Molecular and cytological analyses of large tracks of centromeric DNA reveal the structure and evolutionary dynamics of maize centromeres

We sequenced two maize bacterial artificial chromosome (BAC) clones anchored by the centromere-specific satellite repeat CentC. The two BACs, consisting of approximately 200 kb of cytologically defined centromeric DNA, are composed exclusively of satellite sequences and retrotransposons that can be classified as centromere specific or noncentromere specific on the basis of their distribution in the maize genome. Sequence analysis suggests that the original maize sequences were composed of CentC arrays that were expanded by retrotransposon invasions. Seven centromere-specific retrotransposons of maize (CRM) were found in BAC 16H10. The CRM elements inserted randomly into either CentC monomers or other retrotransposons. Sequence comparisons of the long terminal repeats (LTRs) of individual CRM elements indicated that these elements transposed within the last 1.22 million years. We observed that all of the previously reported centromere-specific retrotransposons in rice and barley, which belong to the same family as the CRM elements, also recently transposed with the oldest element having transposed approximately 3.8 million years ago. Highly conserved sequence motifs were found in the LTRs of the centromere-specific retrotransposons in the grass species, suggesting that the LTRs may be important for the centromere specificity of this retrotransposon family.     

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: nucleotide sequence of long terminal repeats.

Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).     

The heterochromatic copies of the LTR retrotransposons as a record of the genomic events that have shaped the Drosophila melanogaster genome

Transposable elements, which are major components of most genomes, are known to accumulate in heterochromatic regions in which they have progressively diverged in sequence by mutations and internal deletions and insertions (indels) during the course of evolution. They therefore provide a record of the genomic events that have shaped the genomes, some of which could correspond to speciation events. Using the sequence divergence between the long terminal repeats (LTRs), we estimated the date of the insertion events of the LTR retrotransposon copies embedded within the heterochromatin regions of the Drosophila melanogaster genome. We did not detect traces of any specific waves of mobilization of retrotransposons within heterochromatin, apart from a very recent wave, which corresponds to the numerous LTR retrotransposon copies found in euchromatin.     

Fragments of LINE-1 retrotransposons flanked by inverted telomeric repeats are present in the bovine genome. Homology with human LINE-1 elements

In the bovine genome we found two intrachromosomal DNA fragments flanked by inverted telomeric repeats (GenBank Accession Nos. AF136741 and AF136742). The internal parts of the fragments are homologous exclusively to the human sequences and to the consensus sequence of the L1MC4 subfamily of LINE-1 retrotransposons which are widespread among mammalian genomes. We found that distribution of homologous human sequences within our fragments is not random, reflecting a complicated pattern of insertion mechanisms of and maintenance of retrotransposons in mammalian genomes. One of the possible explanations of the origin of LINE-1 truncated elements flanked by inverted telomeric repeats in the bovine genome is that extrachromosomal DNA fragments may be modified by telomerase and subsequently, transferred into chromosomal DNA.     

The changing tails of a novel short interspersed element in Aedes aegypti: genomic evidence for slippage retrotransposition and the relationship between 3' tandem repeats and the poly(dA) tail

A novel family of tRNA-related SINEs named gecko was discovered in the yellow fever mosquito, Aedes aegypti. Approximately 7200 copies of gecko were distributed in the A. aegypti genome with a significant bias toward A + T-rich regions. The 3' end of gecko is similar in sequence and identical in secondary structure to the 3' end of MosquI, a non-LTR retrotransposon in A. aegypti. Nine conserved substitutions and a deletion separate gecko into two groups. Group I includes all gecko that end with poly(dA) and a copy that ends with AGAT repeats. Group II comprises gecko elements that end with CCAA or CAAT repeats. Members within each group cannot be differentiated when the 3' repeats are excluded in phylogenetic and sequence analyses, suggesting that the alterations of 3' tails are recent. Imperfect poly(dA) tail was recorded in group I and partial replication of the 3' tandem repeats was frequently observed in group II. Genomic evidence underscores the importance of slippage retrotransposition in the alteration and expansion of the tandem repeat during the evolution of gecko sequences, although we do not rule out postinsertion mechanisms that were previously invoked to explain the evolution of Alu-associated microsatellites. We propose that the 3' tandem repeats and the poly(dA) tail may be generated by similar mechanisms during retrotransposition of both SINEs and non-LTR retrotransposons and thus the distinction between poly(dA) retrotransposons such as L1 and non-poly(dA) retrotransposons such as I factor may not be informative.     

The Cassandra retrotransposon landscape in sugar beet (Beta vulgaris) and related Amaranthaceae: recombination and re-shuffling lead to a high structural variability

Background and AimsPlant genomes contain many retrotransposons and their derivatives, which are subject to rapid sequence turnover. As non-autonomous retrotransposons do not encode any proteins, they experience reduced selective constraints leading to their diversification into multiple families, usually limited to a few closely related species. In contrast, the non-coding Cassandra terminal repeat retrotransposons in miniature (TRIMs) are widespread in many plants. Their hallmark is a conserved 5S rDNA-derived promoter in their long terminal repeats (LTRs). As sugar beet (Beta vulgaris) has a well-described LTR retrotransposon landscape, we aim to characterize TRIMs in beet and related genomes.MethodsWe identified Cassandra retrotransposons in the sugar beet reference genome and characterized their structural relationships. Genomic organization, chromosomal localization, and distribution of Cassandra-TRIMs across the Amaranthaceae were verified by Southern and fluorescent in situ hybridization.Key resultsAll 638 Cassandra sequences in the sugar beet genome contain conserved LTRs and thus constitute a single family. Nevertheless, variable internal regions required a subdivision into two Cassandra subfamilies within B. vulgaris. The related Chenopodium quinoa harbours a third subfamily. These subfamilies vary in their distribution within Amaranthaceae genomes, their insertion times and the degree of silencing by small RNAs. Cassandra retrotransposons gave rise to many structural variants, such as solo LTRs or tandemly arranged Cassandra retrotransposons. These Cassandra derivatives point to an interplay of template switch and recombination processes – mechanisms that likely caused Cassandra’s subfamily formation and diversification.ConclusionsWe traced the evolution of Cassandra in the Amaranthaceae and detected a considerable variability within the short internal regions, whereas the LTRs are strongly conserved in sequence and length. Presumably these hallmarks make Cassandra a prime target for unequal recombination, resulting in the observed structural diversity, an example of the impact of LTR-mediated evolutionary mechanisms on the host genome.     

Replication of nonautonomous retroelements in soybean appears to be both recent and common

Retrotransposons and their remnants often constitute more than 50% of higher plant genomes. Although extensively studied in monocot crops such as maize (Zea mays) and rice (Oryza sativa), the impact of retrotransposons on dicot crop genomes is not well documented. Here, we present an analysis of retrotransposons in soybean (Glycine max). Analysis of approximately 3.7 megabases (Mb) of genomic sequence, including 0.87 Mb of pericentromeric sequence, uncovered 45 intact long terminal repeat (LTR)-retrotransposons. The ratio of intact elements to solo LTRs was 8:1, one of the highest reported to date in plants, suggesting that removal of retrotransposons by homologous recombination between LTRs is occurring more slowly in soybean than in previously characterized plant species. Analysis of paired LTR sequences uncovered a low frequency of deletions relative to base substitutions, indicating that removal of retrotransposon sequences by illegitimate recombination is also operating more slowly. Significantly, we identified three subfamilies of nonautonomous elements that have replicated in the recent past, suggesting that retrotransposition can be catalyzed in trans by autonomous elements elsewhere in the genome. Analysis of 1.6 Mb of sequence from Glycine tomentella, a wild perennial relative of soybean, uncovered 23 intact retroelements, two of which had accumulated no mutations in their LTRs, indicating very recent insertion. A similar pattern was found in 0.94 Mb of sequence from Phaseolus vulgaris (common bean). Thus, autonomous and nonautonomous retrotransposons appear to be both abundant and active in Glycine and Phaseolus. The impact of nonautonomous retrotransposon replication on genome size appears to be much greater than previously appreciated.     

Both sense and antisense strands of the LTR of the <Emphasis Type="Italic">Schistosoma mansoni Pao</Emphasis>-like retrotransposon <Emphasis Type="Italic">Sinbad</Emphasis> drive luciferase expression

Long terminal repeat (LTR) retrotransposons, mobile genetic elements comprising substantial proportions of many eukaryotic genomes, are so named for the presence of LTRs, direct repeats about 250–600 bp in length flanking the open reading frames that encode the retrotransposon enzymes and structural proteins. LTRs include promotor functions as well as other roles in retrotransposition. LTR retrotransposons, including the Gypsy-like Boudicca and the Pao/BEL-like Sinbad elements, comprise a substantial proportion of the genome of the human blood fluke, Schistosoma mansoni. In order to deduce the capability of specific copies of Boudicca and Sinbad LTRs to function as promotors, these LTRs were investigated analytically and experimentally. Sequence analysis revealed the presence of TATA boxes, canonical polyadenylation signals, and direct inverted repeats within the LTRs of both the Boudicca and Sinbad retrotransposons. Inserted in the reporter plasmid pGL3, the LTR of Sinbad drove firefly luciferase activity in HeLa cells in its forward and inverted orientation. In contrast, the LTR of Boudicca did not drive luciferase activity in HeLa cells. The ability of the Sinbad LTR to transcribe in both its forward and inverted orientation represents one of few documented examples of bidirectional promotor function.     

Diversity of LTR retrotransposons and their role in genome reorganization

Current views of retrotransposons possessing long terminal repeats (LTRs) are described. The existing classification and element types isolated by genome organization are considered. Experimental data are summarized to demonstrate that the replicative cycle of a retrotransposon is not restricted to a single cell and that LTR retrotransposons are transferred between somatic cells with a rate comparable with the element transposition rate within the genome of one cell. The major mechanisms mediating the role of LTR retrotransposons in reorganization of the genome are considered with regard to the strategies of their horizontal and vertical transfer.     

IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques

 The BARE-1 retrotransposon is an active, dispersed, and highly abundant component of the genome of barley (Hordeum vulgare) and other species in its genus. Like all retrotransposons of its kind, BARE-1 is bounded by long terminal repeats (LTRs). We have developed two amplification-based marker methods based on the position of given LTRs within the genome. The IRAP (Inter-Retrotransposon Amplified Polymorphism) markers are generated by the proximity of two LTRs using outward-facing primers annealing to LTR target sequences. In REMAP (REtrotransposon-Microsatellite Amplified Polymorphism), amplification between LTRs proximal to simple sequence repeats such as constitute microsatellites produces markers. The methods can distinguish between barley varieties and produce fingerprint patterns for species across the genus. The patterns indicate that although the BARE-1 family of retrotransposons is disperse, these elements are locally clustered or nested and often found near tandem arrays of a simple sequence repeat. Received: 30 June 1998 / Accepted: 21 August 1998     

Structural organization of transposable element mdg4 from Drosophila melanogaster and a nucleotide sequence of its long terminal repeats

A mobile dispersed genetic element, mdg4 , approximately 7.5 kilobases (kb) long has been cloned from D. melanogaster genome. Chromosomal bands have only few sites of mdg4 , but it always hybridizes to the chromocenter. The location of mdg4 varies among D. melanogaster strains. Blot hybridization shows that, in contrast to other mdg elements, mdg4 sequences are rather heterogeneous. Only few copies are full-length. A strong amplification of mdg4 has occurred during the in vitro cultivation of cells involving only one mdg4 variant. Long terminal repeats (LTRs) and flanking sequences have been sequenced in two cloned copies of transposable element mdg4 . In both cloned copies of mdg4 , LTRs have an identical nucleotide sequence 479 bp long. The mdg4 is flanked by four-base-pair direct repeats, short mismatched palindromes being present at the ends of each LTR. The termini of the mdg4 body contain an oligopurine stretch and a region partially complementary to D. melanogaster tRNA-Lys. Thus, structural organization of mdg4 LTRs is similar to that of several other mdg elements and retroviral proviruses.