic lines of tritordeum

Plants from two transgenic lines of tritordeum (an amphiploid between Triticum turgidum cv. durum and Hordeum chilense) have been analyzed by fluorescence in-situ hybridization (FISH) to characterize the transgene integration sites and chromosome rearrangements. Transgenic lines were transformed in two different events with the genes encoding for the high-molecular-weight glutenin subunits (HMW-GS), 1Ax1 and/or 1Dx5. Three integration sites and four translocations were detected. All three integration sites were located on chromosome segments of Hordeum chilense translocated into wheat chromosomes. No translocations from wheat into H. chilense chromosomes were observed. Both HMW-GS transgenes were expressed at high levels in the endosperm of transgenic plants. The analysis by FISH of transgenic plants allowed the early detection of homozygous and heterozygous plants. The consequences and implications of translocations on breeding are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH

We have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequences, greatly decreasing PCR efficiency selectively across these blocked regions. A second labelling reaction is then done and this product is resuspended in FISH hybridization mixture without further addition of blocking DNA. The hybridization produces little if any non-specific binding on any other chromosomes. We have been able to successfully use this procedure with both human and rat chromosome probes. This technique should be applicable in producing probes for CGH, M-FISH and SKY, as well as reducing the presence of repetitive DNA in genomic libraries     

A comparative ZOO-FISH analysis in bats elucidates the phylogenetic relationships between Megachiroptera and five microchiropteran families

Fluorescence in-situ hybridization with human whole chromosome painting probes (WCPs) was applied to compare the karyotypes of members of five bat families. Twenty-five evolutionarily conserved units (ECUs) were identified by ZOO-FISH analysis. In 10 of these 25 ECUs, thorough GTG-band comparison revealed an identical banding pattern in all families studied. Differences in the remaining ECUs were used as characters to judge the phylogenetic relationships within Chiroptera. Close relations hips were found between Rhinolophidae and Hipposideridae. Also closely related are the representatives of the yangochiropteran families Phyllostomidae (genus studied: Glossophaga, Volleth et al. 1999), Molossidae and Vespertilionidae. All microchiropteran species studied here share four common features not found in the megachiropteran species Eonycteris spelaea. Two of these are considered as derived characters with a high probability of parallel evolution. On the other hand, Eonycteris shares one common, probably derived feature with the rhinolophoid families Rhinolophidae and Hipposideridae and an additional one only with Hipposideridae. At the moment, the relationships between Yangochiroptera, Rhinolophoidea and Megachiroptera must be left in an unsolved trichotomy. Comparison of neighboring segment combinations found in Chiroptera with those found in other mammalian taxa revealed six synapomorphic features for Chiroptera. Therefore, for karyological reasons, monophyly of Chiroptera is strongly supported. This revised version was published online in July 2006 with corrections to the Cover Date.     

Various organizations of the complex repeats in vole sex chromosome heterochromatin

Different patterns of the DNA sequences organization were revealed in the vole (Rodentia) sex chromosome heterochromatin using dual-label fluorescence in-situ hybridization on extended DNA fibers with different repetitive DNA sequences as probes. In Microtus rossiaemeridionalis, the basic type represents the homogeneous relatively short tracks consisting of tandemly reiterated monomers of the MS3 family alternating with similar tracks of MS4 monomers and with non-fluorescent spacers. These tracks varied in the length of both repeats, with an average size of 12-22 kb or 3-5 copies. Apart from this, some continuous tracks of both families spanning 100-200 kb were interrupted by short spacers or single signals from the sequences with homology to LINEs. These results, together with that obtained by the analysis of phage clones of the genomic library, unequivocally demonstrate a variable large-scale DNA structural organization in heterochromatin of the M. rossiaemeridionalis sex chromosome. The dominant type of large-scale DNA organization in M. transcaspicus heterochromatin represents the unicolor relatively long tracks consisting of monotonous but not alternating monomers of MS3 or MS4 with sizes ranging from 15 to 40 kb and separated by extended spacers with an average length of 20 kb. Thus, the formation of the vole sex chromosome heterochromatic regions occurred relatively recently during speciation.     

A probe generated by chromosome microdissection, useful for analyzing Y chromosome evolution in Old World monkeys

We isolated a DNA probe, designated MMDYZ1, using a chromosome microdissection technique from the Y chromosome of the Rhesus monkey. The probe obtained from eight whole Y chromosomes shows higher specificity for the Y short arm of the Rhesus monkey, which consists totally of constitutive heterochromatin. Two microclones (MMY#3 and MMY#4) were constructed from the Y-specific primary PCR products. Sequence analysis of these two microclones revealed that both were essentially identical to each other and the sizes were 870 and 686bp, respectively. From alignment analysis using the Genbank database of primates, the alphoid DNA has the highest affinity with the probe. However, the total composition of this probe has extremely high homology with the Y short arm of the Rhesus monkey, as demonstrated by fluorescence in-situ hybridization (FISH). Comparative FISH-mapping disclosed that this DNA-sequence cluster was located at extremely different sites on the Y chromosome in several species of the Old World monkey. Accordingly, this probe seems to be a high-quality tool, now established for the first time, for investigating Y chromosome evolution of the Old World monkey.     

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.)

Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa. This revised version was published online in July 2006 with corrections to the Cover Date.     

The giant B chromosome of the cyprinid fish Alburnus alburnus harbours a retrotransposon-derived repetitive DNA sequence

The cyprinid fish Alburnus alburnus possesses one of the largest supernumerary chromosomes in all vertebrates. In the present study, amplified fragment length polymorphism analyses (AFLP) and fluorescence in-situ hybridization (FISH) were performed in order to characterize these extraordinary chromosomes in detail. Sequence analysis of the B chromosome-specific DNA revealed a strong homology to a Drosophila Gypsy/Ty3 retrotransposon and also to a medaka (Oryzias latipes) one. The sequence is highly abundant on the B chromosome but undetectable in the normal A chromosome complement. It is also absent from the B chromosome of the closely related species, Rutilus rutilus, suggesting a specific spreading of the mobile element during evolution of the giant supernumerary chromosome within A. alburnus. Meitotic chromosomes were in-situ hybridized with the B chromosome-specific probe, documenting that the additional chromosome behaves as an autopaired ring chromosome in diakineses. Our results suggest that the supernumerary chromosome of A. alburnus is not derived from the normal chromosome complement but has evolved independently.     

High chromosome conservation detected by comparative chromosome painting in chicken, pigeon and passerine birds

Chicken chromosome paints for macrochromosomes 1-10, Z, and the nine largest microchromosomes (Griffin et al. 1999) were used to analyze chromosome homologies between chicken (Gallus gallus domesticus: Galliformes), domestic pigeon (Columba livia: Columbiformes), chaffinch (Fringilla coelebs Passeriformes), and redwing (Turdus iliacus: Passeriformes). High conservation of syntenies was revealed. In general, both macro- and microchromosomes in these birds showed very low levels of interchromosomal rearrangements. Only two cases of rearrangements were found. Chicken chromosome 1 corresponds to chromosome 1 in pigeon, but to chromosomes 3 and 4 in chaffinch and chromosomes 2 and 5 in redwing. Chicken chromosome 4 was shown to be homologous to two pairs of chromosomes in the karyotypes of pigeon and both passerine species. Comparative analysis of chromosome painting data and the results of FISH with (TTAGGG)n probe did not reveal any correlation between the distribution of interstitial telomere sites (ITSs) and chromosome rearrangements in pigeon, chaffinch and redwing. In chaffinch, ITSs were found to co-localize with a tandem repeat GS (Liangouzov et al. 2002), monomers of which contain an internal TTAGGG motif.     

Interstitial telomeric sites and NORs in Hartmann's zebra (Equus zebra hartmannae) chromosomes

Interstitial telomeric sites (ITSs) are considered as signatures of chromosomal rearrangements that take place during karyotype evolution. Understanding that equids have undergone rapid karyotype evolution compared with the average in other mammals, a search of these signatures was carried out in the Hartmann's mountain zebra (Equus zebra hartmannae; EZH) chromosomes. Six consistent ITSs were identified on five of the zebra chromosomes (EZH1p, 1q, 2q, 5q, 6q and 11q). The location of these ITSs coincided with fusion points of some of the evolutionarily conserved human-Hartmann's zebra chromosomal segments suggesting that the sequences are remnants of fusion events between ancestral chromosomes. Incidentally, three of the ITSs also matched with the presence of constitutive heterochromatin. Further, ribosomal gene clusters were localized on five zebra chromosomes and the data were compared with those in other equid species. The findings offer preliminary evidence on the likely evolution of some of the Hartmann's zebra chromosomes and add to the current search for clues that lead to the ancestral chromosomal configuration in equids. This revised version was published online in July 2006 with corrections to the Cover Date.