Characterization of the repetitive sequences in a 200-kb region around the rice waxy locus: diversity of transposable elements and presence of veiled repetitive sequences

Repetitive genomic sequences might have various structural features and properties distinct from those of the known transposable elements (TE). Here, the content and properties of the repetitive sequences present in a 200-kb region around the rice waxy locus were analyzed using the available rice genomic database. In our previous Southern blotting analysis, 70% of the segments in this region showed smeared patterns, but according to the present database analysis, the proportion of repetitive sequences in this region was only 15%. The repetitive segments in this 200-kb region comprised 75 repetitive sequences that we classified into 46 subfamilies: 21 subfamilies were known TEs or repetitive sequences and 25 subfamilies consisted of newly identified TEs or novel types of repetitive sequences. The region contains no long terminal repeat (LTR) retrotransposable elements, but miniature inverted repeat transposable elements (MITEs) constituted a major class among the elements identified. These MITEs showed remarkable structural divergence: 12 elements were found to be new members of known MITE superfamilies, while five elements had novel terminal structures, and did not belong to any known TE families. Interestingly, about 10% of the repetitive sequences, including virus-like sequences did not have any of the usual characteristics of TEs, suggesting that a certain proportion of repetitive sequences that might not share the transpositional mechanisms of known elements are dispersed in the compact rice genome.     

Analysis of 3D genomic interactions identifies candidate host genes that transposable elements potentially regulate

Background

The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.

Results

Using PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes.

Conclusions

4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.

     

Genome dynamics of the major histocompatibility complex: insights from genome paralogy

 It has recently become apparent that the human genome contains at least three regions that are paralogous to the major histocompatibility complex (MHC). The number of gene families with copies in the MHC and these paralogous regions is increasing steadily as genome analysis progresses. This review presents the updated listing of the human gene families that constitute the MHC paralogous group. When genes with multiple copies within the MHC, such as class I and class II genes, are counted as single entities, nearly one-third of the genes residing in the HLA complex have paralogous copies in at least one of the three paralogous regions. The review also discusses the long-term genome dynamics of the MHC, taking into account the rapidly accumulating information on the genomic organizations of the MHCs in various model organisms.     

Characterization of Tpn1 family in the Japanese morning glory: En/Spm-related transposable elements capturing host genes

Some mutant phenotypes are known to be unstable somatically and germinally due to the insertion of transposable elements in the Japanese morning glory (Ipomoea nil). Several transposable elements that cause mutable phenotypes have recently been isolated. All of these elements show characteristic features of the En/Spm (Enhancer/Suppressor-mutator) or CACTA family. They carry common 28 bp terminal inverted repeats and subterminal repetitive regions and are known as the Tpn1 family. All of these elements are thought to be non-autonomous and mobilized by unidentified autonomous element(s). Using a probe corresponding to the subterminal region, we isolated many genomic Tpn clones, 120 of which were classified into 28 types based on their restriction maps. The copy number of the Tpn1 family was estimated to be between 500 and 1,000 copies per haploid genome. We then determined the complete sequences of 28 representative clones from each Tpn type. Most Tpn elements showed a high degree of similarity to plant genes in their internal sequences, suggesting that the Tpn1 family captured host gene sequences during the process of evolution. Detailed analyses of Tpn104 in comparison with an orthologous host gene InAP2B confirmed this assumption.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

Repeat-induced point mutation and the population structure of transposable elements in Microbotryum violaceum

Repeat-induced point mutation (RIP) is a genome defense in fungi that hypermutates repetitive DNA and is suggested to limit the accumulation of transposable elements. The genome of Microbotryum violaceum has a high density of transposable elements compared to other fungi, but there is also evidence of RIP activity. This is the first report of RIP in a basidiomycete and was obtained by sequencing multiple copies of the integrase gene of a copia-type transposable element and the helicase gene of a Helitron-type element. In M. violaceum, the targets for RIP mutations are the cytosine residues of TCG trinucleotide combinations. Although RIP is a linkage-dependent process that tends to increase the variation among repetitive sequences, a chromosome-specific substructuring was observed in the transposable element population. The observed chromosome-specific patterns are not consistent with RIP, but rather suggest an effect of gene conversion, which is also a linkage-dependent process but results in a homogenization of repeated sequences. Particular sequences were found more widely distributed within the genome than expected by chance and may reflect the recently active variants. Therefore, sequence variation of transposable elements in M. violaceum appears to be driven by selection for transposition ability in combination with the context-specific forces of the RIP and gene conversion.     

The origin of interspersed repeats in the human genome

Over a third of the human genome consists of interspersed repetitive sequences which are primarily degenerate copies of transposable elements. In the past year, the identities of many of these transposable elements were revealed. The emerging concept is that only three mechanisms of amplification are responsible for the vast majority of interspersed repeats and that with each autonomous element a number of dependent non-autonomous sequences have co-amplified.     

The clustered and scrambled arrangement of moderately repetitive elements in Drosophila DNA.

An examination of cloned Drosophila DNA has revealed large clusters of densely spaced, short (less than or equal to 1 kb), moderately repetitive elements. Different clusters have many of the same repetitive elements, but these elements are arranged differently in each cluster. It is improbable that this clustered arrangement can be detected by conventional reassociation kinetic and electron microscopic techniques, but it can be detected and features of its fine structure can be determined by a two-dimensional version of Southern's blotting technique. The genomic organization of these clustered repetitive elements was investigated by hybridizing restriction fragments of cloned DNA to polytene chromosomes, to filter-bound recombinant DNA clones and to Southern blots of total Drosophila DNA. These studies demonstrated that clusters occur in euchromatic regions of the chromosomes and that at least one of the clusters has the same repetitive element organization in cloned and in chromosomal DNA. These studies also demonstrated that copies of the elements from one cluster are scattered in at least 1000 chromosomal regions. These regions appear to have differing concentrations of repetitive DNA, but together they account for a large fraction of Drosophila's moderately repetitive DNA. Aside from indicating the genomic organization of cluster elements, this work has identified cluster elements throughout a 9 kb region neighboring one of the heat shock genes, throughout the intron of the major rDNA repeat and within the apparently transposable element, 412.     

Characterization of Stowaway MITEs in pea (Pisum sativum L.) and identification of their potential master elements.

We have investigated miniature inverted-repeat transposable elements (MITEs) of the Stowaway family and corresponding Mariner-like master elements that could potentially facilitate their mobilization in the genome of the garden pea (Pisum sativum L.). The population of pea Stowaway MITEs consists of 103-104 copies dispersed in the genome. Judging from a sequence analysis of 17 isolated Stowaway elements and their flanking genomic regions, the elements are relatively uniform in size and sequence and occur in the vicinity of genes as well as within repetitive sequences. Insertional polymorphism of several elements was detected among various Pisum accessions, suggesting they were still transpositionally active during diversification of these taxa. The identification of several Mariner-like elements (MLEs) harboring intact open reading frames, capable of encoding a transposase, further supports a recent mobilization of the Stowaway elements. Using transposase-coding sequences as a hybridization probe, we estimated that there are about 50 MLE sequences in the pea genome. Among the 5 elements sequenced, 3 distinct subfamilies showing mutual similarities within their transposase-coding regions, but otherwise diverged in sequence, were distinguished and designated as Psmar-1 to Psmar-3. The terminal inverted repeats (TIRs) of these MLE subfamilies differed in their homology to the TIRs of Stowaway MITEs. The homlogy ranged from 9 bp in Psmar-3 to 30 bp in Psmar-1, which corresponds to the complete Stowaway TIR sequence. Based on this feature, the Psmar-1 elements are believed to be the most likely candidates for the master elements of the Stowaway MITEs in pea.     

Short interspersed repeats from Xenopus that contain multiple octamer motifs are related to known transposable elements.

We have identified in an intron of an X. laevis alpha-tubulin gene a member of a novel family of short (226-431 bp) interspersed repetitive elements. We have isolated other members of this family, which we term Ocr, from ovary cDNA and genome libraries and have identified another two in the published sequences of an H1B histone gene cluster and an actin gene intron. The termini of the Ocr elements are formed by a 19 bp inverted repeat that has clear sequence homologies to those of certain large transposable elements, such as 1723 (Xenopus) and Ac (maize). However, the Ocr elements do not appear to be deletion derivatives of larger transposons. The internal regions of the Ocr elements contain multiple copies of the octamer motif (ATTTGCAT) arranged as divergently-orientated dyads. We have shown by a gel mobility shift assay that these octamer dyads specifically bind what is presumably an OTF-type activator protein in oocyte nuclear extracts. We speculate that short interspersed repetitive families of this type may be generated by a mechanism of replicative transposition that uses a DNA intermediate and involves the interaction of DNA-binding proteins also utilised in other cellular processes.     

Transposable elements in mosquitoes

We describe the current state of knowledge about transposable elements (TEs) in different mosquito species. DNA-based elements (class II elements), non-LTR retrotransposons (class I elements), and MITEs (Miniature Inverted Repeat Transposable Elements) are found in the three genera, Anopheles, Aedes and Culex, whereas LTR retrotransposons (class I elements) are found only in Anopheles and Aedes. Mosquitoes were the first insects in which MITEs were reported; they have several LTR retrotransposons belonging to the Pao family, which is distinct from the Gypsy-Ty3 and Copia-Ty1 families. The number of TE copies shows huge variations between classes of TEs within a given species (from 1 to 1000), in sharp contrast to Drosophila, which shows only relatively minor differences in copy number between elements (from 1 to 100). The genomes of these insects therefore display major differences in the amount of TEs and therefore in their structure and global composition. We emphasize the need for more population genetic data about the activity of TEs, their distribution over chromosomes and their frequencies in natural populations of mosquitoes, to further the current attempts to develop a transgenic mosquito unable to transmit malaria that is intended to replace the natural populations.     

Transposable elements in medaka fish

DNA-based transposable elements appear to have been nearly or completely inactivated in vertebrates. Therefore the elements of the medaka fish Oryzias latipes that still have transposition activity provide precious materials for studying transposition mechanisms, as well as the evolution, of transposable elements in vertebrates. Fortunately, the medaka fish has a strong background for genetic and evolutionary studies. The advantages of this host species and their elements, together with results so far obtained, are here described.     

Survey of transposable elements from rice genomic sequences

Oryza sativa L. (domesticated rice) is a monocotyledonous plant, and its 430 Mb genome has been targeted for complete sequencing. We performed a high-resolution computer-based survey for transposable elements on 910 Kb of rice genomic DNA sequences. Both class I and II transposable elements were present, contributing 19.9% of the sequences surveyed. Class II elements greatly outnumbered class I elements (166 versus 22), although class I elements made up a greater percentage (12.2% versus 6.6%) of nucleotides surveyed. Several Mutator-like elements (MULEs) were identified, including rice elements that harbor truncated host cellular genes. MITEs (miniature inverted-repeat transposable elements) account for 71.6% of the mined transposable elements and are clearly the predominant type of transposable element in the sequences examined. Moreover, a putative Stowaway transposase has been identified based on shared sequence similarity with the mined MITEs and previously identified plant mariner-like elements (MLEs). Members of a group of novel rice elements resembling the structurally unusual members of the Basho family in Arabidopsis suggest a wide distribution of these transposons among plants. Our survey provides a preview of transposable element diversity and abundance in rice, and allows for comparison with genomes of other plant species.     

Cloning and characterization of variable-sized gypsy mobile elements in Drosophila melanogaster

A cosmid genomic library from a known gypsy-induced forked mutation, f1, was screened by 32P-labeled gypsy transposable element. Of more than 250 positive clones we randomly selected 21 for in situ hybridization to wild-type polytene chromosomes. Two clones hybridized to region 15F on the X-chromosome, the cytological position of forked. A third clone hybridized to at least 17 sites on the chromosomes indicating the presence of repetitive sequences in the gypsy flanking DNA. All clones labeled the centromeric regions heavily. Ten clones, including the two hybridizing at 15F, were chosen for further analysis, and restriction mapping allowed us to place them into three groups: (1) full-length, (2) slightly diverging, and (3) highly diverging gypsy elements. Group (2) is missing the XbaI site in both their long terminal repeats (LTRs) as well as the middle HindIII site; four of these gypsy elements also have a approximately 100-bp deletion at the 5' LTR. The group (3) gypsy transposons are missing one LTR and also have highly diverging DNA sequences. The restriction analyses further imply that most of these different gypsy elements are present in more than one copy in the genome of the f1 stock used in this study. The results raise intriguing questions regarding the significance of transposable elements in evolution and biological functions.     

Mapping of <Emphasis Type="Italic">Hieracium</Emphasis> (Asteraceae) chromosomes with genus-specific satDNA elements derived from next-generation sequencing data

The highly repetitive DNA fraction of the eukaryotic genome is considered a mobile, rapidly changing entity, thus reflecting trajectories of short-term evolutionary change. It consists of several large classes in which transposable elements and satellite DNA (satDNA) predominate. Despite a growing awareness of its structure and functional significance, the evolutionary dynamics of repetitive elements and, particularly, satDNA remain poorly characterized. Next-generation sequencing (NGS) has opened up new possibilities for high-throughput genome analysis. Here, we applied satDNA repeatome elements derived from NGS data as probes for fluorescence in situ hybridization to characterize the karyotypes of three diploid hawkweed species of the predominantly polyploid apomictic genus Hieracium, namely H. intybaceum, H. prenanthoides and H. alpinum. Three cluster-distributed, genus-specific satDNA elements that are not present in the sister genus Pilosella were identified; notably, one element spans the functional centromeres. Each of the investigated diploids possessed a species-specific assortment of detected repeats. Their utilization as molecular-cytogenetic markers, in combination with ribosomal DNA loci, allowed for the development of a system to identify the individual chromosomes of the Hieracium species, thus providing a basis for the future investigation of karyotype evolution in diploid hawkweeds and for exploring satDNA dynamics in hybrids and apomicts of allopolyploid origin.     

Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.)

Members of a novel Master family of class II transposons were identified in the carrot genome. Two elements, 2.5 kb long DcMaster1 and 4.4 kb long DcMaster-a, are characterized by 22 bp imperfect terminal inverted repeats and by 3 bp target site duplications. GenBank search revealed that related elements are also present in Medicago truncatula, including a 5.1 kb element MtMaster-a. Both DcMaster-a and MtMaster-a contain open reading frames encoding for putative transposases with the complete DDE domain typical for plant class II transposable elements belonging to PIF/Harbinger superfamily, where the Master elements form a distinct group. Less than 10 copies of the DcMaster element containing the DDE domain are present in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in 8 of 14 identified insertion sites. PCR amplification of carrot genomic DNA using a primer complementary to TIRs of DcMaster gave products <400 bp in size. We speculate that these may all represent a MITE-like family of transposable elements that we named Krak, present in the carrot genome in at least 3,600 copies. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers DQ250792 to DQ250807 and DQ353734 to DQ353752.