Structural and evolutionary analysis of the copia-like elements in the Arabidopsis thaliana genome

The analysis of 460 kb of genomic sequence of Arabidopsis thaliana chromosome III allowed us to identify two new transposable elements named AtC1 and AtC2. AtC1 shows identical long terminal repeats (LTRs) and all the structural features characteristic of the copia-like active elements. AtC2 is also a full copia-like element, but a putative stop codon in the open reading frame (ORF) would produce a truncated protein. In order to identify the copia-like fraction of the A. thaliana genome, a careful computer-based analysis of the available sequences (which correspond to 92% of the genome) was performed. Approximately 300 nonredundant copia-like sequences homologous to AtC1 and AtC2 were detected, which showed an extreme heterogeneity in size and degree of conservation. This number of copies would correspond to approximately 1% of the A. thaliana genome. Seventy-one sequences were selected for further analysis, with 23 of them being full complete elements. Five corresponded to previously described ones, and the remaining ones, named AtC3 to AtC18 are new elements described in this work. Most of these elements presented a putative functional ORF, nearly identical LTRs, and the other elements necessary for retrotransposon activity. Phylogenetic trees, supported by high bootstrap values, indicated that these 23 elements could be considered separate families. In turn, these 23 families could be clustered into six major lineages, named copia I-VI. Most of the 71 analyzed sequences clustered into these six main clades. The widespread presence of these copia-like superfamilies throughout plant genomes is discussed.     

Complete nucleotide sequence and genome organization of a Drosophila transposable genetic element, 297

The complete nucleotide sequence of 297, a Drosophila copia-like transposable element, was determined and compared with those of other similar Drosophila elements and mammalian retrovirus proviruses. It was found that 297 contains three long open reading frames, comparable in sizes and locations with gag, pol, and env genes in the proviruses of replication-competent retroviruses in vertebrates. The first and second open reading frames of 297 exhibit sequence homologies to gag and pol, respectively, of Moloney murine leukaemia virus. In particular, as with 17.6, another Drosophila copia-like element, the second open reading frame of 297 was shown to be very similar in its entire organization to the retroviral pol gene and to consist of three enzymatic domains. By contrast, no appreciable homology was found between the third open reading frame of 297 and the retroviral env gene. It is also suggested that 297 and 17.6 are a peculiar pair of copia-like elements recently diverged from a common progenitor.     

Structure of circular copies of the 412 transposable element present in Drosophila melanogaster tissue culture cells, and isolation of a free 412 long terminal repeat

We have isolated, from Drosophila melanogaster tissue culture cells, extrachromosomal circular forms of the transposable element 412, and have cloned some of them in bacteriophage lambda. A total of 24 clones have been analysed in detail by restriction and heteroduplex mapping. Seventeen clones are virtually identical, and contain complete 412 elements with one copy of the long terminal direct repeat (LTR). The remaining seven clones are all different and contain various rearrangements. Four have deletions, two have some 412 sequence substituted by other DNA and one has both an inversion and a deletion. The clone containing the inversion has two LTRs in inverted orientation and separated by a few thousand bases of 412 DNA. The base sequences of the two LTRs in this clone, and of the LTR in one of the 17 clones containing complete elements are very similar to that of the 481 base-pair LTR of a genomic 412 element. We have found no evidence, in either cloned or uncloned material, for 412 elements with two LTRs as a tandem direct repeat. We have found that there are several "free" 412 LTRs in genomic DNA from D. melanogaster strains Canton S and Oregon R, and from D. melanogaster tissue culture cells. We have cloned and sequenced one of these free LTRs. It is 475 base-pairs long and is flanked by a direct repeat four base-pairs long. This sequence differs from that of the 481 base-pair repeat at 16 places including a ten base deletion.     

Truncated and RIP-degenerated copies of the LTR retrotransposon Pholy are clustered in a pericentromeric region of the Leptosphaeria maculans genome

The LMR1 5.2 kb interspersed repeat of Leptosphaeria maculans was described by Taylor and Borgmann [Mol. Plant Microbe Interact. 7 (1994) 181] as an uncharacterized repeated element sharing homologies with both LINEs and SINEs. Here, we used the LMR1 sequence as a template to identify the full-length element within a 184-kb genomic sequence corresponding to the pericentromeric region of the 2.80 Mb chromosome of isolate v23.1.3. This region comprises (i) one 6980-bp full-sized Pholy element bordered by two 275- to 280-bp long terminal repeats (LTRs), (ii) five Pholy-related sequences, usually truncated at their 3' ends, and (iii) five solo-LTRs. Structural features strongly suggested that Pholy corresponds to an ancient copia-like retrotransposon, sharing strong homologies with the Elsa retrotransposon of Stagonospora nodorum. Pholy was also suggested to be specific to pericentromeric regions. Comparative analysis of the structure of the Pholy-like sequences occurring in the 184-kb contig and in other parts of the genome showed that this family of repeats is highly degenerated following extensive repeat induced point mutation (RIP).     

Cloning and characterization of Vine-1, a LTR-retrotransposon-like element in Vitis vinifera L., and other Vitis species.

We report the organization of a grapevine chimeric gene Adhr-Vine-1, composed by an Adhr gene, into which a retroelement, Vine-1, was inserted. Sequence analysis revealed that Adhr is a member of the Adh multigene family, but does not correspond to any other grapevine Adh described to date. Vine-1, albeit defective, is the most complete LTR (long terminal repeat)-retrotransposon-like element described in Vitis vinifera L. It is 2392 bp long, with two almost identical LTRs (287 bp) in the same orientation, and flanked by direct repeats of a 5 bp host DNA. This element presents other features, characteristic of retroviruses and retrotransposons including inverted repeats, a primer binding site, and a polypurine tract. It has a single open reading frame (ORF) of 581 amino acids, potentially encoding for a gag protein and parts of the protease and integrase proteins. Vine-1 is most likely related to the copia-like type family, but with no significant similarity to any previously described plant retrotransposon or inserted element, nor to any eukaryotic element described to date. Vine-1 element has been found in Adhr at the same location in different V. vinifera cultivars, but not in some other analyzed Vitis species. These data suggest that Vine-1 insertion in Adhr is specific to V. vinifera, and has occurred after the Adh isogene separation, but prior to cultivar development. Sequences related to Vine-1 were revealed in multiple copies in the V. vinifera genome and, to a lesser extent, in other analyzed Vitis species. The polymorphism observed prompts us to question the role played by transposition in the evolution of the Vitis genus.     

Extrachromosomal DNA forms of copia-like transposable elements, F elements and middle repetitive DNA sequences in Drosophila melanogaster. Variation in cultured cells and embryos

Drosophila melanogaster embryos and cells in culture were screened for the presence of unintegrated covalently closed circular DNA forms that hybridize to copia-like transposable elements, the F element and uncharacterized dispersed middle repetitive DNA elements. Our results indicate that the majority of copia-like elements (including copia, 297, 412, mdg1, mdg3 and gypsy), the F elements, and 9 of 12 middle repetitive DNA elements are present as free DNA forms in cultured cells and embryos. An 18 base-pair inverted repeat has been reported to flank the long direct repeat of mdg3, implying that mdg3 is not an orthodox copia-like element; however, we have sequenced two independently isolated mdg3 clones and shown that the inverted repeat is not part of the element. The relative abundance with which free DNA forms are found varies between the cultured cells used, and between cultured cells and embryos. This variation, which can be up to 20-fold for some elements, does not correlate well with either the amount of element-specific poly(A)+ RNA present per cell or the number of element-specific sequences integrated in the genome.     

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.