Analysis and location of a rice BAG clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG) n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescence in situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Analysis and location of a rice BAG clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)_n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)_n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.     

Pericentromeric location of the telomeric DNA sequences on the European grayling chromosomes

The chromosomal characteristics, locations and variations of the C-band positive heterochromatin and telomeric DNA sequences were studied in the European grayling karyotype (Thymallus thymallus, Salmonidae) using conventional C-banding, endonucleases digestion banding, silver nitrate (AgNO₃), chromomycin A₃ and 4′,6-diamidino-2-phenylindole staining techniques as well as fluorescence in situ hybridization (FISH) and primed in situ labelling. Original data on the chromosomal distribution of segments resistant to AluI restriction endonuclease and identification of the C-banded heterochromatin presented here have been used to characterize the grayling karyotype polymorphism. Structural and length polymorphism of the chromosome 21 showing a conspicuous heterochromatin block adjacent to the centromere seems to be the result of the deletion and inversion. Two pairs of nuclear organizer regions (NOR)-bearing chromosomes were found to be polymorphic in size and displaying several distinct forms. FISH with telomeric peptide nucleic acid probe enabled recognition of the conservative telomeric DNA sequences. The karyotype of the thymallid fish is thought to experienced numerous pericentric inversions and internal telomeric sites (ITSs) observed at the pericentromeric regions of the six European grayling metacentric chromosomes are likely relics of the these rearrangements. None of the ITS sites matched either chromosome 21 or NOR bearing chromosomes.     

DNA methylation at tobacco telomeric sequences

Majerová et al. (Plant Mol Biol, 2011) have recently reported that a considerable fraction of cytosines at tobacco telomeres is methylated. Although the data presented in this report indicate that tobacco telomeric sequences undergo certain levels of DNA methylation, it is not clear whether the methylated sequences are at telomeres, at internal chromosomal loci or at both.     

Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences.

The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed.     

Stability and structure of telomeric DNA sequences forming quadruplexes containing four G-tetrads with different topological arrangements

Telomeres are DNA-protein structures at the ends of eukaryotic chromosomes, the DNA of which comprise noncoding repeats of guanine-rich sequences. Telomeric DNA plays a fundamental role in protecting the cell from recombination and degradation. Telomeric sequences can form quadruplex structures stabilized by guanine quartets. These structures can be constructed from one, two, or four oligonucleotidic strands. Here, we report the thermodynamic characterization of the stability, analyzed by differential scanning calorimetry, of three DNA quadruplexes of different molecularity, all containing four G-tetrads. The conformational properties of these quadruple helices were studied by circular dichroism. The investigated oligomers form well-defined G-quadruplex structures in the presence of sodium ions. Two have the truncated telomeric sequence from Oxytricha, d(TGGGGT) and d(GGGGTTTTGGGG), which form a tetramolecular and bimolecular quadruplex, respectively. The third sequence, d(GGGGTTGGGGTGTGGGGTTGGGG) was designed to form a unimolecular quadruplex. The thermodynamic parameters of these quadruplexes have been determined. The tetramolecular structure is thermodynamically more stable than the bimolecular one, which, in turn, is more stable than the unimolecular one. The experimental data were discussed in light of the molecular-modeling study.     

Origin-dependent initiation of DNA replication within telomeric sequences

Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.     

Construction of a BAC contig containing the xa5 locus in rice

 The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F₂ population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Received: 12 January 1998 / Accepted: 27 May 1998     

Differential maintenance of DNA sequences in telomeric and centromeric heterochromatin

Repeated DNA in heterochromatin presents enormous difficulties for whole-genome sequencing; hence, sequence organization in a significant portion of the genomes of multicellular organisms is relatively unknown. Two sequenced BACs now allow us to compare telomeric retrotransposon arrays from Drosophila melanogaster telomeres with an array of telomeric retrotransposons that transposed into the centromeric region of the Y chromosome >13 MYA, providing a unique opportunity to compare the structural evolution of this retrotransposon in two contexts. We find that these retrotransposon arrays, both heterochromatic, are maintained quite differently, resulting in sequence organizations that apparently reflect different roles in the two chromosomal environments. The telomere array has grown only by transposition of new elements to the chromosome end; the centromeric array instead has grown by repeated amplifications of segments of the original telomere array. Many elements in the telomere have been variably 5'-truncated apparently by gradual erosion and irregular deletions of the chromosome end; however, a significant fraction (4 and possibly 5 or 6 of 15 elements examined) remain complete and capable of further retrotransposition. In contrast, each element in the centromere region has lost ≥ 40% of its sequence by internal, rather than terminal, deletions, and no element retains a significant part of the original coding region. Thus the centromeric array has been restructured to resemble the highly repetitive satellite sequences typical of centromeres in multicellular organisms, whereas, over a similar or longer time period, the telomere array has maintained its ability to provide retrotransposons competent to extend telomere ends.     

Isolation and mapping of bacterial artificial chromosome (BAC) clone containing telomere-associated sequence

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Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro

Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes. We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands. Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures. Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase. Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.     

Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice

Apomixis in plants is a form of clonal reproduction through seeds. A BAC clone linked to apomictic reproduction in Paspalum simplex was used to locate the apomixis locus on meiotic chromosome preparations. Fluorescent in situ hybridisation revealed the existence of a single locus embedded in a heterochromatin-poor region not adjacent to the centromere. We report here for the first time information regarding the sequencing of a large DNA clone from the apomixis locus. The presence of two genes whose rice homologs were mapped on the telomeric part of the long arm of rice chromosome 12 confirmed the strong synteny between the apomixis locus of P. simplex with the related area of the rice genome at the map level. Comparative analysis of this region with rice as representative of a sexual species revealed large-scale rearrangements due to transposable elements and small-scale rearrangements due to deletions and single point mutations. Both types of rearrangements induced the loss of coding capacity of large portions of the “apomictic” genes compared to their rice homologs. Our results are discussed in relation to the use of rice genome data for positional cloning of apomixis genes and to the possible role of rearranged supernumerary genes in the apomictic process of P. simplex. Ornella Calderini and Song B. Chang have contributed equally to this article     

Rescue of the neural tube defect of loop-tail mice by a BAC clone containing the Ltap gene

The mouse mutant loop-tail (Lp) is an accepted model for the study of neural tube defects (NTDs) in humans. Whereas Lp/+ heterozygotes show a mild tail defect (looped), homozygous Lp/Lp embryos show a very severe form of NTD, with a completely open neural tube from the hindbrain region to the caudal portion of the spinal cord (craniorachischisis). We have recently identified a positional candidate for Lp on chromosome 1, designated as Ltap. Here, we have used an in vivo complementation approach in transgenic mice to attempt to correct the looped-tail phenotype with a bacterial artificial chromosome clone (BAC280A23) that harbors a full-length copy of the Ltap gene. Genotype:phenotype correlations in Lp/+ heterozygotes carrying BAC280A23 show that this clone can rescue the looped-tail phenotype in two independent founder lines (P < 0.05 and P < 0.0001). Importantly, BAC280A23 is also observed to rescue the lethal NTD of Lp/Lp homozygotes, because several viable transgenic Lp/Lp mice could be identified and appeared normal (P < 0.05). Results from these gain-of-function transgenic animals strongly suggest that the positional candidate Ltap present in this BAC is indeed the gene that is defective in loop-tail.     

Protection of DNA ends by telomeric 3' G-tail sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.     

A TRF1-controlled common fragile site containing interstitial telomeric sequences

Mouse telomeres have been suggested to resemble common fragile sites (CFS), showing disrupted TTAGGG fluorescent in situ hybridization signals after aphidicolin treatment. This “fragile” telomere phenotype is induced by deletion of TRF1, a shelterin protein that binds telomeric DNA and promotes efficient replication of the telomeric ds[TTAGGG]n tracts. Here we show that the chromosome-internal TTAGGG repeats present at human chromosome 2q14 form an aphidicolin-induced CFS. TRF1 binds to and stabilizes CFS 2q14 but does not affect other CFS, establishing 2q14 as the first CFS controlled by a sequence-specific DNA binding protein. The data show that telomeric DNA is inherently fragile regardless of its genomic position and imply that CFS can be caused by a specific DNA sequence.     

Genetic and physical maps and a clone bank of mitochondrial DNA from rice

Summary Mitochondrial DNA (mtDNA) was isolated from young green leaves of rice plants. DNA fragments were cloned into lambda DNA, and clones that hybridized to mitochondrial genes from other plants were selected. Distal restriction fragments of these clones were used as probes for the selection of overlapping clones. A genetic map was finally created from the library by walking along the genome. The mitochondrial genome consists of five basic circles, with each circle sharing homologous sequences with one or two other circles. A master circle was constructed from the results of recombination across repeated sequences, and its size was estimated to be 492 kb. A physical map and a bank of overlapping clones were also constructed.