Solo Long Terminal Repeat Research Articles

Retrotransposons and their recognition of pol II promoters: a comprehensive survey of the transposable elements from the complete genome sequence of Schizosaccharomyces pombe.

The complete DNA sequence of the genome of Schizosaccharomyces pombe provides the opportunity to investigate the entire complement of transposable elements (TEs), their association with specific sequences, their chromosomal distribution, and their evolution. Using homology-based sequence identification, we found that the sequenced strain of S. pombe contained only one family of full-length transposons. This family, Tf2, consisted of 13 full-length copies of a long terminal repeat (LTR) retrotransposon. We found that LTR-LTR recombination of previously existing transposons had resulted in extensive populations of solo LTRs. These included 35 solo LTRs of Tf2, as well as 139 solo LTRs from other Tf families. Phylogenetic analysis of solo Tf LTRs reveals that Tf1 and Tf2 were the most recently active elements within the genome. The solo LTRs also served as footprints for previous insertion events by the Tf retrotransposons. Analysis of 186 genomic insertion events revealed a close association with RNA polymerase II promoters. These insertions clustered in the promoter-proximal regions of genes, upstream of protein coding regions by 100 to 400 nucleotides. The association of Tf insertions with pol II promoters was very similar to the preference previously observed for Tf1 integration. We found that the recently active Tf elements were absent from centromeres and pericentromeric regions of the genome containing tandem tRNA gene clusters. In addition, our analysis revealed that chromosome III has twice the density of insertion events compared to the other two chromosomes. Finally we describe a novel repetitive sequence, wtf, which was also preferentially located on chromosome III, and was often located near solo LTRs of Tf elements.

TEOS1, a Novel Transposable Element Family from Oryza Sativa

We have uncovered a novel group of transposable elements TEOS1. (Transposable Element of Oryza 5ativa) by repeat mining of rice genome sequence data We first identified a TEOS1 element. 6,896 bp in length. from a PAC clone of rice chromosome 5 The presence of long terminal repeats (LTRs) and other diagnostic features such as a primer-binding site (PBS) and a polypurine tract (PPT), indicated that TEOS1 is a group of LTR retrotransposon The internal domain of TEOS1 containing two arrays of tandem repeats and a predicated Tf1gene was characteristically distinct from those of other LTR retrotransposons previously described TEOS1 elements identified were flanked by 5-bp direct repeats. some identified as solo LTRs were flanked by 5-7bp direct repeats The PBS sequence vvas homologous to the 3' end region of tRNA (superscript Arg) The deduced amino acid sequence of Tf1 is dissimilar to that of any known mobility related proteins TEOS1 is probable to be trans-activated by other retrotransposons in the rice genome We have estimated that there are about 300 TEOS1 elements per haploid rice genome The presence of repeats in TEOS1 offers the potential to form secondary structure during replication and transcription, and to serve as a hot spot for transposon insertion, augmenting the dynamics of molecular interactions of the genomic sequences.

Isolation and analysis of retroviral integration targets by solo long terminal repeat inverse PCR.

Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.

Copia-Like Retrotransposons in the Rice Genome: Few and Assorted

We performed a comprehensive analysis of Ty1-copia-like retrotransposons in about 10% of the Oryza sativa genome and identieed 100 elements. These were divided into nine classes by their reverse transcriptase similarity and into 39 families according to the sequences of their long terminal repeats (LTRs). The deduced total number of copia-like elements in rice is 1000, comprising 1.7% of the genome. Solo LTRs and truncated elements comprise an additional 2.3% of the genome. This is low compared with other plants, especially grasses, but comparable to Arabidopsis. However, the total number of families in the genome, at least 50, is similar to that in other plants. At least some of these classes are present in the genomes of other grasses, and related elements can be found in dicotyledonous plants. The transcriptional activity of each family was estimated by searching for homologous expressed sequence tags in databases. No correlation was found between the number of homologous expressed sequence tags and family size. In comparison with the copia retrotransposons, 222 gypsy-like and 66 LINE-like retroelements, as well as eve pararetrovirus-like elements, were found, corresponding to about 2220, 660, and 50, respectively, in the genome. A low rate of retrotransposon transcription and a high rate of loss of integrated copies by ectopic, intrachromosomal recombination are proposed as the reasons for the paucity of retrotransposons in the rice genome.

Sequence organization of barley centromeres

By sequencing, fingerprinting and in situ hybridization of a centromere-specific large insert clone (BAC 7), the sequence organization of centromeric DNA of barley could be elucidated. Within 23 kb, three copies of the Ty3/gypsy-like retroelement cereba were present. Two elements of approximately 7 kb, arranged in tandem, include long terminal repeats (LTRs) (approximately 1 kb) similar to the rice centromeric retrotransposon RIRE 7 and to the cereal centromeric sequence family, the primer binding site, the complete polygene flanked by untranslated regions, as well as a polypurine tract 5' of the downstream LTR. The high density (approximately 200 elements/centromere) and completeness of cereba elements and the absence of internally deleted elements and solo LTRs from the BAC 7 insert represent unique features of the barley centromeres as compared to those of other cereals. Obviously, the conserved cereba elements together with barley-specific G+C-rich satellite sequences constitute the major components of centromeric DNA in this species.

Evolutionary history of Cer elements and their impact on the C. elegans genome.

We report the results of sequence analysis and chromosomal distribution of all distinguishable long terminal repeat (LTR) retrotransposons (Cer elements) in the Caenorhabditis elegans genome. Included in this analysis are all readily recognizable full-length and fragmented elements, as well as solo LTRs. Our results indicate that there are 19 families of Cer elements, some of which display significant subfamily structure. Cer elements can be clustered based on their tRNA primer binding sites (PBSs). These clusters are in concordance with our reverse transcriptase- and LTR-based phylogenies. Although we find that most Cer elements are located in the gene depauperate chromosome ends, some elements are located in or near putative genes and may contribute to gene structure and function. The results of RT-PCR analyses are consistent with this prediction.

Insertion of a retroviral solo long terminal repeat in mdr-3 locus disrupts mRNA splicing in mice.

Previously, the abermectin-induced neurotoxicity of subpopulation of CF-1 mice was shown to be caused by the deficiency of mdr-3 P-glycoprotein. Here, we have characterized the molecular nature of the mdr-3 gene mutation in this subpopulation of CF-1 mice. The size of mdr-3 mRNA transcript from ivermectin-sensitive mutant mice was different from that of wild-type mice. Sequence analysis of RT-PCR products isolated from the mutant brain disclosed that the exon 23 of the mdr-3 gene is deleted or altered in the transcripts. The analysis of the genomic locus revealed an insertion of a solo long terminal repeat (LTR) of the ecotropic murine leukemia virus in the reverse orientation in the intron of the mdr-3 gene, causing abnormal splicing and thereby disrupting the mdr-3 gene function. In addition, histopathological analysis of the brains of the ivermectin-treated mutants revealed selective neuronal degeneration in the hippocampal CA3 region. This is the first reported case of a gene mutation induced by a solo retroviral LTR with a phenotypic consequence in the mouse, and may provide new insights into the understanding of the effects of viral solo LTR sequences on mammalian gene expression.

Multiple LTR-retrotransposon families in the asexual yeast Candida albicans.

We have begun a characterization of the long terminal repeat (LTR) retrotransposons in the asexual yeast Candida albicans. A database of assembled C. albicans genomic sequence at Stanford University, which represents 14.9 Mb of the 16-Mb haploid genome, was screened and >350 distinct retrotransposon insertions were identified. The majority of these insertions represent previously unrecognized retrotransposons. The various elements were classified into 34 distinct families, each family being similar, in terms of the range of sequences that it represents, to a typical Ty element family of the related yeast Saccharomyces cerevisiae. These C. albicans retrotransposon families are generally of low copy number and vary widely in coding capacity. For only three families, was a full-length and apparently intact retrotransposon identified. For many families, only solo LTRs and LTR fragments remain. Several families of highly degenerate elements appear to be still capable of transposition, presumably via trans-activation. The overall structure of the retrotransposon population in C. albicans differs considerably from that of S. cerevisiae. In that species, retrotransposon insertions can be assigned to just five families. Most of these families still retain functional examples, and they generally appear at higher copy numbers than the C. albicans families. The possibility that these differences between the two species are attributable to the nonstandard genetic code of C. albicans or the asexual nature of its genome is discussed. A region rich in retrotransposon fragments, that lies adjacent to many of the CARE-2/Rel-2 sub-telomeric repeats, and which appears to have arisen through multiple rounds of duplication and recombination, is also described.

ETn insertion in the mouse Adcy1 gene: transcriptional and phylogenetic analyses.

Early retrotransposons (ETn) are murine transposable elements, bearing some structural similarity to integrated proviruses, and can be insertional mutagens. We have recently identified the causative mutation of the barrelless (Adcy1brl) phenotype as an integration of a 5.7-kb ETn in an intron of the adenylyl cyclase type I (Adcy1) gene. In the present study, Northern blot analysis shows that the ETn insertion results in loss of the normal Adcy1 transcript, a finding consistent with the loss-of-function Adcy1brl mutation, and generation of shorter transcripts. These aberrant transcripts are the products of abnormal RNA splicing and termination owing to the inserted sequence, and transcription initiation within the 3' long terminal repeat (LTR) of the ETn. The DNA sequences of the LTRs were compared in phylogenetic analyses with LTRs from 22 other ETn-related sequences. Three distinct families of ETn sequences can be identified on the basis of their LTRs. The ETn found in Adcy1brl is a member of a family that includes all classified ETn elements known to have recently transposed. Further, of the four known solitary (solo) LTRs, we have identified two that show evidence of recombination between LTRs from different ETn families.

New transposable elements identified as insertions in rice transposon Tnr1.

Tnr1 (235 bp long) is a transposable element in rice. Polymerase chain reactions (PCRs) done with a primer(s) that hybridizes to terminal inverted repeat sequences (TIRs) of Tnr1 detected new Tnr1 members with one or two insertions in rice genomes. Six identified insertion sequences (Tnr4, Tnr5, Tnr11, Tnr12, Tnr13 and RIRE9) did not have extensive homology to known transposable elements, rather they had structural features characteristic of transposable elements. Tnr4 (1767 bp long) had imperfect 64-bp TIRs and appeared to generate duplication of a 9-bp sequence at the target site. However, the TIR sequences were not homologous to those of known transposable elements, indicative that Tnr4 is a new transposable element. Tnr5 (209 bp long) had imperfect 46-bp TIRs and appeared to generate duplication of sequence TTA like that of some elements of the Tourist family. Tnr11 (811 bp long) had 73-bp TIRs with significant homology to those of Tnr1 and Stowaway and appeared to generate duplication of sequence TA, indicative that Tnr11 is a transposable element of the Tnr1/Stowaway family. Tnr12 (2426 bp long) carried perfect 9-bp TIRs, which began with 5'-CACTA- -3' from both ends and appeared to generate duplication of a 3-bp target sequence, indicative that Tnr12 is a transposable element of the En/Spm family. Tnr13 (347 bp long) had 31-bp TIRs and appeared to generate duplication of an 8-bp target sequence. Two sequences, one the transposon-like element Crackle, had partial homology in the Tnr13 ends. All five insertions appear to be defective elements derived from autonomous ones encoding the transposase gene. All had characteristic tandem repeat sequences which may be recognized by transposase. The sixth insertion sequence, named RIRE9 (3852 bp long), which begins with 5'-TG- -3' and ends with 5'- -CA-3', appeared to generate duplication of a 5-bp target sequence. These and other structural features indicate that this insertion is a solo LTR (long terminal repeat) of a retrotransposon. The transposable elements described above could be identified as insertions into Tnr1, which do not deleteriously affect the growth of rice cells.

Retrotransposon BARE-1 and Its Role in Genome Evolution in the Genus Hordeum.

The replicative retrotransposon life cycle offers the potential for explosive increases in copy number and consequent inflation of genome size. The BARE-1 retrotransposon family of barley is conserved, disperse, and transcriptionally active. To assess the role of BARE-1 in genome evolution, we determined the copy number of its integrase, its reverse transcriptase, and its long terminal repeat (LTR) domains throughout the genus Hordeum. On average, BARE-1 contributes 13.7 x 10(3) full-length copies, amounting to 2.9% of the genome. The number increases with genome size. Two LTRs are associated with each internal domain in intact retrotransposons, but surprisingly, BARE-1 LTRs were considerably more prevalent than would be expected from the numbers of intact elements. The excess in LTRs increases as both genome size and BARE-1 genomic fraction decrease. Intrachromosomal homologous recombination between LTRs could explain the excess, removing BARE-1 elements and leaving behind solo LTRs, thereby reducing the complement of functional retrotransposons in the genome and providing at least a partial "return ticket from genomic obesity."

Characterization of a novel class of interspersed LTR elements in primate genomes: structure, genomic distribution, and evolution.

Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human, chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13 family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus.

Retroviral sequences located within an intron of the dilute gene alter dilute expression in a tissue-specific manner.

The murine dilute coat color locus encodes an unconventional myosin heavy chain that is thought to be required for the elaboration or maintenance of dendrites or organelle transport in melanocytes and neurons. In previous studies we showed that the d mutation carried by many inbred strains of mice (now referred to as dilute viral, dv), is caused by the integration of an ecotropic murine leukemia virus (Emv-3) into the dilute gene and that phenotypic revertants of dv (termed d+) result from viral excision; a solo viral long terminal repeat (LTR) is all that remains in revertant DNA. In the studies described here we show that Emv-3 sequences are located within an intron of the dilute gene in a region of the C-terminal tail that is differentially spliced. We also show that these Emv-3 sequences result in the production of shortened and abnormally spliced dilute transcripts and that the level of this effect varies among tissues. This tissue-specific effect on dilute expression likely accounts for the absence of neurological abnormalities observed in dv mice. Surprisingly, we also found that the solo viral LTR present in revertant d+ DNA produces a tissue-specific effect on dilute expression, although this effect is less dramatic than with the full-length provirus and produces no obvious mutant phenotype. These findings have important implications for understanding the effects of viral sequences on mammalian gene expression.

A novel family of retrotransposon-like elements in Xenopus laevis with a transcript inducible by two growth factors.

A cDNA clone named 1A11 was isolated in a screen for genes that are activated by both mesoderm inducing factors FGF and activin in animal explants of Xenopus laevis embryos. In undisturbed embryos, 1A11 is expressed during the gastrula stage in the entire marginal zone where mesoderm originates, and later in the somites, the tailbud, and at much lower levels in lateral mesoderm. The 1A11 sequence of 4.5 Kb has a 220 bp repeat at its ends, indicative of a retrotransposon-like structure. A long open reading frame encodes a predicted protein with only short homologies to the gag and protease regions of retroviruses and retrotransposons. Multiple copies of 1A11-related sequences were found in the Xenopus genome, constituting solo LTRs (long terminal repeats) of 1267 bp, and unique region copies (i.e., sequences internal to the repeats in the cDNA). Inverted repeats of 5 bp and apparent target site duplications of 5 bp surround the sequenced solo LTR. Thus, 1A11 is a new retrotransposon-like element in Xenopus laevis.

Endogenous retroviral long terminal repeats within the HLA-DQ locus.

Two endogenous retroviral long terminal repeats (LTRs) were found in the human major histocompatibility complex locus HLA-DQ. The solo LTRs, unlinked to retrovirus structural genes, are located approximately 5 kilobases apart from each other and in the same transcriptional orientation, which is opposite to that for the HLA-DQB1 gene. These elements exhibit greater than 90% homology to the LTRs of the human endogenous retrovirus HERV-K10. The conservation of putative regulatory elements found within the LTRs and their position relative to the HLA-DQB1 gene suggest that these elements may confer distinct regulatory properties on genes in the HLA-DQ region. Polymorphic variation between different HLA haplotypes for the presence of the LTRs at this location and of the molecular architecture within this subregion is supported by polymerase chain reaction and Southern blot analysis. Comparisons of chromosomes with and without the LTRs in this region will provide a unique opportunity in the human genome to analyze transposition or integration of retroviral sequences.

Autonomous expression of RTVL-H endogenous retroviruslike elements in human cells

Northern (RNA) blot analysis of RNA from various human cell lines and tissues has demonstrated that elements belonging to the RTVL-H family of human endogenous retroviruslike sequences are expressed in several cell types. The highest levels of RTVL-H-related RNAs were observed in teratocarcinoma cell line NTera2D1, HeLa cells, two bladder carcinoma cell lines, and normal amniotic tissue. Expression was also observed in normal chorion and in some other cell lines. The RTVL-H transcription pattern varied among the different cell types, but several expressed a unit-length 5.6-kilobase transcript. Characterization of cDNA clones corresponding to transcripts present in NTera2D1 cells indicates that the complex transcription pattern observed in these cells is generated by the following: (i) transcription of both full-length and deleted genomic elements, which is initiated within the 5' long terminal repeat (LTR) and, in all but one case, polyadenylated in the 3' LTR; (ii) the splicing of both unit-length transcripts and transcripts from a deleted element; (iii) transcription involving solo LTR sequences; and (iv) transcription which, in one case, reads through the 3' LTR into flanking cellular sequences. Sequence data obtained from 25 cDNA clones revealed that at least 13 RTVL-H elements are expressed in NTera2D1 cells. The positions of several termination codons within the pol region are the same among nine different elements, indicating that an ancestral RTVL-H element bearing these mutations dispersed within the genome. We also found that RTVL-H expression varied among samples of amnion and chorion tissue isolated from different individuals. These findings demonstrate that regulated autonomous expression of RTVL-H sequences occurs in human cells.

Mobile genetic elements in Drosophila melanogaster (recent experiments)

Recent data obtained in the authors' laboratories concerning the behaviour of mobile genetic elements of Drosophila melanogaster are reviewed. It was found that the mobile element jockey represents the typical LINE element. It is efficiently transcribed in D. melanogaster cells in flies and in culture. Transcription is initiated from the +1 nucleotide of jockey and depends on an internal promoter. This is the first case of an internal promoter being used by RNA polymerase II. Several events which take place during the transposition bursts in ctMR2 family of strains were described. Among them are the removal of mobile dispersed genetics (mdg) elements (with solo long terminal repeat (LTR) remaining at the site of excision), complete removal of an mdg element, and reinsertion of the same mdg to the same place either in the presence or in absence of solo LTR sequence. Finally, the formation of deletions was observed. A 462-bp deletion destroying the white locus can be further repaired (w+ reversion). Thus, transposition bursts include many different genetic events. A novel system of prolonged genome destabilization was described. It depends on mobilization of a new mobile element called Stalker. After certain crosses Stalker actively moves for dozens of generations giving rise to large numbers of insertion mutations. Several novel genes were detected using mobilized Stalker. They include a modifier of mdg4 and six enhancers of yellow mutations.

Reversion of a gypsy-induced mutation at the yellow (y) locus of Drosophila melanogaster is associated with the insertion of a newly defined transposable element.

To understand the molecular basis of the phenotype of gypsy-induced mutations, we have analyzed the structure of phenotypic revertants of the y2 allele, which is caused by the insertion of the gypsy element into the 5' region of the yellow (y) locus. Seven spontaneous revertants examined fall into two different classes. Three of these revertants arose by homologous recombination between the two gypsy long terminal repeats (LTRs), leaving behind a solo LTR. Four additional revertants contain an intact 3' LTR and half of the 5' LTR, but the central portion of gypsy has been replaced by a different 6.5-kilobase transposable element that contains a poly(A) tail. These results suggest that the mutagenic effect of the gypsy element is not due to its insertion into sequences necessary for transcription or to the distancing between the yellow promoter and remote regulatory sequences but is a consequence of idiosyncratic properties of the element itself.

Molecular analysis of elements inserted into mouse gamma-actin processed pseudogenes.

DNA from ten mouse genomic clones, each containing distinct gamma-actin processed pseudogenes, was subjected to electron microscopic heteroduplex analysis, and in three cases (lambda mA36, lambda mA118 and lambda mA119) the heteroduplex formed with the DNA of a reference clone was found to be interrupted by a single-stranded loop. The genomic regions corresponding to these loops were subjected to structural analysis and they were found to represent different elements (IEs) inserted into the pseudogenes in a manner that gave rise to short target-site direct repeats. IE 36 (500 base-pairs in length) was found to be an intercisternal A-particle solo long terminal repeat (LTR), a 46 nucleotide region of which had undergone five-fold tandem amplification and subsequent mutation. IE 119 (501 base-pairs in length) was also a solo LTR, bearing similarity to the recently-described GLN-3 class of murine retroviral-like elements. IE 118 (865 base-pairs in length) is repeated 1000-2000 times in the mouse genome. It is not related to any known class of mobile elements, but does possess some sequence motifs that suggest it may be an LTR of a hitherto unrecognized family of retroviral-like elements. It also possesses a 26 out of 27 nucleotide identity to a region of the flanking pseudogene, suggesting that it may have suffered gene conversion.

Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites.

A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22-base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription.