Structure and Origin of a Novel Dimeric Retroposon B1-dID

Here we describe a new short retroposon family of rodents. Like the primate Alu element consisting of two similar monomers, it is dimeric, but the left and right monomers are different and descend from B1 and ID short retroposons, respectively. Such elements (B1-dID) were found in the genomes of Gliridae, Sciuridae, Castoridae, Caviidae, and Hystricidae. Nucleotide sequences of this retroposon can be assigned to several structural variants. Phylogenetic analysis of B1-dID and related sequences suggests a possible scenario of B1-dID evolution in the context of rodent evolution. Received: 30 August 1999 / Accepted: 20 March 2000     

Locating transposable element polymorphisms in bacterial genomes

Although whole-genome sequencing is greatly extending our knowledge of the genetic capacity of those bacterial species, it is only directly informative for the particular strain sequenced. Many bacterial species exhibit more or less genetic polymorphism within their populations and characterising this variety is an extremely important way of elucidating the biology of these species. Often genomic polymorphisms are associated with multicopy elements, particularly transposable elements. We describe a novel method that efficiently characterises the sequences of such polymorphisms. We have optimised heminested inverse PCR (hINVPCR) to assess the diversity of insertional polymorphisms of a transposable element (IS6110) in clinical isolates of Mycobacterium tuberculosis. To increase the yield of information, genomic DNA was digested with different endonucleases (Bsp1286I, HaeII or PvuI), and primers based on both the 5' and 3' ends of IS6110 were used to amplify and determine the genomic sequence upstream (or downstream) of the transposable element. We found that both the choice of restriction enzyme and the use of primers at both ends of the transposable element significantly increased the diversity of the insertion sites identified. Band stabbing was incorporated into the method as an alternative to cloning in order to screen large number of isolates at a sequence level in a rapid and labour-efficient fashion. We describe some of the purposes to which such data can be put.     

Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.     

The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60% are complete, and 7% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5'-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5'-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.     

Identification and characterization of a new member of the SINE Au retroposon family (GmAu1) in the soybean, Glycine max (L.) Merr., genome and its potential application

A plant short interspersed element (SINE) was identified in Glycine max after re-sequencing of the soybean sequence characterized amplified region (SCAR) markers. Detailed analysis revealed that this newly recognized SINE element consisted of a tRNA-related region, a tRNA non-related region, direct flanking repeat sequences, and a short stretch of Ts at the 3′-terminal region. These features are similar to previously characterized SINEs. To investigate the evolution of the SINE retroposon, BLASTN was used to search against genome sequences of other plants. Since it is homologous with the retroposon Au in Aegilops umbellulata (wheat) and its homology in soybean, the SINE is named as GmAu1. Genome analysis of the Glycine max var. Willimas 82 uncovered more than 847 copies of GmAu1 per haploid genome of soybean. Examination of the regions flanking the inserted GmAu1 sequences indicated a preference for introns over exons or other noncoding regions. Considering the flanking insertion sequences, 146 primers were designed in order to detect insertion mutations by a PCR-based method. Seventy-seven primers displayed polymorphism and were used to develop corresponding GmAu1-based SCAR markers. The retroposon GmAu1 and its related SCAR markers identified in this study will prove valuable to future investigations into the genetic mapping, phylogeny, and evolution of the Glycine genus.     

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.     

First open reading frame protein (ORF1p) of the Blattella germanica R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains

The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was islated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established by us for R1 ORF1p of B. germanica.     

Acquisition of an intracisternal A-particle element by a translocated c-myc gene in a murine plasma cell tumor.

The J558 plasma cell tumor contains two forms of a translocated c-myc gene which are distinguished by virtue of their 3' flanking sequences. The J558 alpha 4 and alpha 25 myc genes are broken by a 12;15 translocation which links c-myc exon 1 to C alpha switch sequences. Comparative restriction mapping and DNA sequence analyses demonstrated that an intracisternal A-particle (IAP) element inserted approximately 2 kilobases 3' of an alpha 4-type myc gene to generate the alpha 25 gene copy. The steady-state level of truncated myc RNAs in J558 was comparable to that in another plasma cell tumor line (MPC-11) which harbors a translocated c-myc locus without an IAP element. The significance of these observations for the putative role of IAP elements in the genesis or progression or both of plasma cell tumors is discussed.     

A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.     

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).     

Nucleotide sequence of a repetitive element isolated from Leptospira interrogans serovar hardjo type hardjo-bovis.

A repetitive element from the genome of Leptospira interrogans serovar hardjo type hardjo-bovis ('L. hardjo-bovis') was identified, cloned and sequenced. Similar sequences were shown by hybridization to be encoded by a further eight of 32 other leptospiral serovars tested. An undefined number of repetitive elements were located in the L. hardjo-bovis genome; sequence degeneracy of the elements was observed and no significant open reading frames were identified within the AT-rich (60%) 1467 bp repetitive element. The termini encoded a GC-rich 8 bp repeat motif and two variants showed rearrangements centred on these motifs. The nucleotide sequences of the chromosomal regions flanking the repetitive elements were determined but showed no similarities, with one exception which had a GAAC repeat directly adjacent to both termini. Similar hybridization patterns were shown by Southern transfers of L. hardjo-bovis total genomic digests probed with the repetitive element. Oligonucleotide primer pairs designed from sequences internal to the repetitive element and adjacent chromosomal regions were used in polymerase chain reaction experiments. With one primer pair all L. hardjo-bovis isolates, but no other serovar, gave identical amplified products. Evidence that the repetitive element may have derived from an acquired insertion sequence that is now inactive and chromosomally fixed is discussed.     

Short Retroposons of the B2 Superfamily: Evolution and Application for the Study of Rodent Phylogeny

Short retroposons can be used as natural phylogenetic markers. By means of hybridization and PCR analysis, we demonstrate that B2 retroposon copies are present only in the three rodent families: Muridae, Cricetidae, and Spalacidae. This observation highlights the close phylogenetic relation between these families. Two novel B2-related retroposon families, named DIP and MEN elements, are described. DIP elements are found only in the genomes of jerboas (family Dipodidae) and birch mice (family Zapodidae), demonstrating the close relationship between these rodents. MEN element copies were isolated from the squirrel, Menetes berdmorei, but were not detected in three other species from the family Sciuridae. The MEN element has an unusual dimeric structure: the left and right monomers are B2- and B1-related sequences, respectively. Comparison of the B2, DIP, MEN, and 4.5S₁ RNA elements revealed an 80-bp core sequence located at the beginning of the B2 superfamily retroposons. This observation suggests that these retroposon families descended from a common progenitor. A likely candidate for this direct progenitor could be the ID retroposon. Received: 20 December 1996 / Accepted: 17 June 1997     

First open reading frame protein (ORF1p) of the <Emphasis Type="Italic">Blattella germanica</Emphasis> R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains

The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was isolated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established for R1 ORF1p of B. germanica.     

Identification ofTnr3, aSuppressor-Mutator/Enhancer-like transposable element from rice

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion. A 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1536 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5′-CACTA--- -3′; these repeats are similar to those found in members of theEn/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to theEn/Spm family and is thus namedTnr3 (transposable element inrice no.3). In fact,Tnr3 carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of theEN/Spm family. The subterminal repeat sequences inTnr3 are, however, of two kinds, although they share homology with each other.Tnr3 and its relatives were present in multiple copies in rice. Considering the length ofTnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.