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PubMed1997

The Ac-st2 element of maize exhibits a positive dosage effect and epigenetic regulation.

A novel derivative of the maize transposable element Ac, termed Ac-st2, that displays a positive dosage effect in maize has been identified. Although identical in sequence to other Ac elements, increasing the copy number of the element in the endosperm results in earlier and more frequent Ds excision. Ac-st2 autonomously transposes and catalyzes somatic excision of Ds elements. Germinal transpositions of either Ac-st2 or Ds, however, were not observed. The Ac-st2 phenotype includes a reduction in Ac transcript accumulation that is associated with increased methylation at specific sites in the promoter region of the major transcriptional start site within Ac (ORFa). This element differs from metastable (cycling) Ac derivatives in that Ac-st2 conditions a uniform transposition pattern throughout endosperm and plant development. Ac-st2 undergoes frequent increases in activity after its association with an active Ac element. This change in activity correlates with reduced levels of methylation in the ORFa promoter region. Using a competitive PCR assay, Ac transcript accumulation was followed through endosperm development. From these data, a model is proposed to explain the patterns of variegation associated with both "wild type" active Ac and Ac-st2 elements.

DNA Methylation↗
PubMed1999

Identification of a full-size hobo element and deletion-derivatives in Korean populations of Drosophila melanogaster.

We have isolated and characterized several members of the hobo transposable element family from Korean populations of Drosophila melanogaster. All of the Korean lines tested appeared to have 3.0 kb hobo elements and a high copy number of smaller derivatives of the element. To determine whether a 3.0 kb hobo element of these populations is consistent with the role of an autonomous hobo element, we cloned and sequenced this hobo element. Based on the result of the entire DNA sequence, a cloned 3.0 kb element called HKN96, it was found to be the same as a fully-functional 2959 bp HFL1-type sequence. Each small element appeared to have arisen from the HFL1 element by a different internal deletion. A specific 1.7 kb Kh hobo element, which is the most abundant in the Korean lines tested, seems to have originated from the HFL1 hobo element by an internal deletion of 1253 bp by the removal of nucleotides between positions 939 and 2191. The sequences of the Th1 and Th2 elements appeared to be identical to that of the HFL1 with the exception of internal deletions of 1442 bp and 1455 bp removing nucleotides 940-2381 and 923-2377, respectively. Based on the number of TPE repeats, all of the members of the hobo element family in Korean lines tested have three perfect S repeats. The widespread presence of identical copies of the Kh deletion derivative suggests that it might have a role in the regulation of hobo-induced hybrid dysgenesis.

Animals↗
PubMed1988

A yeast sigma composite element, TY3, has properties of a retrotransposon.

Sigma is a 340- or 341-base pair repetitive element which is located almost exclusively within 19 base pairs of the 5' ends of various tRNA genes in the Saccharomyces cerevisiae genome. Although most sigma elements characterized to date are isolated insertions, a few of the elements occur relatively closely spaced. One of these pairs is a direct repeat of the sigma element separated by an internal domain 4.7 kilobase pairs in length. Not only does this structure resemble a composite transposable element, but regions within the sigma elements and intervening domain are homologous to conserved regions in retroviruses and retrotransposons of yeast and other organisms. Two features suggest that the sigma elements and intervening DNA transposed in a concerted event: only one of the two sigma elements is associated with a tRNA gene, and only the outside ends of the two elements are flanked by the 5-base pair direct repeats that usually flank individual sigma insertions. Examination of genomic DNA from five laboratory strains indicates that the 4.7 kilobase pair internal domain is present in one to four copies per haploid genome and that the genomic location of this domain differs from strain to strain. In addition, Northern blot analysis showed the presence of a 5.2 kilobase poly(A) transcript which hybridizes to both sigma and internal domain-specific probes. The existence of this composite element may suggest new ways to consider the mechanisms by which retrotransposons select their targets.

Base Sequence↗
PubMed1981

Site-specific properties of Tn7 transposition into the E. coli chromosome.

A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and spectinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extreme specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA. To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristic of the E. coli chromosome.

Chromosome Mapping↗
PubMed1989

Characterization of 5' truncated transposed copies of the I factor in Drosophila melanogaster.

I factors in Drosophila melanogaster are transposable elements structurally related to Mammalian LINEs. Their transposition is activated at high frequencies during I-R hybrid dysgenesis and is associated with the production of mutations of various sorts. Very few of these mutations have been studied at the molecular level; those reported so far result either from chromosomal rearrangements or from insertions of complete I factors. We have analysed three I-R induced yellow mutations and have found that one of them is due to the insertion of an I element very similar to the complete I factor, whereas the other two are due to insertions of I elements that are truncated at their 5' ends; one of them exhibits an unusual 3' end. We discuss possible mechanisms of production of such modified I elements.

Animals↗
PubMed2006

Mapped Ds/T-DNA launch pads for functional genomics in barley.

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non-autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single-copy lines after crossing with AcTPase-expressing plants or by Agrobacterium-mediated transformation. Genomic DNA flanking Ds and T-DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T-DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non-redundant, gene-containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T-DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.

Base Composition↗
PubMed1986

Enhancer-like stimulation of yeast tRNA gene expression by a defined region of the Ty element micro-injected into Xenopus oocytes.

In the yeast Saccharomyces cerevisiae, the majority of the tRNA genes are found associated with transposable elements one of which is the Ty element. We noticed that the transcriptional activity of several of these genes was rather different depending on the type of the accompanying element(s) [Nelböck et al. (1985) Biol. Chem. Hoppe-Seyler 366, 1041-1051]. In order to study the influence of a Ty element on tRNA gene expression, we used the method of micro-injection into Xenopus oocytes taking advantage of a Ty+/Ty- allelic pair of a tRNALys1 gene recently isolated in our laboratory. A direct comparison showed that the expression of the plus allele was c. sixfold higher than that of the minus allele. In order to determine sequences of the Ty responsible for this effect, we constructed a number of variants in which distinct segments of the Ty were deleted or rearranged. The activating ability was localized to a 590-bp segment of the Ty containing the 'promoter delta', independent of its orientation or location towards the tRNA gene thus resembling the effects described for transcriptional enhancers. This is the first time that a long-range effect has been observed on the expression of a gene transcribed by RNA polymerase III.

Animals↗
PubMed1989

Organization of complex transposon Tn2610 carrying two copies of tnpA and tnpR.

Transposon Tn2610 has two elements of 3.5 kilobase pairs as inverted repeats, one set at each end. This unique terminal element contained the transposition genes tnpA and tnpR. Only the tnpA gene in the left element was functional for transposition, whereas both tnpR genes were active. Possible evolutionary relationships among class II transposable elements are proposed on the basis of the genetic and structural organization of Tn2610.

Anti-Bacterial Agents↗
PubMed2004

Evolution of P elements in natural populations of Drosophila willistoni and D. sturtevanti.

To determine how population structure of the host species affects the spread of transposable elements and to assess the strength of selection acting on different structural regions, we sequenced P elements from strains of Drosophila willistoni and Drosophila sturtevanti sampled from across the distributions of these species. Elements from D. sturtevanti exhibited considerable sequence variation, and similarity among them was correlated to geographic distance between collection sites. By contrast, all D. willistoni elements sampled were essentially identical (pi < 0.2%) and exhibited patterns typical of a recent population expansion. While the canonical P elements sampled from D. sturtevanti appear to be long-time residents in that species, a rapid expansion of a very young canonical P-element lineage is suggested in D. willistoni, overcoming barriers such as large geographical distances and moderate levels of population subdivision. Between-species comparisons reveal selective constraints on P-element evolution, as indicated by significantly different substitution rates in noncoding, silent, and replacement sites. Most remarkably, in addition to replacement sites, selection pressure appears to be strong in the first and third introns and in the 3' and 5' flanking regions.

Animals↗
PubMed1982

Molecular cloning of sequences from a Drosophila RNA polymerase II locus by P element transposon tagging.

We have identified a lethal mutation in the D. melanogaster RNA polymerase II locus, RpIIC4, caused by insertion of a transposable element associated with the phenomenon of hybrid dysgenesis (P element). Using previously cloned P element sequences as a hybridization probe we have isolated a hybrid lambda phage clone carrying a 10 kb genomic DNA fragment containing a 1.3 kb P element insert and flanking sequences from the RpII locus. The non-P sequences in this clone (lambda DmRpII-1) hybridize to polytene chromosome band region 10C, the cytogenetic location of RpIIC4, and revertants which lose the lethal RNA polymerase II mutation also lose P element sequences from the locus. We have generated several additional P element insertions into the locus and shown that they can occur at two or more different sites. These experiments illustrate that mutagenesis by P element insertion and use of cloned P DNA to retrieve the DNA sequences into which insertion has occurred may be a general method for cloning genetically defined loci in Drosophila.

Alleles↗
PubMed2004

Epigenetic control of CACTA transposon mobility in Arabidopsis thaliana.

Epigenetic mutation, heritable developmental variation not based on a change in nucleotide sequence, is widely reported in plants. However, the developmental and evolutionary significance of such mutations remains enigmatic. On the basis of our studies of the endogenous Arabidopsis transposon CACTA, we propose that the inheritance of epigenetic gene silencing over generations can function as a transgenerational genome defense mechanism against deleterious movement of transposons. We previously reported that silent CACTA1 is mobilized by the DNA hypomethylation mutation ddm1 (decrease in DNA methylation). In this study, we report that CACTA activated by the ddm1 mutation remains mobile in the presence of the wild-type DDM1 gene, suggesting that de novo silencing is not efficient for the defense of the genome against CACTA movement. The defense depends on maintenance of transposon silencing over generations. In addition, we show that the activated CACTA1 element transposes throughout the genome in DDM1 plants, as reported previously for ddm1 backgrounds. Furthermore, the CACTA1 element integrated into both the ddm1-derived and the DDM1-derived chromosomal regions in the DDM1 wild-type plants, demonstrating that this class of transposons does not exhibit targeted integration into heterochromatin, despite its accumulation in the pericentromeric regions in natural populations. The possible contribution of natural selection as a mechanism for the accumulation of transposons and evolution of heterochromatin is discussed.

Arabidopsis↗
PubMed1986

Isolation and characterization of a Ty element inserted into the ribosomal DNA of the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has about 30 to 50 copies of a transposable element Ty. Most of these elements are located at the 5' ends of protein coding sequences and are flanked by a 5 bp duplication. We report below an insertion of a Ty element into one of the repeated ribosomal RNA (rRNA) genes of yeast. The element is located between the 3' ends of the divergentally transcribed 37S and 5S rRNA's and is not flanked by a 5 bp duplication. In addition, one end of the Ty insertion is contiguous with a 306 bp deletion of the sequences of the rRNA gene. We find that this insertion, unlike most Ty insertions, is mitotically unstable.

Base Sequence↗
PubMed2025

The Demographic History of Populations and Genomic Imprinting have Shaped the Transposon Patterns in Arabidopsis lyrata.

Purifying selection is expected to prevent the accumulation of transposable elements (TEs) within their host, especially when located in and around genes and if affected by epigenetic silencing. However, positive selection may favor the spread of TEs, causing genomic imprinting under parental conflict, as genomic imprinting allows parent-specific influence over resource accumulation to the progeny. Concomitantly, the number and frequency of TE insertions in natural populations are conditioned by demographic events. In this study, we aimed to test how demography and selective forces interact to affect the accumulation of TEs around genes, depending on their epigenetic silencing, with a particular focus on imprinted genes. To this aim, we compared the frequency and distribution of TEs in Arabidopsis lyrata from Europe and North America. Generally, we found that TE insertions showed a lower frequency when they were inserted in or near genes, especially TEs targeted by epigenetic silencing, suggesting purifying selection at work. We also found that many TEs were lost or got fixed in North American populations during the colonization and the postglacial range expansion from refugia of the species in North America, as well as during the transition to selfing, suggesting a potential "TE load." Finally, we found that silenced TEs increased in frequency and even tended to reach fixation when they were linked to imprinted genes. We conclude that in A. lyrata, genomic imprinting has spread in natural populations through demographic events and positive selection acting on silenced TEs, potentially under a parental conflict scenario.

DNA Transposable Elements↗
PubMed2003

Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements.

We describe a technique to tag Drosophila proteins with GFP at their native genomic loci. This technique uses a new, small P transposable element (the Wee-P) that is composed primarily of the green fluorescent protein (GFP) sequence flanked by consensus splice acceptor and splice donor sequences. We demonstrate that insertion of the Wee-P can generate GFP fusions with native proteins. We further demonstrate that GFP-tagged proteins have correct subcellular localization and can be expressed at near-normal levels. We have used the Wee-P to tag genes with a wide variety of functions, including transmembrane proteins. A genetic analysis of 12 representative fusion lines demonstrates that loss-of-function phenotypes are not caused by the Wee-P insertion. This technology allows the generation of GFP-tagged reagents on a genome-wide scale with diverse potential applications.

Animals↗
PubMed1990

Regulation of transposition in bacteria.

Bacterial transposons are subject to a variety of regulatory processes that affect the quantity, quality, and timing of transposition events. Many of these processes seem specifically designed to provide features that favor the evolutionary success of the element. The most important conclusion reached from the identification and characterization of these regulatory mechanisms is that transposable elements are not mechanistic accidents of recent origin, but instead are highly evolved entities that have adapted to their ecological niche with a degree of sophistication comparable to that exhibited by plasmids and bacterial viruses.

Bacteria↗
PubMed2003

Characterization of hobo element copy number and integrity in Brasilian populations of Drosophila melanogaster.

We have determined the copy number and the presence of full-size hobo transposable elements in eight Brasilian strains of Drosophila melanogaster. Genomic DNA was digested with AvaII and XhoI restriction enzymes, respectively, and probed with a 963 bp sequence of the hobo element. Variable numbers of full-sized and defective elements were detected in all strains. The range of the copy number was 22.13 +/- 4.52. Blots showed the presence of a 2.6 kb fragment, corresponding to the complete element, in all strains exception of one and the 1.0 kb sequence, correponding to the Th1 and Th2 repressor elements. There was neither association among copy numbers of hobo elements and latitude nor the mean annual temperatures in the original geographical region of each strain.

Animals↗
PubMed1999

A tourist element in the 5'-flanking region of the catalase gene CatA reveals evolutionary relationships among Oryza species with various genome types.

Tourist-OsaCatA, a transposable element, was found in the 5'-flanking region of the rice gene CatA. The characteristics of this element are similar to those of the other Tourist elements so far found in Oryza sativa. PCR and sequence analyses of 37 accessions of 18 species revealed that all the Oryza species examined, except for one accession, have either a full-length or a partial Tourist element at this locus. Unlike the Tourist elements previously reported, this Tourist element is found in all four Oryza species complexes in the Oryzeae tribe. All AA genome Oryza species, except O. longistaminata, contain the full-length Tourist element. O. longistaminata and the species of the O. officinalis, O. meyeriana and O. ridleyi complexes contain the partial element. A phylogenetic tree of Oryza species based on the nucleotide sequences of these Tourist elements was constructed. The O. longistaminata accessions were placed near the neighboring cluster of the officinalis complex. We propose that the ancestor of O. longistaminata and that of other species with the AA genome diverged, and the ancestor(s) of the O. officinalis, O. ridleyi and O. meyeriana complexes then diverged from the ancestor of O. longistaminata in the course of the evolution of the Oryza species. The Tourist elements associated with CatA and its orthologs thus provide useful tools for examining evolutionary relationships among Oryza species.

Base Sequence↗
PubMed1999

Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio.

The Tol2 element is found in the genome of the medaka fish, Oryzias latipes, and contains DNA sequences similar to those of transposons of the hAT family. Previously, we have developed a transient embryonic excision assay in zebrafish, in which zebrafish embryos were injected with a plasmid DNA harboring the Tol2 element, and have shown that the Tol2 element is excisable from the injected plasmid DNA (Kawakami, K., Koga, A., Hori, H., Shima, A., 1998. Excision of the Tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17-22). Although the Tol2 element is thought to be autonomous, an active transposase encoded by the Tol2 element has not been identified. Here we report the identification and analysis of mRNA transcribed from the Tol2 element in zebrafish embryos. The Tol2 transcript has the capacity to encode a protein of 649 amino acids, whose amino acid sequence is similar to those of transposases of the hAT family. To determine whether the transcript encodes an active enzyme, we developed a novel transient embryonic excision assay in which zebrafish fertilized eggs were co-injected with RNA transcribed in vitro using the Tol2 cDNA as a template and a plasmid DNA harboring a nonautonomous Tol2 element, which has a deletion in the transposase coding region. The nonautonomous Tol2 element could be efficiently excised in the zebrafish only when co-injected with the Tol2 RNA. This result indicates that the Tol2 transcript encodes an active enzyme, a probable transposase, that can catalyze the excision reaction in trans. Further, by the co-injection analysis, we found that the Tol2 sequence lacking the first intron sequence of the transposase gene could not be excised, suggesting that it may contain essential cis-elements.

Amino Acid Sequence↗