A 41–42 bp tandemly repeated sequence isolated from nuclear envelopes of chicken erythrocytes is located predominantly on microchromosomes

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41–42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.     

Presence of female-specific bent-repetitive DNA sequences in the genomes of turkey and pheasant and their interactions with W-protein of chicken

Two female-specific repetitive DNA units, the 0.4 kb PstI and 0.5 kb TaqI sequences, were detected in the genomic DNA of turkey and pheasant, respectively, by Southern blot hybridization under non-stringent conditions with the W chromosome-specific 0.7 kb XhoI repetitive unit of chicken as a probe. Cloning and sequencing of these two repetitive units revealed that they shared features with the XhoI family repetitive unit of chicken although the overall similarities of the nucleotide sequences were less than 60%. In common with the chicken XhoI family they consisted of tandem repeats of about 21 bp, the majority of which contained (A)_3–5 and (T)_3–5 clusters separated by six or seven relatively G+C-rich sequences, and they behaved as bent DNA molecules on polyacrylamide gel electrophoresis at room temperature. W-protein, purified from chicken liver nuclei and shown to bind with high affinity to the XhoI family repetitive unit, also bound with the cloned repetitive units from turkey and pheasant. DNase I footprint analysis suggested that the mode of interaction of W-protein with these units was similar to that with the 0.7 kb XhoI sequence. On the other hand, W-protein did not bind to the female-specific 0.4 kb BamHI repetitive unit from the Bobwhite quail. The 0.4 kb BamHI sequence contained some A and T clusters but these clusters did not appear in phase with the pitch of DNA helix and the repetitive unit did not show DNA bending.     

Occupancy of the majority of DNA in the chicken W chromosome by bent-repetitive sequences

Another family of repetitive sequences, designated the EcoRI family, was found in the DNA of the chicken W chromosome by hybridization with the W chromosome-specific XhoI family probe under conditions of low stringency. A 1.2 kb EcoRI fragment, the major repeating unit of the family, was cloned and sequenced. The 1.2 kb unit showed an overall sequence similarity of about 68% to the 0.7 kb XhoI family repeating unit and it consisted of tandem repeats of average length 21 bp, most of which contained (A)_3–5 and (T)_3–4 clusters separated by 6–8 G+C-rich sequences. These features and its behavior as a strongly bent molecule in solution were very similar to those found for other W chromosome-specific repetitive sequences in the order Galliformes: XhoI family of chicken, PstI family of turkey and TaqI family of pheasant. The cloned 1.2 kb unit contained 78 CpG dinucleotide sequences and those that were in HapII, HhaI and BstUI sites were shown to be extensively methylated in the genomic DNA. Repetition frequencies of the 1.2 kb unit among the female population of chicken fell into high- and low-level classes, which accounted for about 30% and 10%, respectively, of the DNa in the W chromosome. Thus, 70% to 90% of the DNA in the chicken W chromosome was shown to be occupied by bent-repetitive sequences. The EcoRI and XhoI family sequences were not intermingled over the short range but each family formed a unique domain ranging from one to several million base pairs.by H.C. Macgregor     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

Microsatellites from the linkage groups E26C13 and E50C23 are located on the Gallus gallus domesticus microchromosomes 20 and 21

Using the method of dual color fluorescence in situ hybridization and a set of chromosome-specific BAC clones, localization of microsatellites LEI0345 and LEI0336 on chicken (Gallus gallus domesticus) mitotic chromosomes was performed. Microsatellite LEI0345 (TAM 32, BAC clones r49A10 and r55M23) from the linkage group E26C13 was mapped to microchromosome 20, while microsatellite LEI0336 (TAM 32, BAC clones r19E22 and r13C08) from the linkage E50C23 was assigned to microchromosome 21. Using the PCR technique, an attempt to assign the suitable markers to chromosome-specific BAC clones was made. The PCR data confirmed the microsatellite localization performed with the help of FISH technique and showed the presence of the LEI0345 microsatellite sequence on many other chicken microchromosomes, except for microchromosomes 19 and 22. Linkage groups E26C13 and E50C23 were assigned to microchromosomes 20 and 21, respectively.     

FISH mapping of 57 BAC clones reveals strong conservation of synteny between Galliformes and Anseriformes

Karyotypes of chicken (Gallus gallus domesticus; 2n = 78) and mallard duck (Anas platyrhynchos; 2n = 80) share the typical organization of avian karyotypes including a few macrochromosome pairs, numerous indistinguishable microchromosomes, and Z and W sex chromosomes. Previous banding studies revealed great similarities between chickens and ducks, but it was not possible to use comparative banding for the microchromosomes. In order to establish precise chromosome correspondences between these two species, particularly for microchromosomes, we hybridized 57 BAC clones previously assigned to the chicken genome to duck metaphase spreads. Although most of the clones showed similar localizations, we found a few intrachromosomal rearrangements of the macrochromosomes and an additional microchromosome pair in ducks. BAC clones specific for chicken microchromosomes were localized to separate duck microchromosomes and clones mapping to the same chicken microchromosome hybridized to the same duck microchromosome, demonstrating a high conservation of synteny. These results demonstrate that the evolution of karyotypes in avian species is the result of fusion and/or fission processes and not translocations.     

Characterization of the turkey MHC chromosome through genetic and physical mapping

Previous studies in the chicken have identified a single microchromosome (GGA16) containing the ribosomal DNA (rDNA) and two genetically unlinked MHC regions, MHC-B and MHC-Y. Chicken DNA sequence from these loci was used to develop PCR primers for amplification of homologous fragments from the turkey (Meleagris gallopavo). PCR products were sequenced and overgo probes were designed to screen the CHORI 260 turkey BAC library. BAC clones corresponding to the turkey rDNA, MHC-B and MHC-Y were identified. BAC end and subclone sequencing confirmed identity and homology of the turkey BAC clones to the respective chicken loci. Based on subclone sequences, single-nucleotide polymorphisms (SNPs) segregating within the UMN/NTBF mapping population were identified and genotyped. Analysis of SNP genotypes found the B and Y to be genetically unlinked in the turkey. Silver staining of metaphase chromosomes identified a single pair of microchromosomes with nucleolar organizer regions (NORs). Physical locations of the rDNA and MHC loci were determined by fluorescence in situ hybridization (FISH) of the BAC clones to metaphase chromosomes. FISH clearly positioned the rDNA distal to the Y locus on the q-arm of the MHC chromosome and the MHC-B on the p-arm. An internal telomere array on the MHC chromosome separates the B and Y loci.     

Chromosome reshuffling in birds of prey: the karyotype of the world's largest eagle (Harpy eagle, Harpia harpyja) compared to that of the chicken (Gallus gallus)

Like various other diurnal birds of prey, the world's largest eagle, the Harpy (Harpia harpyja), presents an atypical bird karyotype with 2n=58 chromosomes. There is little knowledge about the dramatic changes in the genomic reorganization of these species compared to other birds. Since recently, the chicken provides a “default map” for various birds including the first genomic DNA sequence of a bird species. Obviously, the gross division of the chicken genome into relatively gene-poor macrochromosomes and predominantly gene-rich microchromosomes has been conserved for more than 150 million years in most bird species. Here, we present classical features of the Harpy eagle karyotype but also chromosomal homologies between H. harpyja and the chicken by chromosome painting and comparison to the chicken genome map. We used two different sets of painting probes: (1) chicken chromosomes were divided into three size categories: (a) macrochromosomes 1–5 and Z, (b) medium-sized chromosomes 6–10, and (c) 19 microchromosomes; (2) combinatorially labeled chicken chromosome paints 1–6 and Z. Both probe sets were visualized on H. harpyja chromosomes by multicolor fluorescence in situ hybridization (FISH). Our data show how the organization into micro- and macrochromosomes has been lost in the Harpy eagle, seemingly without any preference or constraints.     

Heterochromatin and highly repeated DNA sequences in rye (Secale cereale)

Secale cereale DNA, of mean fragment length 500 bp, was fractionated by hydroxylapatite chromatography to allow recovery of a very rapidly renaturing fraction (C₀t 0–0.02). This DNA fraction was shown to contain several families of highly repeated sequence DNA. Two highly repeated families were purified; (1) a fraction which renatured to a density of 1.701 g/ cc and comprised 2–4% of the total genome, and (2) polypyrimidine tract DNA which comprised 0.1% of the total genome. The 1.701 g/cc DNA consisted of short sequence repeat units (5–50 bp long) tandemly repeated in blocks 30 kb long, while a portion of the polypyrimidine tract DNA behaved as part of a much larger block of tandemly repeated sequences. The chromosomal location of these sequences was determined by the in situ hybridisation of radioactive, complementary RNA to root tip mitotic chromosomes and showed the 1.701 g/cc sequences to be largely limited to the telomeric blocks of heterochromatin, accounting for 25–50% of the DNA present in these parts of the chromosomes. The polypyrimidine tracts were distributed at interstitial locations with 20–30% of the sequences at three well defined sites. The combined distributions of the 1.701 g/cc DNA sequences and polypyrimidine tracts effectively individualised each rye chromosome thus providing a sensitive means of identifying these chromosomes. The B chromosomes present in Secale cereale cv. Unevita, did not show defined locations for the sequences analysed. — The data are discussed in terms of the structure of the rye genome and the generality of the observed genomic arrangement of highly repeated sequence DNA.     

Banded krait minor satellite (Bkm) contains sex and species-specific repetitive DNA

A novel class of repetitive DNA was isolated from a Bkm DNA library by exclusion hybridization. This sequence was mapped to the short arm of the W chromosome of banded krait, Bungarus fasciatus. Southern blot hybridization showed that these sequences are sex and species specific. Sequence analysis of a 206 bp long clone, BR87, revealed the presence of a tandem array of two internal repeat units of 18–19 bp alternating with each other with a gap of 1,2 or 3 nucleotides. To our knowledge, this is the first report of an exclusively W chromosome-and species-specific repeat isolated from any reptile. The functional significance of this sequence based on its organisation is discussed.     

Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis,Lacertidae) and elucidation of karyotype evolution in lacertid lizards

The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n?=?38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11–18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

Isolation and chromosomal localization of a germ line-specific highly repetitive DNA family in Acricotopus lucidus (Diptera, Chironomidae)

. In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line. Received: 12 April 1997; in revised form 26 June 1997 / Accepted: 29 June 1997     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes)

We isolated a new family of satellite DNA sequences from Hae III- and Eco RI-digested genomic DNA of the Blakistons fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.An erratum to this article can be found at Communicated by Y. Hiraoka     

Tetraploid-octoploid relationships and karyological evolution in the order Acipenseriformes (Pisces) karyotypes,nucleoli, and nucleolus-organizer regions in four acipenserid species

The karyotypes of four Acipenseriformes species, Acipenser gueldenstaedti, 2n=250±8, A. ruthenus, A. stellatus and Huso huso, 2n=118±2, are described. In all four karyotypes the majority of chromosomes are meta- and submetacentric macrochromosomes, and microchromosomes of different morphology make up about one third of the set. In A. ruthenus the NORs are located in the telomeric region of a pair of microchromosomes and at least in one pair of middle-size acrocentrics, and in A. stellatus and Huso huso also in the telomeric regions of at least one pair of microchromosomes. The modal number of active nucleoli in A. gueldenstaedti nuclei amounts to 6–8 (range 2–12), in A. ruthenus, A. stellatus and H. huso nuclei to 2–3 (range 1–6). The data obtained point to the tetraploid origin of Acipenseriformes species with 120 chromosomes and to the octoploid origin of species with 240–260 chromosomes.     

Association and in Silico Assignment of Sequences from Turkey BACs

Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.     

Association and in silico assignment of sequences from turkey BACs

Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.     

FISH on avian lampbrush chromosomes produces higher resolution gene mapping

Giant lampbrush chromosomes, which are characteristic of the diplotene stage of prophase I during avian oogenesis, represent a very promising system for precise physical gene mapping. We applied 35 chicken BAC and 4 PAC clones to both mitotic metaphase chromosomes and meiotic lampbrush chromosomes of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Fluorescence in situ hybridization (FISH) mapping on lampbrush chromosomes allowed us to distinguish closely located probes and revealed gene order more precisely. Our data extended the data earlier obtained using FISH to chicken and quail metaphase chromosomes 1–6 and Z. Extremely low levels of inter- and intra-chromosomal rearrangements in the chicken and Japanese quail were demonstrated again. Moreover, we did not confirm the presence of a pericentric inversion in Japanese quail chromosome 4 as compared to chicken chromosome 4. Twelve BAC clones specific for chicken chromosome 4p and 4q showed the same order in quail as in chicken when FISH was performed on lampbrush chromosomes. The centromeres of chicken and quail chromosomes 4 seem to have formed independently after centric fusion of ancestral chromosome 4 and a microchromosome.     

Chromosomal mapping of chicken mega-telomere arrays to GGA9, 16, 28 and W using a cytogenomic approach

Four mega-telomere loci were mapped to chicken chromosomes 9, 16, 28, and the W sex chromosome by dual-color fluorescence in situ hybridization using a telomeric sequence probe and BAC clones previously assigned to chicken chromosomes. The in-common features of the mega-telomere chromosomes are that microchromosomes are involved rather than macrochromosomes; in three cases (9, 16, 28) acrocentrics are involved with the mega-telomeres mapping to the p arms. Three of the four chromosomes (9, 16, W) encode tandem repeats which in two cases (9 and 16) involve the ribosomal DNA arrays (the 5S and 18S-5.8S-28S gene repeats, respectively). All involved chromosomes have a typical-sized telomere on the opposite terminus. Intra- and interindividual variation for mega-telomere distribution are discussed in terms of karyotype abnormalities and the potential for mitotic instability of some telomeres. The diversity and distribution of telomere array quantity in the chicken genome should be useful in contributing to research related to telomere length regulation - how and by what mechanism genomes and individual chromosomes establish and maintain distinct sets of telomere array sizes, as well as for future studies related to stability of the chicken genome affecting development, growth, cellular lifespan and disease. An additional impact of this study includes the listing of BAC clones (26 autosomal and six W BACs tested) that were cytogenetically verified; this set of BACs provide a useful tool for future cytogenetic analyses of the microchromosomes.