Repetitive DNA Component Research Articles

Evolutionary Dynamics of the Repetitive DNA in the Karyotypes of Pipa carvalhoi and Xenopus tropicalis (Anura, Pipidae).

The large amphibian genomes contain numerous repetitive DNA components that have played an important role in the karyotypic diversification of this vertebrate group. Hypotheses based on the presumable primitive karyotype (2n = 20) of the anurans of the family Pipidae suggest that they have evolved principally through intrachromosomal rearrangements. Pipa is the only South American pipid, while all the other genera are found in Africa. The divergence of the South American lineages from the African ones occurred at least 136 million years ago and is thought to have had a strong biogeographic component. Here, we tested the potential of the repetitive DNA to enable a better understanding of the differentiation of the karyotype among the family Pipidae and to expand our capacity to interpret the chromosomal evolution in this frog family. Our results indicate a long history of conservation in the chromosome bearing the H3 histone locus, corroborating inferences on the chromosomal homologies between the species in pairs 6, 8, and 9. The chromosomal distribution of the microsatellite motifs also provides useful markers for comparative genomics at the chromosome level between Pipa carvalhoi and Xenopus tropicalis, contributing new insights into the evolution of the karyotypes of these species. We detected similar patterns in the distribution and abundance of the microsatellite arrangements, which reflect the shared organization in the terminal/subterminal region of the chromosomes between these two species. By contrast, the microsatellite probes detected a differential arrangement of the repetitive DNA among the chromosomes of the two species, allowing longitudinal differentiation of pairs that are identical in size and morphology, such as pairs 1, 2, 4, and 5. We also found evidence of the distinctive composition of the repetitive motifs of the centromeric region between the species analyzed in the present study, with a clear enrichment of the (CA) and (GA) microsatellite motifs in P. carvalhoi. Finally, microsatellite enrichment in the pericentromeric region of chromosome pairs 6, 8, and 9 in the P. carvalhoi karyotype, together with interstitial telomeric sequences (ITS), validate the hypothesis that pericentromeric inversions occurred during the chromosomal evolution of P. carvalhoi and reinforce the role of the repetitive DNA in the remodeling of the karyotype architecture of the Pipidae.

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization

BackgroundSatellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA.ResultsSatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26–28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific.ConclusionsWe conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.

Highly GC-rich heterochromatin in chili peppers ( Capsicum -Solanaceae): A cytogenetic and molecular characterization

Abstract Capsicum comprises 35 species of chili peppers and five of them are cultivated worldwide as spices or vegetables. Diploid karyotypes based in x = 12 and x = 13 are common in Capsicum and the constitutive heterochromatin (cHet) is of particular interest in the genus since it is largely variable, particularly its highly GC-rich fraction. However, the repetitive DNA components of this heterochromatic regions are unknown. Given the co-localization of rDNA loci with the CMA+DAPI– heterochromatic bands, we tested the hypothesis that the highly GC-rich cHet fraction is composed of the whole 18S-25S ribosomal DNA (rDNA) unit or some of its components. Here we report on a novel satellite for Capsicum and Solanaceae composed of the complete rDNA unit. We physically mapped six Capsicum derived specific 18S-25S rDNA probes that covered the entire span of the rDNA unit and analysed a DraI restriction product on eight chromosomally different taxa of Capsicum, representative of the major phylogenetic clades of chili peppers. The co-localization of every gene and spacer probes of the 18S-25S rDNA unit suggest their structural function as a major repetitive component of the highly GC-rich cHet in Capsicum species with x = 12. In addition, analyses of the clones derived from restriction assays in C. pubescens suggested that the differential functional status of 18S-25S rDNA loci (nucleolar organizer regions -NORs- or cHet) in this species is related to a divergence in a short sequence upstream the regulatory transcription initiation site (TIS) of the intergenic spacer (IGS). The results here provided evidence that an rDNA mega satellite played a significant role in the evolution of the karyotype features of x = 12 Capsicum species. The finding of a mega satellite family derived from the whole rDNA unit is a novelty for plant genomes.

Structural and functional liaisons between transposable elements and satellite DNAs.

Transposable elements (TEs) and satellite DNAs (satDNAs) are typically identified as major repetitive DNA components in eukaryotic genomes. TEs are DNA segments able to move throughout a genome while satDNAs are tandemly repeated sequences organized in long arrays. Both classes of repetitive sequences are extremely diverse, and many TEs and satDNAs exist within a genome. Although they differ in structure, genomic organization, mechanisms of spread, and evolutionary dynamics, TEs and satDNAs can share sequence similarity and organizational patterns, thus indicating that complex mutual relationships can determine their evolution, and ultimately define roles they might have on genome architecture and function. Motivated by accumulating data about sequence elements that incorporate features of both TEs and satDNAs, here we present an overview of their structural and functional liaisons.

Matrin 3 Augments the Transcriptional Activity of an SV40 Promoter-Mediated Luciferase Gene with a Highly Repetitive DNA Component

We examined the transcriptional augmentation of matrin 3, a nuclear matrix protein, of the SV40 promotermediated luciferase gene (pGL3) following transient transfection of recombinant plasmids into cells. It has been reported that the interaction of the Xmn I fragment, a highly repetitive DNA component as one of a typical matrix- or scaffold-attachment regions (MAR/SAR) tethered upstream from the SV40 promoter (pGL3-Xmn I) with matrin 3 appeared to be required for augmentation of luciferase gene transcription. In this study, we investigated the levels of induction in cells overexpressing the wild type and several deletion mutants of matrin 3. It appeared that pGL3-Xmn I augmented luciferase production to 4-times the control level in Ac2F cells, but 23-fold in cells overexpressing matrin 3. Electrophoretic mobility shift assay showed that the Xmn I fragment augmented luciferase gene transcription through interaction with matrin 3. Furthermore, our findings suggest that all of the functional domains tested in matrin 3 were necessary for transcriptional augmentation. We aim not only to describe the transcriptional augmentation of matrin 3 with MAR/SAR, but also to strengthen interest in their use to mediate the expression of therapeutic transgenes.

Discovery and assembly of repeat family pseudomolecules from sparse genomic sequence data using the Assisted Automated Assembler of Repeat Families (AAARF) algorithm

BackgroundHigher eukaryotic genomes are typically large, complex and filled with both genes and multiple classes of repetitive DNA. The repetitive DNAs, primarily transposable elements, are a rapidly evolving genome component that can provide the raw material for novel selected functions and also indicate the mechanisms and history of genome evolution in any ancestral lineage. Despite their abundance, universality and significance, studies of genomic repeat content have been largely limited to analyses of the repeats in fully sequenced genomes.ResultsIn order to facilitate a broader range of repeat analyses, the Assisted Automated Assembler of Repeat Families algorithm has been developed. This program, written in PERL and with numerous adjustable parameters, identifies sequence overlaps in small shotgun sequence datasets and walks them out to create long pseudomolecules representing the most abundant repeats in any genome. Testing of this program in maize indicated that it found and assembled all of the major repeats in one or more pseudomolecules, including coverage of the major Long Terminal Repeat retrotransposon families. Both Sanger sequence and 454 datasets were appropriate.ConclusionThese results now indicate that hundreds of higher eukaryotic genomes can be efficiently characterized for the nature, abundance and evolution of their major repetitive DNA components.

Phylogenetic relationships within the Mysticeti (whalebone whales) based upon studies of highly repetitive DNA in all extant species

The suborder Mysticeti (whalebone whales) comprises ten extant species. Taxonomic relationships within the suborder were studied by using three non-cross hybridizing highly repetitive DNA components as probes in molecular hybridizations including all ten species. The first component had the same repeat length, 1760 bp, in all species and constituted a large portion of all genomes. The second component occurred in all species of the family Balaenopteridae. rorquals, with a repeat length of = 420 bp. The same repeat length was also found in the gray whale, Eschrichtius robustus. of the monotypic family Eschrichtüdae and in the pygmy right whale, Caperea marginata, a species that has usually been included in the family Balaenidae, right whales. The component was also found in the bowhead, Balaena mysticerus. and in the right whale, Eubalaena glacialis, however, in these species the = 420 bp repeal length did not occur. The third component was present in all balaenopterid genomes and also in the gray whale and the pygmy right whale. The component was virtually nonexistent in the right whale and the bowhead. Thus, at the DNA level, there is a major distinction between the bowhead and the right whale vis-æ-vis all other mysticetes including the pygmy right whale. The results support the exclusion of the pygmy right whale from the family Balaenidae.

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.

Organization and variability of the maize genome

With a size approximating that of the human genome, the maize genome is about to become the largest plant genome yet sequenced. Contributing to that size are a whole-genome duplication event and a retrotransposition explosion that produced a large amount of repetitive DNA. This DNA is greatly under-represented in cDNA collections, so analysis of the maize transcriptome has been an expedient way of assessing the gene content of maize. Over 2 million maize cDNA sequences are now available, making maize the third most widely studied organism, behind mouse and man. To date, the sequencing of large-sized DNA clones has been largely driven by the genetic interests of different investigators. The recent construction of a physical map that is anchored to the genetic map will aid immensely in the maize genome-sequencing effort. However, studies showing that the repetitive DNA component is highly polymorphic among maize inbred lines point to the need to sample vertically a few specific regions of the genome to evaluate the extent and importance of this variability.

Why repetitive DNA is essential to genome function

There are clear theoretical reasons and many well-documented examples which show that repetitive, DNA is essential for genome function. Generic repeated signals in the DNA are necessary to format expression of unique coding sequence files and to organise additional functions essential for genome replication and accurate transmission to progeny cells. Repetitive DNA sequence elements are also fundamental to the cooperative molecular interactions forming nucleoprotein complexes. Here, we review the surprising abundance of repetitive DNA in many genomes, describe its structural diversity, and discuss dozens of cases where the functional importance of repetitive elements has been studied in molecular detail. In particular, the fact that repeat elements serve either as initiators or boundaries for heterochromatin domains and provide a significant fraction of scaffolding/matrix attachment regions (S/MARs) suggests that the repetitive component of the genome plays a major architectonic role in higher order physical structuring. Employing an information science model, the 'functionalist' perspective on repetitive DNA leads to new ways of thinking about the systemic organisation of cellular genomes and provides several novel possibilities involving repeat elements in evolutionarily significant genome reorganisation. These ideas may facilitate the interpretation of comparisons between sequenced genomes, where the repetitive DNA component is often greater than the coding sequence component.

An assessment of the relationships among species of Camelidae by satellite DNA comparisons.

Tandem satellite arrays and interspersed repetitive DNA components of the New World camelids guanaco, llama, alpaca, and vicuña and the Old World bactrian camel have been identified and compared. Southern hybridizations, using camel restriction fragments as probes, indicated that satellite DNAs in all camelids examined have been conserved since the last common ancestor about 5-10 MY ago. The hybridization profiles, however, varied from totally identical (MspI-sat) to highly differentiated (PstI-sat and EcoRI-sat) between Old and New World species. Repetitive DNA patterns specific of South American camelids were identified by most of the vicuña and guanaco probes and (a) llama and guanaco have undifferentiable patterns, supporting the view that the former is a domesticated form of the latter; (b) vicuña patterns were species-specific and in agreement with its position in a separate taxonomic unit; (c) the presence in alpaca of BamHI, TaqI and EcoRI patterns that are intermediate between those of the species above, suggested that the origin of the alpaca may be found in a cross-breed between the guanaco and vicuña.

Transcription Modulation by a Rat Nuclear Scaffold Protein, P130, and a Rat Highly Repetitive DNA Component or Various Types of Animal and Plant Matrix or Scaffold Attachment Regions

The XmnI fragment, a highly repetitive DNA component, and animal and plant matrix or scaffold attachment region (MAR/SAR) were examined for similarity in interaction with nuclear scaffold. As the XmnI fragment bound a 130 kDa scaffold protein (P130) in vitro, various types of MAR/SAR fragments could bind 130 and 123 kDa scaffold proteins. The native XmnI and MAR/SAR fragments clearly augmented SV40 promoter-mediated luciferase gene transcription following transient transfection of recombinant plasmids into various types of recipient cells. In contrast, the XmnI fragment methylated at the cytosine base of the unique HindIII site, and a synthetic variant DNA deficient in base unpairing characteristic of MAR/SAR, could neither bind P130 nor augment this transcription. These two types of genomic regions appeared to have similar properties of interaction with nuclear scaffold, by which the activity of appropriately positioned promoter can be modulated.

Evolution of a functionally related lactate dehydrogenase and pyruvate decarboxylase pseudogene complex in maize

A large proportion of the maize genome is repetitive DNA (60-80%) with retrotransposons contributing significantly to the repetitive DNA component. The majority of retrotransposon DNA is located in intergenic regions and is organized in a nested fashion. Analysis of an 8.2-kb segment of maize genomic DNA demonstrated the presence of three retrotransposons of different reiteration classes in addition to lactate dehydrogenase and pyruvate decarboxylase pseudogenes. Both of the pseudogenes were located within a defective retrotransposon element (LP-like element) which possessed identical long terminal repeats (LTRs) with inverted repeats at each end, a primer binding site, a polypurine tract, and generated a 5-bp target site duplication. A model describing the events leading to the formation of the LP-like element is proposed.

Involvement of DNA Methylation in Binding of a Highly Repetitive DNA Component to Nuclear Scaffold Proteins from Rat Liver

Experimental reduction of the amount of CpG methylation in a highly repetitive DNA component was achieved by growth of Ac2F cells in the presence of 5-aza-2′-deoxycytidine or procainamide, as judged by the results of methyl-sensitive restriction endonuclease digestion and colony hybridization. Modification of genomic DNA with these DNA methylation inhibitors increased the release of 370-bp highly repetitive DNA from rat chromosomal DNA byHindIII digestion. This result indicated that highly repetitive DNA components in the nuclear scaffold fraction are hypermethylated. On the other hand, methylated DNA was used for southwestern analysis to investigate the protein(s) which bind specifically to the DNA in the nuclear scaffold fraction. The introduction of additional methylated cytosines within a highly repetitive DNA component affected the binding of DNA to the nuclear scaffold proteins. Thus, cytosine methylation may be involved in the regulation of gene expression and construction of the higher-order structure of chromatin.

Involvement of phosphorylation in binding of nuclear scaffold proteins from rat liver to a highly repetitive DNA component

The results of our previous work [Hibino et al., Biochim. Biophys. Acta 1174 (1993) 162–170] suggested that a highly repetitive DNA component facilitates bending of the helix axis to be recognized by the nuclear scaffold proteins from rat liver, P123 and P130. In the present experiment, it was shown that binding of these proteins to such a repetitive DNA component from rat liver nuclei (370-bp XmnI fragment) is based on a cooperative mode of interaction, although the binding activity of P130 is much higher than that of P123. The immunoblot analysis with anti-phosphoamino acid antibodies suggested that phosphorylation of serine and threonine residues occurs on P123 and P130, but also of tyrosine residue(s) on P130. The phosphatase assay showed that phosphoryl groups on these proteins may be involved in altering the DNA binding activities of the proteins. Thus, the results in the present study imply that phosphorylation of a nuclear scaffold protein in addition to the degree of bending of the DNA helix axis plays an important role in anchoring chromatin to the nuclear scaffold and in construction of a higher-order chromatin structure.

Purification and characterization of a DNA binding protein in a nuclear scaffold fraction from rat ascites hepatoma cells.

Our most recent work [Hibino et al. (1995) Cancer Lett., 88, 49-55] has shown that the selective binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat ascites hepatoma cells (P230) depend on the degree of sequence-directed bending of the helix axis. In the present experiment, this protein has been highly purified and isolated by a series of column chromatographic procedures to migrate as a single band to a molecular weight position of 230 kd on a SDS-polyacrylamide gel. A filter binding assay showed that the binding of a repetitive AT-rich component (369 bp XmnI fragment) from the hepatoma nucleus, which has a strongly bent overall structure, to isolated P230 is based on a cooperative mode of interaction. Distamycin A, which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding to this protein. These results suggest that AT-rich regions in highly repetitive DNA cause bending of the helix axis to be recognized by nuclear scaffold protein(s). Moreover, it has been shown that the nuclear scaffold fraction from rat liver or an actively growing hepatocyte cell line (Ac2F cells) does not contain P230, but does have a repetitive bent DNA binding protein (P130), which has an apparent molecular weight of 130 kd. In addition, the immunoblot analysis showed that mouse anti-P130 antiserum reacts with P230. Thus, the results in the present study imply that there is some difference in the higher order structure of the nuclear DNA attachment region between rat liver or actively growing hepatocytes and the hepatoma, although P230 appears to be immunochemically similar to P130.

Binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat-ascites hepatoma cells.

Separate samples of a self-ligated tandem dimer of a highly repetitive DNA component (369-bp HindIII fragment) from rat-ascites hepatoma nuclei were digested with different restriction enzymes that cleave only once in the monomer. The resulting 369-bp sequence-permuted monomers showed anomalously slow gel electrophoretic mobility. Of them, the XmnI fragment had the slowest mobility. This suggests that bending of the helix axis is the strongest in this fragment. Our previous work has shown that such a repetitive bent DNA has selective affinities for two nuclear scaffold proteins from rat liver that have molecular weights of 123,000 and 130,000 Hibino et al. (1992) Biochem. Biophys. Res. Commun., 184, 853-858; Hibino et al. (1993) Biochim. Biophys. Acta, 1174, 162-170). In the present experiment, it has been found that the nuclear scaffold fraction from rat-ascites hepatoma cells does not contain these proteins, but does have a repetitive bent DNA-binding protein that has a molecular weight of about 230,000. These results imply that there is some difference in the structure of nuclear DNA attachment region between rat liver and the hepatoma.

DNA class organization on maize Adh1 yeast artificial chromosomes.

The organization of higher plant genomes is poorly understood. These genomes are typified by their large size and extensive repetitive DNA component. To further our understanding of the composition and arrangement of genomic DNA sequences, we have performed a detailed analysis of a contiguous interval of 280 kb surrounding the Adh1 locus of maize. A series of overlapping lambda subclones was isolated, and individual fragments were characterized with respect to their genomic copy number. Cross-hybridization analyses were used to define a minimum of 37 repetitive DNA classes within the 280-kb interval. Hybridizations with highly repetitive DNAs cloned from other regions of the maize genome suggested that > 50% of all highly repetitive elements in maize are represented on this single yeast artificial chromosome. These repeated sequences were found in an organizational pattern not previously observed; individual repetitive elements are interspersed with one another in an apparently random fashion and are spatially separate from single copy number sequences. Extensive tandem arrays were not found. Sequences from one end of the 280-kb interval were used to isolate overlapping yeast artificial chromosome clones, representing the first step in a chromosome walk.

Purification and characterization of nuclear scaffold proteins which bind to a highly repetitive bent DNA from rat liver

Our previous work (Hibino et al. (1992) Biochem. Biophys. Res. Commun. 184, 853–858) has shown that the binding affinities of a highly repetitive DNA component for rat nuclear scaffold proteins, P123 and P130, depend on the degree of sequence-directed bending of the helix axis. In the present experiment, these proteins have been purified and finally isolated by DNA-Sepharose column chromatography. The p I values of P123 and P130 were 7.2 and 8.1, respectively. The southwestern blotting revealed that a highly repetitive bent DNA (370-bp XmnI fragment) from rat liver binds readily to the isolated proteins under a hypotonic condition (50 mM NaCl) and that the level of the binding affinity for each protein was lowered with increasing NaCl concentration. The sedimentation analysis predicted that direct interaction between the XmnI fragment and P123 or P130 results in the formation of a complex which consists of two of the fragments and one molecule of the protein, alternatively, one of the fragment and three molecules of the proteins. Distamycin A, an antibiotic which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding of the fragment to P123 or P130, whereas neither removal of the bend nor binding inhibition was observed with chromomycin A 3, an antibiotic specific for GC-rich sites in DNA. These results imply that AT-rich regions in a highly repetitive DNA component cause bending of the helix axis to be recognized by some of nuclear scaffold proteins.

Evolution of the common cetacean highly repetitive DNA component and the systematic position of Orcaella brevirostris.

The common cetacean highly repetitive DNA component was analyzed with respect to its evolution and value for establishing phylogenetic relationships. The repeat length of the component, which is tandemly organized, is approximately 1750 bp in all cetaceans except the delphinids, in which the repeat length is approximately 1580 bp. The evolution of the component was studied after sequencing the component in different odontocetes representing the Delphinidae (delphinids), Monodontidae (narwhals), and Ziphiidae (beaked whales). The evolution of this component is very slow, and comparisons showed that sequence divergence among species corresponds closely to their generally accepted phylogenetic relationships and that the component evolves in a concerted manner. The phylogenetic information obtained in this study identified the Irrawaddy dolphin (Orcaella brevirostris) as a delphinid and did not support a close relationship of this species with the Monodontidae.