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Stability study of sorghum BAC 13I16. DNA was isolated from 38 single colonies derived from one-day (a) and five-day (b) cultures and digested with HindIII. (a) Four clones (lanes a1, a2, a3, a4) showed a modified but apparently identical restriction pattern . (b) Seven clones (lanes b1, b2, b3, b4, b5, b6, b7) showed modified restriction patterns. The pattern in lanes b6 and b7 is similar to the one in lanes a1 to a4. Lanes a0 and b0 are HindIII-digested λDNA.
Source publication
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 B...
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Citations
... Despite the increasing evidence pointing to a functional significance of the repetitive DNA fraction, there are still limitations to characterizing their role in different biological processes due to their high diversity, genomic abundance, complex evolution mode, and difficulty in isolation and sequencing [29][30][31]. Thus, the real biological relevance of this fraction of eukaryotic genomes is yet to be revealed. ...
Eukaryotic genomes are rich in repetitive DNA sequences grouped in two classes regarding their genomic organization: tandem repeats and dispersed repeats. In tandem repeats, copies of a short DNA sequence are positioned one after another within the genome, while in dispersed repeats, these copies are randomly distributed. In this review we provide evidence that both tandem and dispersed repeats can have a similar organization, which leads us to suggest an update to their classification based on the sequence features, concretely regarding the presence or absence of retrotransposons/transposon specific domains. In addition, we analyze several studies that show that a repetitive element can be remodeled into repetitive non-coding or coding sequences, suggesting (1) an evolutionary relationship among DNA sequences, and (2) that the evolution of the genomes involved frequent repetitive sequence reshuffling, a process that we have designated as a "DNA remodeling mechanism". The alternative classification of the repetitive DNA sequences here proposed will provide a novel theoretical framework that recognizes the importance of DNA remodeling for the evolution and plasticity of eukaryotic genomes.
... Despite the increasing evidence pointing to a functional significance of the repetitive DNA fraction, there are still limitations to characterizing their role in different biological processes due to their high diversity, genomic abundance, complex evolution mode, and difficulty in isolation and sequencing [29][30][31]. Thus, the real biological relevance of this fraction of eukaryotic genomes is yet to be revealed. ...
Abstract: Eukaryotic genomes are rich in repetitive DNA sequences grouped in two classes regarding their genomic organization: tandem repeats and dispersed repeats. In tandem repeats, copies of a short DNA sequence are positioned one after another within the genome, while in dispersed repeats, these copies are randomly distributed. In this review we provide evidence that both tandem and dispersed repeats can have a similar organization, which leads us to suggest an update to their classification based on the sequence features, concretely regarding the presence or absence of retrotransposons/transposon specific domains. In addition, we analyze several studies that show that a repetitive element can be remodeled into repetitive non-coding or coding sequences, suggesting (1) an evolutionary relationship among DNA sequences, and (2) that the evolution of the genomes involved frequent repetitive sequence reshuffling, a process that we have designated as a “DNA remodeling mechanism”. The alternative classification of the repetitive DNA sequences here proposed will provide a novel theoretical framework that recognizes the importance of DNA remodeling for the evolution and plasticity of eukaryotic genomes.
... Despite the increasing evidence pointing to a functional significance of the repetitive DNA fraction, there are still limitations to characterizing their role in different biological processes due to their high diversity, genomic abundance, complex evolution mode, and difficulty in isolation and sequencing [29][30][31]. Thus, the real biological relevance of this fraction of eukaryotic genomes is yet to be revealed. ...
... Sequences identical or similar to two parts (corresponding to each flank of the C42 transgene) of such an 'empty' site are interspersed in supercontig 1.836, but rather than forming an expected contiguous DNA stretch, they are spaced by a minimum 4 kb. Thus, the supercontig, or other currently available A. aegypti genome data (Nene et al., 2007), do not provide information on a larger genomic context of the transgene integration, perhaps because of instability of shotgun clones containing tandemly repeated sequences (Song et al., 2001) and/or inadequate genome sampling during the A. aegypti genome project. Lack of the SDR-linked contigs within the A. aegypti genome has been highlighted earlier (Hall et al., 2014). ...
In many taxa, sex chromosomes are heteromorphic and largely non-recombining. Evolutionary models predict that spread of recombination suppression on the Y chromosome is fueled by the accumulation of sexually antagonistic alleles in close linkage to the sex determination region. However, empirical evidence for the existence of sexually antagonistic alleles is scarce. In the mosquito Aedes aegypti, the sex-determining chromosomes are homomorphic. The region of suppressed recombination, which surrounds the male-specific sex-determining gene, remains very small, despite ancient origin of the sex chromosomes in the Aedes lineage. We conducted a genetic analysis of the A. aegypti chromosome region tightly linked to the sex locus. We used a strain with an enhanced green fluorescent protein (EGFP)-tagged transgene inserted near the male-determining gene to monitor crossing-over events close to the boundary of the sex-determining region (SDR), and to trace the inheritance pattern of the transgene in relation to sex. In a series of crossing experiments involving individuals with a recombinant sex chromosome we found developmental abnormalities leading to 1:2 sex biases, caused by lethality of half of the male or female progeny. Our results suggest that various factors causing sex-specific lethal effects are clustered within the neighborhood of the SDR, which in the affected sex are likely lost or gained through recombination, leading to death. These may include genes that are recessive lethal, vital for development and/or sexually antagonistic. The sex chromosome fragment in question represents a fascinating test case for the analysis of processes that shape stable boundaries of a non-recombining region.Heredity advance online publication, 3 August 2016; doi:10.1038/hdy.2016.57.
... Clones displaying distinct restriction digestion patterns and positive for pSc200 or pSc250 were chosen for finer analyses. Namely, these clones were first subjected to a stability analysis [69], then restriction mapped using either partial digestion with one enzyme or a complete digestion with two [70]. ...
Background
A prominent and distinctive feature of the rye (Secale cereale) chromosomes is the presence of massive blocks of subtelomeric heterochromatin, the size of which is correlated with the copy number of tandem arrays. The rapidity with which these regions have formed over the period of speciation remains unexplained.
Results
Using a BAC library created from the short arm telosome of rye chromosome 1R we uncovered numerous arrays of the pSc200 and pSc250 tandem repeat families which are concentrated in subtelomeric heterochromatin and identified the adjacent DNA sequences. The arrays show significant heterogeneity in monomer organization. 454 reads were used to gain a representation of the expansion of these tandem repeats across the whole rye genome. The presence of multiple, relatively short monomer arrays, coupled with the mainly star-like topology of the monomer phylogenetic trees, was taken as indicative of a rapid expansion of the pSc200 and pSc250 arrays. The evolution of subtelomeric heterochromatin appears to have included a significant contribution of illegitimate recombination. The composition of transposable elements (TEs) within the regions flanking the pSc200 and pSc250 arrays differed markedly from that in the genome a whole. Solo-LTRs were strongly enriched, suggestive of a history of active ectopic exchange. Several DNA motifs were over-represented within the LTR sequences.
Conclusion
The large blocks of subtelomeric heterochromatin have arisen from the combined activity of TEs and the expansion of the tandem repeats. The expansion was likely based on a highly complex network of recombination mechanisms.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2667-5) contains supplementary material, which is available to authorized users.
... When cloning of genomic DNA fragments (in a vector such as a plasmid, fosmid, bacterial artificial chromosome or yeast artificial chromosome) is included in the analysis of repetitive DNA, another factor makes it difficult to obtain accurate nucleotide sequences, that is, the low reliability of sequence reads due to the possible rearrangements occurring during the maintenance or amplification in cells of host bacteria or yeast. Degradation of cloned tandemrepeat DNA is a widely known phenomenon with a number of illustrations in the literature (Brutlag et al., 1977;Neil et al., 1990;Song et al., 2001). In the present study, we examined the effect of the culturing temperature of the host bacteria on the degree of degradation of a repetitive DNA fragment cloned into a bacterial plasmid. ...
For accurate analyses of eukaryotic tandem-repeat DNA, it is often required to clone a genomic DNA frag-ment into a bacterial plasmid. It is, however, a serious problem that tandem-repeat DNA is frequently sub-jected to structural changes during maintenance or amplification in the host bacteria. Here, we show an example of a clear difference in the instability of tandem-repeat DNA between different culturing tempera-tures. A fragment of monkey centromeric DNA carried by pUC19 was considerably degraded by culturing bacteria at 37 °C, but the damage was reduced at 25 °C. Thus, culturing temperature is a significant factor for avoiding degradation, in addition to the genotype of the host bacteria. Genomes of higher eukaryotes generally contain large amounts of tandem-repeat DNA, such as centromeric, telomeric and ribosomal DNA sequences. For accu-rate and fine structural analyses of such DNA, sequence data obtained from shotgun sequencing (even in the case of a hierarchical shotgun strategy) are often inadequate because of the difficulty of as-sembling contig sequences from sequence reads. This is considered a serious problem, especially for repeat DNA with high sequence homogeneity among repeat units. As a reflection of this problem, great portions of large-scale tandem-repeat regions are often put aside from the genome browsers built by genome sequen-cing projects. When cloning of genomic DNA fragments (in a vector such as a plasmid, fosmid, bacterial artificial chromosome or yeast artificial chromosome) is in-cluded in the analysis of repetitive DNA, another fac-tor makes it difficult to obtain accurate nucleotide sequences, that is, the low reliability of sequence reads due to the possible rearrangements occurring during the maintenance or amplification in cells of host bacteria or yeast. Degradation of cloned tandem-repeat DNA is a widely known phenomenon with a
... though BACs are considered to be stable cloning vectors, it has been reported that tandem repeats can be unstable in BAC clones (Song et al., 2001). In conclusion, comparisons between BACs and the genome sequence data suggest that small arrays of satellite units can be well-resolved, while larger satellite blocks may www.frontiersin.org ...
Subtelomeric regions in eukaryotic organisms are known for harboring species-specific tandemly repeated satellite sequences. However, studies on the molecular organization and evolution of subtelomeric repeats are scarce, especially in plants. Khipu is a satellite DNA of 528-bp repeat unit, specific of the Phaseolus genus, with a subtelomeric distribution in common bean, P. vulgaris. To investigate the genomic organization and the evolution of khipu, we performed genome-wide analysis on the complete genome sequence of the common bean genotype G19833. We identified 2,460 khipu units located at most distal ends of the sequenced regions. Khipu units are arranged in discrete blocks of 2–55 copies and are heterogeneously distributed among the different chromosome ends of G19833 (from 0 to 555 khipus units per chromosome arm). Phylogenetically related khipu units are spread between numerous chromosome ends, suggesting frequent exchanges between non-homologous subtelomeres. However, most subclades contain numerous khipu units from only one or few chromosome ends indicating that local duplication is also driving khipu expansion. Unexpectedly, we also identified 81 khipu units located at centromeres. All the centromeric khipu units belong to a single divergent clade also comprised of a few units from several subtelomeres, suggesting that a few sequence exchanges between centromeres and subtelomeres took place in the common bean genome. The divergence and low copy number of these centromeric units from the subtelomeric units could explain why they were not detected by FISH (Fluorescence in situ Hybridization) although it can not be excluded that these centromeric units may have resulted from errors in the pseudomolecule assembly. Altogether our data highlight extensive sequence exchanges in subtelomeres between non-homologous chromosomes in common bean and confirm that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes.
... It should also be possible to introduce large (up to 160 kb) centromeric BACs into plant genomes using Agrobacterium and specialized T-DNA vectors (Hamilton et al., 1996;Liu et al., 1999). However, given the instability of repeat arrays in E. coli and Agrobacterium, there are inherent limitations to what can be accomplished using BAC clones (Song et al., 2001(Song et al., , 2003Nakano et al., 2005;Chang et al., 2011). In addition, a major unaddressed biological limitation is the fact that centromeres are specified in a sequence-independent (epigenetic) fashion (Birchler and Han, 2009). ...
Plant genome engineering as a practical matter will require stable introduction of long and complex segments of DNA sequence into plant genomes. Here we show that it is possible to synthetically engineer and introduce centromere-sized satellite repeat arrays into maize. We designed a synthetic repeat monomer of 156 bp that contains five DNA-binding motifs (LacO, TetO, Gal4, LexA, and CENPB), and extended it into tandem arrays using an overlapping PCR method similar to that commonly used in gene synthesis. The PCR products were then directly transformed into maize using biolistic transformation. We identified three resulting insertion sites (arrayed binding sites), the longest of which is at least 1100 kb. The LacI DNA-binding module is sufficient to efficiently tether YFP to the arrayed binding sites. We conclude that synthetic repeats can be delivered into plant cells by omitting passage through Escherichia coli, that they generally insert into one locus, and that great lengths may be achieved. It is anticipated that these experimental approaches will be useful for future applications in artificial chromosome design.
... Additionally, the number of successfully generated clones decreases when attempting to achieve higher insert sizes, and there has been suggestion that there are species-specific library insert-size limitations based on base-pair content and sequence dissimilarities [60]. In addition, BAC insert rearrangement can occur in the early stage of library construction and duplication [61]. Furthermore, BAC clones containing tandemly repeated DNA sequences are not stable in E. coli during routine maintenance and propagation. ...
Pulse crops are considered minor on a global scale despite their nutritional value for human consumption. Therefore, they are relatively less extensively studied in comparison with the major crops. The need to improve pulse crop production and quality will increase with the increasing global demand for food security and people's awareness of nutritious food. The improvement of pulse crops will require fully utilizing all their genetic resources. Bacterial artificial chromosome (BAC) libraries of pulse crops are essential genomic resources that have the potential to accelerate gene discovery and enhance molecular breeding in these crops. Here, we review the availability, characteristics, applications, and potential applications of the BAC libraries of pulse crops.
... The power of NGS, which can generate up to gigabases of sequence data in a single run, has presented new opportunities for the investigation of highly complex populations of repetitive elements in plant genomes101112. The procedure is cloning-free, thus avoiding the potential bias caused by difficulties in propagating some repeat types in bacteria [13], and provides random sequence sampling across the genome. In principle, repeat detection is then based on evaluating the frequencies of identical or similar sequence reads, which increase as the genomic copy numbers of corresponding repetitive elements increase. ...
Silene latifolia is a dioecious [corrected] plant with well distinguished X and Y chromosomes that is used as a model to study sex determination and sex chromosome evolution in plants. However, efficient utilization of this species has been hampered by the lack of large-scale sequencing resources and detailed analysis of its genome composition, especially with respect to repetitive DNA, which makes up the majority of the genome.
We performed low-pass 454 sequencing followed by similarity-based clustering of 454 reads in order to identify and characterize sequences of all major groups of S. latifolia repeats. Illumina sequencing data from male and female genomes were also generated and employed to quantify the genomic proportions of individual repeat families. The majority of identified repeats belonged to LTR-retrotransposons, constituting about 50% of genomic DNA, with Ty3/gypsy elements being more frequent than Ty1/copia. While there were differences between the male and female genome in the abundance of several repeat families, their overall repeat composition was highly similar. Specific localization patterns on sex chromosomes were found for several satellite repeats using in situ hybridization with probes based on k-mer frequency analysis of Illumina sequencing data.
This study provides comprehensive information about the sequence composition and abundance of repeats representing over 60% of the S. latifolia genome. The results revealed generally low divergence in repeat composition between the sex chromosomes, which is consistent with their relatively recent origin. In addition, the study generated various data resources that are available for future exploration of the S. latifolia genome.
