Novel non-autonomous transposable elements on W chromosome of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The sex chromosomes of the silkworm Bombyx mori are designated ZW(XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Bm1 do not occur in the WGS data, we suggest that the Bm1 sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.     

Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori

The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome.     

The sunflower (Helianthus annuus L.) genome reflects a recent history of biased accumulation of transposable elements

Aside from polyploidy, transposable elements are the major drivers of genome size increases in plants. Thus, understanding the diversity and evolutionary dynamics of transposable elements in sunflower (Helianthus annuus L.), especially given its large genome size (～3.5 Gb) and the well‐documented cases of amplification of certain transposons within the genus, is of considerable importance for understanding the evolutionary history of this emerging model species. By analyzing approximately 25% of the sunflower genome from random sequence reads and assembled bacterial artificial chromosome (BAC) clones, we show that it is composed of over 81% transposable elements, 77% of which are long terminal repeat (LTR) retrotransposons. Moreover, the LTR retrotransposon fraction in BAC clones harboring genes is disproportionately composed of chromodomain‐containing Gypsy LTR retrotransposons (‘chromoviruses’), and the majority of the intact chromoviruses contain tandem chromodomain duplications. We show that there is a bias in the efficacy of homologous recombination in removing LTR retrotransposon DNA, thereby providing insight into the mechanisms associated with transposable element (TE) composition in the sunflower genome. We also show that the vast majority of observed LTR retrotransposon insertions have likely occurred since the origin of this species, providing further evidence that biased LTR retrotransposon activity has played a major role in shaping the chromatin and DNA landscape of the sunflower genome. Although our findings on LTR retrotransposon age and structure could be influenced by the selection of the BAC clones analyzed, a global analysis of random sequence reads indicates that the evolutionary patterns described herein apply to the sunflower genome as a whole.     

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori

Summary Three -type early chorion gene copies (6F76.1, 6F76.2, and 6F76.3) are dispersed in the late region of chorion locus Chl-2. Detailed analysis of the 5-flanking region and the intron of 6176.1 shows that they contain sequences that are homologous to Bombyx mori Bm l repeat elements. Interestingly, the Bm l -type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6176.1 5-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion -gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.Offprint requests to: G. Rodakis     

Comparative analysis of complete orthologous centromeres from two subspecies of rice reveals rapid variation of centromere organization and structure

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. This function is conserved across species, but the DNA components that are involved in kinetochore formation differ greatly, even between closely related species. To shed light on the nature, evolutionary timing and evolutionary dynamics of rice centromeres, we decoded a 2.25‐Mb DNA sequence covering the centromeric region of chromosome 8 of an indica rice variety, ‘Kasalath’ (Kas‐Cen8). Analysis of repetitive sequences in Kas‐Cen8 led to the identification of 222 long terminal repeat (LTR)‐retrotransposon elements and 584 CentO satellite monomers, which account for 59.2% of the region. A comparison of the Kas‐Cen8 sequence with that of japonica rice ‘Nipponbare’ (Nip‐Cen8) revealed that about 66.8% of the Kas‐Cen8 sequence was collinear with that of Nip‐Cen8. Although the 27 putative genes are conserved between the two subspecies, only 55.4% of the total LTR‐retrotransposon elements in ‘Kasalath’ had orthologs in ‘Nipponbare’, thus reflecting recent proliferation of a considerable number of LTR‐retrotransposons since the divergence of two rice subspecies of indica and japonica within Oryza sativa. Comparative analysis of the subfamilies, time of insertion, and organization patterns of inserted LTR‐retrotransposons between the two Cen8 regions revealed variations between ‘Kasalath’ and ‘Nipponbare’ in the preferential accumulation of CRR elements, and the expansion of CentO satellite repeats within the core domain of Cen8. Together, the results provide insights into the recent proliferation of LTR‐retrotransposons, and the rapid expansion of CentO satellite repeats, underlying the dynamic variation and plasticity of plant centromeres.     

A computational study of the dynamics of LTR retrotransposons in the Populus trichocarpa genome

Retrotransposons are an ubiquitous component of plant genomes, especially abundant in species with large genomes. Populus trichocarpa has a relatively small genome, which was entirely sequenced; however, studies focused on poplar retrotransposons dynamics are rare. With the aim to study the retrotransposon component of the poplar genome, we have scanned the complete genome sequence searching full-length long-terminal repeat (LTR) retrotransposons, i.e., characterised by two long terminal repeats at the 5′ and 3′ ends. A computational approach based on detection of conserved structural features, on building multiple alignments, and on similarity searches was used to identify 1,479 putative full-length LTR retrotransposons. Ty1-copia elements were more numerous than Ty3-gypsy. However, many LTR retroelements were not assigned to any superfamily because lacking of diagnostic features and non-autonomous. LTR retrotransposon remnants were by far more numerous than full-length elements, indicating that during the evolution of poplar, large amplification of these elements was followed by DNA loss. Within superfamilies, Ty3-gypsy families are made of more members than Ty1-copia ones. Retrotransposition occurred with increasing frequency following the separation of Populus sections, with different waves of retrotransposition activity between Ty3-gypsy and Ty1-copia elements. Recently inserted elements appear more frequently expressed than older ones. Finally, different levels of activity of retrotransposons were observed according to their position and their density in the linkage groups. On the whole, the results support the view of retrotransposons as a community of different organisms in the genome, whose activity (both retrotransposition and DNA loss) has heavily impacted and probably continues to impact poplar genome structure and size.     

Evidence for a relationship between the Bombyx mori middle repetitive Bm1 sequence family and U1 snRNA

Several genomic library equivalents of Bombyx mori were constructed in the EMBL-4 lambda derivative. The genomic bank was screened with purified Bombyx mori U1 RNA and twenty positive clones for the U1 gene were isolated. Three U1-related sequences were subcloned and sequenced. Two of the sequences are U1 pseudogenes while a third sequence represents a member of the Bm1 family of repetitive elements of B.mori with significant sequence similarity to U1 small nuclear RNA. The U1-related Bm1 element exhibits 82% sequence similarity with the Bm1 consensus sequence and, under less stringent computer comparison parameters, 60% similarity with a composite B.mori/Drosophila melanogaster U1 gene. The Bm1 family consensus sequence exhibits 53% sequence similarity with the composite U1 gene. The two pseudogenes possess highly conserved sequences with the B.mori U1 gene only for the first 101 nucleotides. These findings are indicative of at least two different categories of U1-related sequences in B.mori, one with a possible evolutionary relationship to the Bm1 family of repetitive elements and the other representing characteristic processed pseudogenes with retroposon mode of dispersion and target selection for the TTTA hotspot. In addition, the U1-related Bm1 element may demonstrate for the first time that a family of retroposons is ultimately derived from a U snRNA.This article is dedicated in memory of Ms. Deborah Lampert who helped so much in the preparation of this paper.     

A Helitron-Like Transposon Superfamily from Lepidoptera Disrupts (GAAA)n Microsatellites and is Responsible for Flanking Sequence Similarity within a Microsatellite Family

Transposable elements (TEs) are mobile DNA regions that alter host genome structure and gene expression. A novel 588 bp non-autonomous high copy number TE in the Ostrinia nubilalis genome has features in common with miniature inverted-repeat transposable elements (MITEs): high A + T content (62.3%), lack of internal protein coding sequence, and secondary structure consisting of subterminal inverted repeats (SIRs). The O. nubilalis TE has inserted at (GAAA)_n microsatellite loci, and was named the microsatellite-associated interspersed nuclear element (MINE-1). Non-autonomous MINE-1 superfamily members also were identified downstream of (GAAA)_n microsatellites within Bombyx mori and Pectinophora gossypiella genomes. Of 316 (GAAA)_n microsatellites from the B. mori whole genome sequence, 201 (63.6%) have associated autonomous or non-autonomous MINE-1 elements. Autonomous B. mori MINE-1s a encode a helicase and endonuclease domain RepHel-like protein (BMHELp1) indicating their classification as Helitron-like transposons and were renamed Helitron1_BM. Transposition of MINE-1 members in Lepidoptera has resulted in the disruption of (GAAA)_n microsatellite loci, has impacted the application of microsatellite-based genetic markers, and suggests genome sequence that flanks TT/AA dinucleotides may be required for target site recognition by RepHel endonuclease domains.     

The retrotransposon RTip1 is integrated into a novel type of minisatellite, MiniSip1, in the genome of the common morning glory and carries another new type of minisatellite, MiniSip2

 Transposable elements have often been discovered as new insertion sequences in known genes, and minisatellites are often employed as molecular markers in diagnostic and mapping studies. We compared the genes for flower pigmentation in a line of the common morning glory bearing fully colored flowers with those in two anthocyanin ^flaked mutable lines producing variegated flowers and found RFLPs at the region of the ANS gene for anthocyanin biosynthesis. The DNA rearrangements detected by the RFLPs are due to integration of a novel type of minisatellite, MiniSip1, having a long LTR retrotransposon, RTip1, inserted in the mutable lines. The structural analysis of the rearranged region revealed that the 12.4-kb RTip1 element is flanked by 5-bp target duplications within the MiniSip1 sequence and contains two LTR sequences of about 590 bp, a primer binding site for tRNA^Lys, a typical polypurine tract and another new type of minisatellite, MiniSip2. Since no long open reading frame corresponding to the gag and pol genes was found, RTip1 appears to be a defective Ty3/gypsy-like element. Interestingly, the 269-bp-long MiniSip1 element comprises two alternating motifs of 41 bp and 19 bp, whereas the 962 bp long MiniSip2 element consists of two partially alternating motifs of 86 bp and 90 bp which are partially homologous to each other. Possible evolutionary processes that may have generated the rearranged structure at the ANS gene region are also discussed. Received: 25 April 1997 / Accepted: 16 May 1997     

Probing the W chromosome of the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis>, with sequences from microdissected sex chromatin

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.     

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.     

Genomic organization of repetitive DNAs in the cichlid fish <Emphasis Type="Italic">Astronotus ocellatus</Emphasis>

To contribute to the knowledge of fish genomes, we identified and characterized by means of nucleotide sequencing and physical chromosome mapping, three classes of repetitive DNAs in the genome of the South American cichlid fish Astronotus ocellatus. The first class corresponds to a satellite DNA family (AoSat) that shares similarity with a centromeric satellite DNA of the pufferfish Tetraodon nigroviridis. The second repetitive DNA class (AoRex3) is related to the retrotransposon Rex3, which is widely distributed among teleost fishes. The last repetitive element (AoLINE) shows a high similarity to the CR1-like LINE element of other teleosts. The three isolated repetitive elements are clustered in the centromeric heterochromatin of all chromosomes of the complement. The repetitive sequences are not randomly distributed in the genome, suggesting a pattern of compartmentalization on chromosomes.     

MAX, a novel retrotransposon of the BEL-Pao family, is nested within the Bari1 cluster at the heterochromatic h39 region of chromosome 2 in Drosophila melanogaster

A homogeneous array of 80 tandem repeats of the Bari1 transposon is located in the pericentromeric h39 region of chromosome 2 of Drosophila melanogaster. Here, we report that the Bari1 cluster is interrupted by an 8556-bp insertion. DNA sequencing and database searches identified this insertion as a previously unannotated retrotransposon that we have named MAX. MAX possesses two ORFs; ORF1 putatively encodes a polyprotein comprising GAG and RT domains, while ORF2 could encode a 288-amino acid protein of unknown function. Alignment with the RT domains of known LTR retrotransposons shows that MAX belongs to the BEL-Pao family, which remarkable for its widespread presence in different taxa, including lower chordates. We have analyzed the distribution of MAX elements within representative species of the Sophophora subgroup and found that they are restricted to the species of the melanogaster complex, where they are heavily represented in the heterochromatin of all autosomes and on the Y chromosome.Communicated by G. P. Georgiev     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

Molecular paleontology of transposable elements from Arabidopsis thaliana

We report results of a comprehensive computer-assisted analysis of new transposable elements (TEs) from Arabidopsis thaliana. Our analysis revealed several previously unknown pogo- and En/Spm-like families and two novel superfamilies of DNA transposons, Arnold and Harbinger. One of the En/Spm-like families (Atenspm) was found to be involved in generating satellite arrays in paracentromeric regions. Of the two superfamilies reported, Harbinger is distantly related to bacterial IS5-like insertion elements, and Arnold contains DNA transposons without terminal inverted repeats (TIRs), which were never reported in eukaryotes before. Furthermore, we report a large number of young and diverse copia-like autonomous and nonautonomous retroelements and discuss their potential evolutionary relationship with mammalian retroviruses. The A.thaliana genome harbors copia-like retroelements which encode a putative env-like protein reported previously in the SIRE-1 retrotransposon from soybean. Finally, we demonstrate a nonrandom chromosomal distribution of the most abundant A.thaliana TEs clustered in the first half of chromosome II, which includes the centromeric region. The families of TEs from A.thaliana are relatively young, extremely diverse and much smaller than those from mammalian genomes. We discuss the potential factors determining similarities and differences in the evolution of TEs in mammals and A. thaliana. This revised version was published online in July 2006 with corrections to the Cover Date.