How repeated retroelements format genome function

Genomes operate as sophisticated information storage systems. Generic repeated signals in the DNA format expression of coding sequence files and organize additional functions essential for genome replication and accurate transmission to progeny cells. Retroelements comprise a major fraction of many genomes and contain a surprising diversity of functional signals. In this article, we summarize some features of the taxonomic distribution of retroelements, especially mammalian SINEs, tabulate functional roles documented for different classes of retroelements, and discuss their potential roles as genome organizers. In particular, the fact that certain retroelements serve as boundaries for heterochromatin domains and provide a significant fraction of scaffolding/matrix attachment regions (S/MARs) suggests that the reversed transcribed 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 organization of cellular genomes and provides several novel possibilities involving retroelements in evolutionarily significant genome reorganization.     

A new middle repetitive sequence of Nicotiana plumbaginifolia genome

A middle repetitive sequence NPR18 was isolated from Nicotiana plumbaginifolia nuclear genome [8]. Sequences homologous to the repeat are dispersed through genomes of several Nicotiana species. compute-assisted data analysis of NPR18 primary sequence reveals several features attributed to mobile genetic elements: an AT content higher than average for nuclear DNA of genus Nicotiana plants; a number of direct and inverted repeats. Some of the repeats displayed homology to the terminal and subterminal repeats of Ac/Ds-like plant elements.     

Role of recombination and replication fork restart in repeat instability

Eukaryotic genomes contain many repetitive DNA sequences that exhibit size instability. Some repeat elements have the added complication of being able to form secondary structures, such as hairpin loops, slipped DNA, triplex DNA or G-quadruplexes. Especially when repeat sequences are long, these DNA structures can form a significant impediment to DNA replication and repair, leading to DNA nicks, gaps, and breaks. In turn, repair or replication fork restart attempts within the repeat DNA can lead to addition or removal of repeat elements, which can sometimes lead to disease. One important DNA repair mechanism to maintain genomic integrity is recombination. Though early studies dismissed recombination as a mechanism driving repeat expansion and instability, recent results indicate that mitotic recombination is a key pathway operating within repetitive DNA. The action is two-fold: first, it is an important mechanism to repair nicks, gaps, breaks, or stalled forks to prevent chromosome fragility and protect cell health; second, recombination can cause repeat expansions or contractions, which can be deleterious. In this review, we summarize recent developments that illuminate the role of recombination in maintaining genome stability at DNA repeats.     

Improved repeat identification and masking in Dipterans

Repetitive sequences are a major constituent of many eukaryote genomes and play roles in gene regulation, chromosome inheritance, nuclear architecture, and genome stability. The identification of repetitive elements has traditionally relied on in-depth, manual curation and computational determination of close relatives based on DNA identity. However, the rapid divergence of repetitive sequence has made identification of repeats by DNA identity difficult even in closely related species. Hence, the presence of unidentified repeats in genome sequences affects the quality of gene annotations and annotation-dependent analyses (e.g. microarray analyses). We have developed an enhanced repeat identification pipeline using two approaches. First, the de novo repeat finding program PILER-DF was used to identify interspersed repetitive elements in several recently finished Dipteran genomes. Repeats were classified, when possible, according to their similarity to known elements described in Repbase and GenBank, and also screened against annotated genes as one means of eliminating false positives. Second, we used a new program called RepeatRunner, which integrates results from both RepeatMasker nucleotide searches and protein searches using BLASTX. Using RepeatRunner with PILER-DF predictions, we masked repeats in thirteen Dipteran genomes and conclude that combining PILER-DF and RepeatRunner greatly enhances repeat identification in both well-characterized and un-annotated genomes.     

Characterizing the composition and evolution of homoeologous genomes in hexaploid wheat through BAC-end sequencing on chromosome 3B

Bread wheat (Triticum aestivum) is one of the most important crops worldwide. However, because of its large, hexaploid, highly repetitive genome it is a challenge to develop efficient means for molecular analysis and genetic improvement in wheat. To better understand the composition and molecular evolution of the hexaploid wheat homoeologous genomes and to evaluate the potential of BAC-end sequences (BES) for marker development, we have followed a chromosome-specific strategy and generated 11 Mb of random BES from chromosome 3B, the largest chromosome of bread wheat. The sequence consisted of about 86% of repetitive elements, 1.2% of coding regions, and 13% remained unknown. With 1.2% of the sequence length corresponding to coding sequences, 6000 genes were estimated for chromosome 3B. New repetitive sequences were identified, including a Triticineae-specific tandem repeat (Fat) that represents 0.6% of the B-genome and has been differentially amplified in the homoeologous genomes before polyploidization. About 10% of the BES contained junctions between nested transposable elements that were used to develop chromosome-specific markers for physical and genetic mapping. Finally, sequence comparison with 2.9 Mb of random sequences from the D-genome of Aegilops tauschii suggested that the larger size of the B-genome is due to a higher content in repetitive elements. It also indicated which families of transposable elements are mostly responsible for differential expansion of the homoeologous wheat genomes during evolution. Our data demonstrate that BAC-end sequencing from flow-sorted chromosomes is a powerful tool for analysing the structure and evolution of polyploid and highly repetitive genomes.     

Swine liver methylomes of Berkshire,Duroc and Landrace breeds by MeDIPS

Using a methyl‐DNA immunoprecipitation technique in combination with next‐generation deep sequencing, we conducted comprehensive DNA methylation profiling of liver genomes from three pig breeds: Berkshire, Duroc and Landrace. The profiles revealed that the distribution patterns of methylation signals along the genome are conserved among the three pig breeds. Specifically, many signals in coding genes were found in introns, and most signals in the repetitive elements were identified in non‐long terminal repeat (LTR) retrotransposons such as long and short interspersed repetitive elements, implying a significant association with alternative splicing and expression of retrotransposable elements respectively. Differentially methylated regions among the three pig breeds were identified in the non‐LTR retrotransposons, suggesting that they may lead to differential retrotransposable element activity. Altogether, this study provides advanced swine methylome data and valuable resources for understanding the function of DNA methylation in the evolutionary divergence of different pig breeds.     

DNA, chromosomes, and in situ hybridization.

In situ hybridization is a powerful and unique technique that correlates molecular information of a DNA sequence with its physical location along chromosomes and genomes. It thus provides valuable information about physical map position of sequences and often is the only means to determine abundance and distribution of repetitive sequences making up the majority of most genomes. Repeated DNA sequences, composed of units of a few to a thousand base pairs in size, occur in blocks (tandem or satellite repeats) or are dispersed (including transposable elements) throughout the genome. They are often the most variable components of a genome, often being species and, occasionally, chromosome specific. Their variability arises through amplification, diversification and dispersion, as well as homogenization and loss; there is a remarkable correlation of molecular sequence features with chromosomal organization including the length of repeat units, their higher order structures, chromosomal locations, and dispersion mechanisms. Our understanding of the structure, function, organization, and evolution of genomes and their evolving repetitive components enabled many new cytogenetic applications to both medicine and agriculture, particularly in diagnosis and plant breeding.     

Structural organization of the termini of the L and S components of the genome of pseudorabies virus.

The sequences of several hundred nucleotides around the junctions between the L and S components in concatemeric DNA and in mature virion DNA were ascertained. The two ends of the mature genome (which are joined in concatemeric DNA) show no sequence homology. Several directly repeated elements are present near both ends of the genome. Furthermore, the last 82 nucleotides at the left end of the L component (and of the genome) are repeated in inverted form (inverted repeat within the L component [IRL]) approximately 350 to 600 nucleotides downstream (depending on the virus isolate) bracketing the UL2 component. A comparison between the sequences at the right and left ends of the L component of the genome showed patchy homology, probably representing a vestigial inverted repeat bracketing the L component (IRL). Furthermore, less than 5% of the genomes have an L component that is in the orientation opposite to that of most of the viral genomes, indicating that the vestigial IRL that brackets the UL sequence may be sufficient to mediate inversion of the L component in some of the genomes. On the other hand, the UL2 component, which is bracketed by a perfect IRL, does not invert to a greater extent than does the L component (if it inverts at all). Analysis of the nucleotide sequence at the concatemeric junction of three different pseudorabies virus isolates showed almost complete sequence conservation. The sequence and organization of the repeated elements in the different isolates were almost identical, despite their different histories and origins. The high degree of conservation of these repeated elements implies that they may fulfill an essential function in the life cycle of the virus.     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

Nucleotide sequence and characterization of a repetitive DNA element from the genome of Bordetella pertussis with characteristics of an insertion sequence

A repeating element of DNA has been isolated and sequenced from the genome of Bordetella pertussis. Restriction map analysis of this element shows single internal ClaI, SphI, BstEII and SalI sites. Over 40 DNA fragments are seen in ClaI digests of B. pertussis genomic DNA to which the repetitive DNA sequence hybridizes. Sequence analysis of the repeat reveals that it has properties consistent with bacterial insertion sequence (IS) elements. These properties include its length of 1053 bp, multiple copy number and presence of 28 bp of near-perfect inverted repeats at its termini. Unlike most IS elements, the presence of this element in the B. pertussis genome is not associated with a short duplication in the target DNA sequence. This repeating element is not found in the genomes of B. parapertussis or B. bronchiseptica. Analysis of a DNA fragment adjacent to one copy of the repetitive DNA sequence has identified a different repeating element which is found in nine copies in B. parapertussis and four copies in B. pertussis, suggesting that there may be other repeating DNA elements in the different Bordetella species. Computer analysis of the B. pertussis repetitive DNA element has revealed no significant nucleotide homology between it and any other bacterial transposable elements, suggesting that this repetitive sequence is specific for B. pertussis.     

Molecular analysis of a novel tandemly organized repetitive DNA sequence in<Emphasis Type="Italic">Citrus limon</Emphasis> (L.) Burm

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and molecular analysis of a novel tandemly organized repetitive DNA sequence from the genome of Citrus limon. Digestion of C. limon DNA with Hinf I produced a prominent fragment of approximately 300 bp. Southern blotting revealed a ladder composed of DNA fragments that were multimers of the 300-bp Hinf I band. Thus, Hinf I digestion revealed a novel satellite, which we have called the C. limon satellite DNA 300 (CL300). Sequence analysis shows significant homology between a portion of the CL300 monomer and the transposase box of an En/Spm-like element. The CL300 satellite was also detected in grapefruit, sour orange, trifoliate orange and kumquat. These results suggest that the CL300 repeat is an ancient satellite, and we propose that a significant portion originated by amplification of a genomic region containing the En/Spm-like transposase element.     

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

Background  

Higher 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.     

Dialect-I,species-specific repeated DNA sequence from barley,Hordeum vulgare

Summary Dialect-1, species-specific repetitive DNA sequence of barley Hordeum vulgare, was cloned and analysed by Southern blot and in situ hybridization. Dialect-1 is dispersed through all barley chromosomes with copy number 5,000 per genome. Two DNA fragments related to Dialect-1 were revealed in phage library, subcloned and mapped. All three clones are structurally heterogenous and it is suggested that the full-length genomic repeat encompassing Dialect-1 is large in size. The Dialect-1 DNA repeat is represented in the genomes of H. vulgare and ssp. agriocrithon and spontaneum in similar form and copy number; it is present in rearranged form with reduced copy number in the genomes of H. bulbosum and H. murinum, and it is absent from genomes of several wild barley species as well as from genomes of wheat, rye, oats and maize. Dialect-1 repeat may be used as a molecular marker in taxonomic studies and for identification of barley chromosomes in interspecies hybrids.     

Mitochondrial genomes of the genus Ephydatia Lamouroux, 1816: can palindromic elements be used in species-level studies?

Poriferan mitochondrial DNA (mtDNA), especially large intergenic regions, is a target for the insertion of repetitive hairpin-forming elements. These elements are responsible for the large mt genome size differences observed even among closely related sponge taxa. In this study, we present the new, nearly complete, mt genome sequence of Ephydatia fluviatilis and compare it with previously published mt genomes of freshwater sponges. Special emphasis was placed on comparison with the closely related species Ephydatia muelleri, thereby comparing the only two species of the genus Ephydatia on the western Balkan Peninsula. In particular, we analyzed repetitive palindromic elements within the mitochondrial intergenic regions. The genomic distribution of these repetitive elements was analyzed and their potential role in the evolution of mt genomes discussed. We show here that palindromic elements are widespread through the whole mt genome, including the protein coding genes, thus introducing genetic variability into mt genomes.     

Next generation sequencing reveals genome downsizing in allotetraploid Nicotiana tabacum, predominantly through the elimination of paternally derived repetitive DNAs

We used next generation sequencing to characterize and compare the genomes of the recently derived allotetraploid, Nicotiana tabacum (<200,000 years old), with its diploid progenitors, Nicotiana sylvestris (maternal, S-genome donor), and Nicotiana tomentosiformis (paternal, T-genome donor). Analysis of 14,634 repetitive DNA sequences in the genomes of the progenitor species and N. tabacum reveal all major types of retroelements found in angiosperms (genome proportions range between 17-22.5% and 2.3-3.5% for Ty3-gypsy elements and Ty1-copia elements, respectively). The diploid N. sylvestris genome exhibits evidence of recent bursts of sequence amplification and/or homogenization, whereas the genome of N. tomentosiformis lacks this signature and has considerably fewer homogenous repeats. In the derived allotetraploid N. tabacum, there is evidence of genome downsizing and sequences loss across most repeat types. This is particularly evident amongst the Ty3-gypsy retroelements in which all families identified are underrepresented in N. tabacum, as is 35S ribosomal DNA. Analysis of all repetitive DNA sequences indicates the T-genome of N. tabacum has experienced greater sequence loss than the S-genome, revealing preferential loss of paternally derived repetitive DNAs at a genome-wide level. Thus, the three genomes of N. sylvestris, N. tomentosiformis, and N. tabacum have experienced different evolutionary trajectories, with genomes that are dynamic, stable, and downsized, respectively.     

Genome-wide comparative analysis of simple sequence coding repeats among 25 insect species

We present a detailed genome-scale comparative analysis of simple sequence repeats within protein coding regions among 25 insect genomes. The repetitive sequences in the coding regions primarily represented single codon repeats and codon pair repeats. The CAG triplet is highly repetitive in the coding regions of insect genomes. It is frequently paired with the synonymous codon CAA to code for polyglutamine repeats. The codon pairs that are least repetitive code for polyalanine repeats. The frequency of hexanucleotide and dinucleotide motifs of codon pair repeats is significantly (p<0.001) different in the Drosophila species compared to the non-Drosophila species. However, the frequency of synonymous and non-synonymous codon pair repeats varies in a correlated manner (r(2)=0.79) among all the species. Results further show that perfect and imperfect repeats have significant association with the trinucleotide and hexanucleotide coding repeats in most of these insects. However, only select species show significant association between the numbers of perfect/imperfect hexamers and repeat coding for single amino acid/amino acid pair runs. Our data further suggests that genes containing simple sequence coding repeats may be under negative selection as they tend to be poorly conserved across species. The sequences of coding repeats of orthologous genes vary according to the known phylogeny among the species. In conclusion, the study shows that simple sequence coding repeats are important features of genome diversity among insects.     

Molecular analysis of the genomic organization of the Hymenoptera Diadromus pulchellus and Eupelmus vuilleti

The genomic organization of two parasitic wasps was analyzed by DNA reassociation. Cot curves revealed a pattern with three types of components. A highly repetitive DNA, accounting for 15 to 25% of the genome, was identified as satellite DNA. The moderately repetitive DNA corresponds to 26 to 42% of the genome in both species, and shows large variations in complexity, repetitive frequency and a number of sub-components between males and females. These variations are seen as resulting from DNA amplification during somatic and sexual differentiation. Dot blot analyses show that such DNA amplifications concern several types of structural and regulatory genes. The presence of repeated mobile elements was studied by the Roninson method to compare the repeated sequence patterns of Diadromus pulchellus and Eupelmus vuilleti with those of Drosophila melanogaster. The occurrence and organization of mobile elements in these Hymenoptera differ from those of the neighboring order of Diptera. The repetitive and unique components define very large genomes (1 to 3 × 10⁹ base pairs). The genomic organization in Parasitica appears to be an extreme drosophilan type. We propose that the germinal genome of these parasitic wasps is primarily composed of satellite DNA blocks and very long stretches of unique sequences, separated by a few repeated and/or variously deleted, interspersed elements of each mobile element family.     

Rickettsial evolution in the light of comparative genomics

Rickettsia are best known as strictly intracellular vector‐borne bacteria that cause mild to severe diseases in humans and other animals. Recent advances in molecular tools and biological experiments have unveiled a wide diversity of Rickettsia spp. that include species with a broad host range and some species that act as endosymbiotic associates. Molecular phylogenies of Rickettsia spp. contain some ambiguities, such as the position of R. canadensis and relationships within the spotted fever group. In the modern era of genomics, with an ever‐increasing number of sequenced genomes, there is enhanced interest in the use of whole‐genome sequences to understand pathogenesis and assess evolutionary relationships among rickettsial species. Rickettsia have small genomes (1.1–1.5 Mb) as a result of reductive evolution. These genomes contain split genes, gene remnants and pseudogenes that, owing to the colinearity of some rickettsial genomes, may represent different steps of the genome degradation process. Genomics reveal extreme genome reduction and massive gene loss in highly vertebrate‐pathogenic Rickettsia compared to less virulent or endosymbiotic species. Information gleaned from rickettsial genomics challenges traditional concepts of pathogenesis that focused primarily on the acquisition of virulence factors. Another intriguing phenomenon about the reduced rickettsial genomes concerns the large fraction of non‐coding DNA and possible functionality of these “non‐coding” sequences, because of the high conservation of these regions. Despite genome streamlining, Rickettsia spp. contain gene families, selfish DNA, repeat palindromic elements and genes encoding eukaryotic‐like motifs. These features participate in sequence and functional diversity and may play a crucial role in adaptation to the host cell and pathogenesis. Genome analyses have identified a large fraction of mobile genetic elements, including plasmids, suggesting the possibility of lateral gene transfer in these intracellular bacteria. Phylogenetic analyses have identified several candidates for horizontal gene acquisition among Rickettsia spp. including tra, pat2, and genes encoding for the type IV secretion system and ATP/ADP translocase that may have been acquired from bacteria living in amoebae. Gene loss, gene duplication, DNA repeats and lateral gene transfer all have shaped rickettsial genome evolution. A comprehensive analysis of the entire genome, including genes and non‐coding DNA, will help to unlock the mysteries of rickettsial evolution and pathogenesis.