Characterisation of a short,highly repeated and centromerically localised DNA sequence in crested and marbled newts of the genus Triturus

A 32–33 bp highly repeated DNA sequence, TkS1, has been isolated from genomic DNA of the newt Triturus karelini digested with the restriction endonucleases HaeIII or AluI. TkS1 is known to be localised in the centromeric heterochromatin of all the chromosomes in T. karelini and the related species T. cristatus TkS1 has been shown to be present in varying amounts in the genomic DNA of a range of species of Triturus, including representatives of the two main subgenera Triturus and Palaeotriton. A programme of sequencing of monomers, dimers and trimers of TkS1 was carried out in order to determine the level of conservation of the sequence within and between species of Triturus. Altogether 204 monomer (32/33 bp) clones were made of TkS1 from three individuals of T. karelini, and one individual each of T. cristatus, T. carnifex, T. dobrogicus and T. marmoratus, all members of the subgenus Triturus and the cristatus species group. A number of dimer (64 bp) and trimer (96 bp) clones were also made from DNA of a single specimen of T. karelini digested with HaeIII or AluI. Three distinct types of TkS1 were identified in all species examined, except for T. marmoratus where only two of the types were found. The types were distinguished on the basis of certain recurring divergent patterns in monomers sequenced from T. karelini. Type 1 is mainly characterised by the presence of an AluI site at positions 24–27 and type 3 mainly by the presence of an additional base (C) at position 14. Type 2 normally lacks the AluI site and the C at position 14, as well as having a number of other distinguishing features. TkS1 and its three types have remained remarkably constant in sequence since before the divergence of T. marmoratus from other species in the cristatus species group, about 10 million years ago. Examination of all 204 monomer clones and comparison with consensus sequences for the three types shows less than 5% divergence at any one position in the sequence. There is good evidence from examination of dimer and trimer clones of TkS1 that the different types are intermingled with each other, and all three types are likely to be present on all chromosomes. Dimeric (64 bp) TkS1 clones constructed from AluI fragments of T. karelini DNA show evidence of a trimeric (96 bp) supertype with the pattern type 1-type 3-type 1 that is much more common than would be expected on a random basis. The properties of TkS1 are discussed in relation to models for the involvement of highly repeated satellite DNA in chromosomal growth and evolution.W. Hennig     

Chromosome 1 in crested and marbled newts (Triturus)

The heteromorphic chromosomes 1 of Triturus cristatus carnifex and T. marmoratus were studied in mitotic metaphase after staining with the Giemsa C-banding technique and with the fluorochromes, DAPI (AT-specific) and mithramycin (GC-specific). They were also examined in the lampbrush form under phase-contrast before fixation and after fixation and staining with Giemsa. Chromosomes 1 of T.c. carnifex are asynaptic and achiasmatic throughout most of their long arms. They are also heteromorphic in most of their long arms for the patterns of Giemsa and fluorochrome staining and the distribution of distinctive lampbrush loops. The heteromorphic regions correspond to the regions that are asynaptic and achiasmatic. They stain more strongly with mithramycin and more weakly with DAPI than the remainder of the chromosomes, signifying that their DNA is relatively rich in GC. The patterns of staining with Giemsa and fluorochromes and the distributions of distinctive lateral loops vary from one animal to another in the same species and even in the same population. The asynaptic and achiasmatic regions of chromosomes 1 in T. marmoratus extend throughout the whole of the long arms and well beyond the heterochromatic region. Chiasmata form only in the short arm and occasionally in the short euchromatic segment at the tip of the long arms. The staining patterns of chromosomes 1 in T. marmoratus differ from those in T.c. carnifex although, like carnifex, their DNA is relatively GC-rich. The chromosomes 1 of T. marmoratus are more submetacentric than those of T.c. carnifex. In T. marmoratus chromosome 1B is about 12% shorter than 1A. There is a short paracentric inversion heterozygosity in the long arm of chromosome 1B in T. marmoratus which probably accounts for the lack of chiasmata in the euchromatin that separates the centromere from the start of the heterochromatin. In both carnifex and marmoratus, embryos that are homomorphic for chromosome 1 arrest and die at the late tailbud stage of development. The same applies to F₁ hybrid embryos T.c. carnifex x T. marmoratus, and this has permitted identification of chromosomes 1A and 1B in both species. There is no correspondence between patterns of Giemsa or fluorochrome staining of the heteromorphic regions of chromosome 1 and any feature of the lampbrush chromosomes. However, the short euchromatic ends of the long arms of chromosomes 1 in both species are distinguished in the lampbrush form by a series of uniformly small loops of fine texture associated with very small chromomeres. The Giemsa C-staining patterns of both chromosomes 1A and 1B are different in each of the four subspecies of T. cristatus. T.c. karelinii stands out by having unusually large masses of Giemsa C-staining centromeric heterochromatin on all but 1 of its 12 chromosomes. A scheme is proposed for the evolution of chromosome 1 in T. cristatus and T. marmoratus, based on all available cytological and molecular data.     

Complete meiotic pairing of crested newt chromosomes.

The longest chromosome (number 1) of Trituturus cristatus carries a heteromorphic segment, a heterozygosity perpetuated by a balanced lethal system. The heteromorphic segment is regarded as achiasmate and has been claimed to be asynaptic. Direct observations of chromosome pairing in spermatocytes and oocytes yield some cases where all homologous chromosomes appear to be completely paired, but the individual bivalents could not be identified as pachytene is not particularly clear in this species. The long arms of bivalent 1 usually remain attached by a terminal chiasma in spermatocytes of T. c. cristatus but the corresponding chiasma is only rarely present in T. c. carnifex spermatocytes. Synaptonemal complexes have been measured in both spermatocytes and oocytes of T. c. cristatus. A karyotype constructed from these measurements matches the main features of somatic and lampbrush chromosome karyotypes, indicating that all chromosomes must be completely paired and proportionately represented as synaptonemal complex. The total length of synaptonemal complex is much the same in spermatocytes and oocytes and is similar to the length in spermatocytes of Xenopus laevis. These two amphibian examples supplement a recent survey of other vertebrate classes to reinforce its conclusion that synaptonemal complex length is not related to genome size in vertebrates.     

Distribution of satellite DNA TkS1 in genomes of salamanders (Salamandridae)

Genomes of 22 species and subspecies of salamandrids (Triturus, Cynops, Neurergus, Notophthalmus, Pachytriton, and Pleurodeles ) were studied. Satellite TkS1 (32-33 bp) was found in all examined species, both in direct and reversed orientations. The use of single-primer PCR with oligonucleotides, which are homologous to the most conservative part of satellite TkS1, allowed us to reveal DNA fragments, flanked by single copies (or small tandem repeats) of satellites TkS1 having various (5' and 3') orientation. Such fragments were observed in Pleurodeles waltl, Triturus a. alpestris, T. vulgaris lantzi, T. v. vulgaris, Neurergus crocatus and T. vulgaris graecus. The length of amplified DNA fragments in three subspecies of T. vulgaris differed. This might be connected with unequal amounts and different distribution of 5' and 3' copies of satellite TkS1 in their genomes. Patterns of DNA amplification in Triturus a. alpestris and Neurergus crocatus were quite similar. Only two species (Triturus a. alpestris and T. v. vulgaris) had approximately equal amounts and similar distribution of 5' and 3' copies of satellite TkS1 in their genomes.     

Satellite DNA of the red flour beetle Tribolium castaneum--comparative study of satellites from the genus Tribolium

A highly abundant satellite DNA comprising 17% of the Tribolium castaneum (Insecta, Coleoptera) genome was cloned and sequenced. The satellite monomer is 360 bp long, has a high A+T content of 73%, and lacks significant internal substructures. The sequence variability is 3.6%, essentially due to random distribution of single-point mutations. The satellite is evenly distributed in the regions of centromeric heterochromatin of all 20 chromosomes, as shown by fluorescent in situ hybridization. Comparison of T. castaneum satellite with those from three different but congeneric species reveals the highest sequence similarity of 47.1% with the satellite from the sibling species Tribolium freemani. The phylogenetic relationships among Tribolium species deduced from satellite sequence agree with those based on karyological, chemotaxonomic, and hybridization data. This indicates a parallel in the divergence of satellites and some genetic and cytogenetic characters. Despite low mutual sequence similarity, which makes them species-specific, Tribolium satellites have a common structural characteristic: a block of about 95% A+T content, 20 to 42 bp long, flanked at one side by an inverted repeat which can potentially form a thermodynamically stable dyad structure. Since similar structural features are found in centromeric DNA of Saccharomyces cerevisiae and Chironomus pallidivittatus, their possible importance in centromere function may be inferred.      

Conflict begets complexity: the evolution of centromeres

Centromeres mediate the faithful segregation of eukaryotic chromosomes. Yet they display a remarkable range in size and complexity across eukaryotes, from approximately 125 bp in budding yeast to megabases of repetitive satellites in human chromosomes. Mapping the fine-scale structure of complex centromeres has proven to be daunting, but recent studies have provided a first glimpse into this unexplored bastion of our genomes and the evolutionary pressures that shape it. Evolutionary studies of proteins that bind centromeric DNA suggest genetic conflict as the underlying basis of centromere complexity, drawing interesting parallels with the myriad selfish elements that employ centromeric activity for their own survival.     

Identification of xenopus CENP-A and an associated centromeric DNA repeat

Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors such as binding of the centromeric histone centromere protein A (CENP-A). Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homologue of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with frog centromeric repeat 1 (Fcr1), a 174-base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

Structure and properties of a highly repetitive DNA sequence in sheep

Cleavage of sheep DNA with the restriction endonuclease EcoR I yields three discrete size classes (370, 435 and 800bp) of highly repetitive DNA. The 435bp long fragment was cloned and its nucleotide sequence determined. All three classes of repetitive DNA are related to each other as seen by cross-hybridisation. They are tandemly arranged in the genome and in situ hybridisation to sheep lymphocyte chromosomes show their location mainly in the centromere region of all chromosomes. The primary sequence of the repetitive DNA shows a close structural similarity to the bovine 1.715 satellite DNA, however only poor cross-hybridisation between the sheep and cattle repetitive DNA could be shown.     

Satellite DNA sequences in the human acrocentric chromosomes: information from translocations and heteromorphisms.

Satellite III DNA has been located by in situ hybridization in chromosomes 1, 3--5, 7, 9, 10, 13--18, 20--22, and Y and ribosomal DNA (rDNA) in the acrocentric chromosomes 13--15, 21, and 22. In the acrocentric chromosomes, the satellite DNA is located in the short arm. Here we report comparisons by in situ hybridization of the amount of satellite DNA in Robertsonian translocation and "normal variant" chromosomes with that in their homologs. In almost all dicentric Robertsonian translocations, the amount of satellite DNA is less than that in the normal homologs, but it is rarely completely absent, indicating that satellite DNA is located between the centromere and the nucleolus organizer region (NOR) and that the breakpoints are within the satellite DNA. The amount of satellite DNA shows a range of variation in "normal" chromosomes, and this is still more extreme in "normal variant" chromosomes, those with large short arm (p+ or ph+) generally having more satellite DNA than those with small short arms (p- or ph-). The cytological satellites are heterogeneous in DNA content; some contain satellite DNA, others apparently do not, and the satellite DNA content is not related to the size or intensity of fluorescence of the satellites. The significance of these variations for the putative functions of satellite DNA is discussed.     

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.     

Localisation of the classical DNA satellites on human chromosomes as determined by primed in situ labelling (PRINS)

The chromosomal localisation and relative amounts in humans of the classical DNA satellites I, II and III have been determined by using the primed in situ labelling reaction with a variety of oligonucleotide primers. The centromeres of seven of the human chromosomes, viz. nos 6, 8, 11, 12, 18, 19 and X, are not identifiably marked by any of the primers. A possible phylogenetic explanation of this is suggested and the possible relationship of the classical satellites to the function of the centromere is discussed. Received: 5 November 1996 / Accepted: 25 March 1997     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.