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PubMed2006

Excision and transposition of piggyBac transposable element in tobacco budworm embryos.

The TTAA-specific lepidopteran transposon piggyBac has already proved useful as a gene-transfer vector for efficient transformation of a wide variety of insects. Transposable element excision and transposition assays are useful indicators of an element's ability to be mobilized in vivo and, thus, potentially serve as a transforming vector. Here, we report that this transposon is capable of excision and transposition in tobacco budworm embryos with relatively low frequency.

Animals↗
PubMed2005

Fixation of transposable elements in the Drosophila melanogaster genome.

We have investigated at the molecular level four cases in which D. melanogaster middle repetitive DNA probes consistently hybridized to a particular band on chromosomes sampled from a D. melanogaster natural population. Two corresponded to true fixations of a roo and a Stalker element, and the others were artefacts of the in situ hybridization technique caused by the presence of genomic DNA flanking the transposable elements (TEs) in the probes. The two fixed elements are located in the beta-heterochromatin (20A and 80B, respectively) and are embedded in large clusters of other elements, many of which may also be fixed. We also found evidence that this accumulation is an ongoing process. These results support the hypothesis that TEs accumulate in the non-recombining part of the genome. Their implications for the effects of TEs on determining the chromatin structure of the host genomes are discussed in the light of recent evidence for the role of TE-derived small interfering-RNAs as cis -acting determinants of heterochromatin formation.

Animals↗
PubMed1997

The maize transposable element Ac induces recombination between the donor site and an homologous ectopic sequence.

The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby beta-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5' end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5' end of the GUS open reading frame and had a deletion at the 3' end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.

Base Sequence↗
PubMedDate not supplied

Accumulation of transposable elements in the genome of Drosophila melanogaster is associated with a decrease in fitness.

Replicates of the two isogenic laboratory strains of Drosophila melanogaster, 2b and Harwich, contain different average transposable element (TE) copy numbers in the same genetic background. These lines were used to analyze the correlation between TE copy number and fitness. Assuming a weak deleterious effect of each TE insertion, a decrease in fitness is expected with an increase in genomic TE copy number. Higher rates of ectopic exchanges and, consequently, chromosomal rearrangements resulting in early embryonic death are also predicted from an increase in TE copy number. Therefore egg hatchability is expected to decrease as the genomic TE copy number increases. In 2b, where replicate lines have diverged up by 90 TE copies per haploid genome, a negative correlation between the number of TE insertions and both fitness and egg hatchability were found. Neither correlation was significant for the Harwich replicates, which have only diverged by 30 TE copies. The average deleterious effect of a TE insertion on fitness and its components was estimated as 0.004. Both homozygous and heterozygous TE insertions were shown to have deleterious effects on fitness and its components.

Analysis of Variance↗
PubMed1988

Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Genetic and molecular evidence presented in this paper demonstrate that the Mos factor for inherited mosaicism is a special copy of the transposable element mariner. Mosaicism observed in the presence of the Mos (Mosaic) factor results from a high frequency of excision of the mariner element from an insertion site near the white-eye gene in Drosophila mauritiana. The Mos factor promotes the excision of mariner elements from genomic insertion sites other than the site in wpch, and it also promotes its own loss from the genome. Putative transpositions of Mos to new genomic sites have also been observed. A copy of mariner present at a particular site in a Mos strain has been shown to be missing in derived strains in which the Mos factor has been lost, and in strains with putative transpositions. We propose that this copy of mariner is identical to the Mos factor.

Animals↗
PubMed1995

Transposable elements map in a conserved pattern of distribution extending from beta-heterochromatin to centromeres in Drosophila melanogaster.

In situ hybridisation to mitotic chromosomes shows that sequences homologous to different Drosophila melanogaster transposable elements are widely distributed not only in beta but also in alpha-heterochromatin. Clusters of these sequences are detected in most proximal positions. They colocalise with known satellite sequences in several regions, but are also located in places where no known sequence has been mapped so far. The pattern of hybridisation is dinstinctive and specific for each element, and presents constant features in six different D. melanogaster strains studied. The entirely heterochromatic Y chromosome contains large amounts of these sequences. Additionally, some of these sequences appear to be present in substantial quantities in the smallest minichromosome of Drosophila, Dp(1;f)1187.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant.

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.

Base Sequence↗
PubMed1993

The transposable element En/Spm-encoded TNPA protein contains a DNA binding and a dimerization domain.

The En/Spm-encoded TNPA protein binds to 12-bp DNA sequence motifs that are present in the subtermini of the transposable element. DNA binding of TNPA to monomeric and dimeric forms of the binding motif was analyzed by gel retardation and cross-linking studies. A DNA binding domain at the N-terminal and a dimerization domain at the C-terminal portion of TNPA were localized using deletion derivatives of TNPA. These domains are novel since no apparent homology has been found in the data bases. The stoichiometry of the TNPA-DNA complexes was analyzed. A special complex is formed with a tail-to-tail dimeric DNA binding motif, most probably involving two DNA-bound TNPA molecules that interact via their dimerization domains. In redox reactions the requirement for one or two disulfide bonds for DNA binding of TNPA was shown. The implications of these findings for the excision mechanism of En/Spm are discussed.

Animals↗
PubMed2005

Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations.

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed.

DNA Transposable Elements↗
PubMed1987

Influence of transposable elements on the structure and function of the A1 gene of Zea mays.

The structure of the A1 gene of Zea mays was determined by sequencing cDNA and genomic clones. The gene is composed of four exons and three short introns. The 40.1-kd A1 protein is an NADPH-dependent reductase. Germinal derivatives of the mutable a1-m1 allele with either recessive or wild-type phenotype have been isolated. Sequence analysis of these revertant alleles indicates that frame-shift mutations abolish A1 gene function, whereas one additional amino acid within the protein sequence still allows wild-type gene expression. The presence of a second, promoter-like structure, upstream of the functional A1 gene promoter is discussed with respect to its possible involvement in differential expression of the A1 gene. The structure of the a1-m2 8004, 3456 and 4412 alleles, featuring distinguishable phenotypes in the presence of Spm(En), was also determined. In all alleles the 1080-bp-long inhibitor (I) element is located 15 bp upstream of the CAAT box of the A1 gene promoter. The unusual response of a1-m2 alleles to trans-active signals of the Spm(En) element is discussed with respect to the position of the I inserts. Also presented are data on the structure and insertion sites of transposable elements determined by cloning and sequencing of the mutable a1 alleles a1-mpapu, a1-mr 102 and a1-ml.

Alcohol Oxidoreductases↗
PubMed1987

Transcription of transposable element Activator (Ac) of Zea mays L.

Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize lines. A 3.5-kb mRNA with an abundance of 1-3 x 10 of the poly(A) RNA, however, is found exclusively in those lines that carry an active Ac. Plants with two Ac elements contain slightly more 3.5-kb Ac transcript than those with only one Ac. Overlapping cDNA clones spanning most of the message have been isolated and sequenced. The 5'-end of the transcript was determined by Northern hybridization and S1 mapping. It starts at several sites over a distance of nearly 100 bases, contains an AUG-free leader 600-700 nucleotides long, has a long open reading frame encoding 807 amino acids and an untranslated 3'-sequence of 239 nucleotides. Four introns with a combined length of 654 bases are removed from the primary transcript. Radiosequencing of in vitro translation products shows that translation of the long open reading frame begins at the first AUG, even though it is located in an unfavourable sequence context. The transcript is found in all organs investigated, provided an active Ac is present in the stock.

Journal Article↗
PubMed2002

Gene transfer and cloning of flanking chromosomal regions using the medaka fish Tol2 transposable element.

For the ultimate purpose of developing genetic tools using the medaka fish Tol2 transposable element, we examined whether it can transfer a marker gene into the fish genome and also be applied for cloning of chromosomal regions adjacent to insertion points. An internal region of Tol2 was removed and replaced with the green fluorescent protein (GFP) gene and a bacterial plasmid replication origin. This modified Tol2 clone was microinjected into fertilized eggs together with messenger RNA for the Tol2 transposase. The GFP gene was found to be integrated into chromosomes and transmitted to subsequent generations. Restriction enzyme digestion of genomic DNA of a transformant fish, followed by ligation and introduction into bacteria, produced a plasmid containing the entire element and flanking chromosomal regions. Sequencing analysis of this clone demonstrated transposition of the element in the germline of the first generation. Thus, the basic requirements for a gene transfer vector and gene tagging system were fulfilled.

Journal Article↗
PubMed1983

Enhanced susceptibility of a transposable-element-bearing strain of Drosophila melanogaster to somatic eye-color mutations by ethyl nitrosourea, methyl nitrosourea, and X-rays.

A strain of Drosophila with the genes z and w+ plus a transposable element (TE) is about 3 times more sensitive than a strain without TE toward somatic eye-color mutations after larval exposure to ethyl nitrosourea, methyl nitrosourea and X-rays. The assay system with TE is simple, reliable, and sensitive for detecting somatic mutations induced in vivo by mutagens.

Animals↗
PubMed2004

[Transposable element mariner].

Mariner and mariner-like elements (MLEs) have been found in a wide range of organisms including human since its discovery in Drosophila mauritiana. MLEs belong to the mariner/Tc1 superfamily, one of the most diverse and widespread Class II transposable elements. MLEs have a conserved "D,D(34)D" motif in their transposases and they transpose by cut-and-paste mechanisms. Their extraordinarily wide host range and horizontal transmission in distantly related species indicate that they do not need additional host-specific factors for transposition. The evidence that MLEs could transform a wide variety of organisms especially the vertebrates supported the host-independent mechanism and suggested the availability as a kind of potential transforming vector.

Animals↗
PubMed1982

Genetic transformation of Drosophila with transposable element vectors.

Exogenous DNA sequences were introduced into the Drosophila germ line. A rosy transposon (ry1), constructed by inserting a chromosomal DNA fragment containing the wild-type rosy gene into a P transposable element, transformed germ line cells in 20 to 50 percent of the injected rosy mutant embryos. Transformants contained one or two copies of chromosomally integrated, intact ry1 that were stably inherited in subsequent generations. These transformed flies had wild-type eye color indicating that the visible genetic defect in the host strain could be fully and permanently corrected by the transferred gene. To demonstrate the generality of this approach, a DNA segment that does not confer a recognizable phenotype on recipients was also transferred into germ line chromosomes.

Animals↗
PubMed2001

Loss of transposase-DNA interaction may underlie the divergence of mariner family transposable elements and the ability of more than one mariner to occupy the same genome.

Mariners are a large family of eukaryotic DNA-mediated transposable elements that move via a cut-and-paste mechanism. Several features of the evolutionary history of mariners are unusual. First, they appear to undergo horizontal transfer commonly between species on an evolutionary timescale. They can do this because they are able to transpose using only their own self-encoded transposase and not host-specific factors. One consequence of this phenomenon is that more than one kind of mariner can be present in the same genome. We hypothesized that two mariners occupying the same genome would not interact. We tested the limits of mariner interactions using an in vitro transposition system, purified mariner transposases, and DNAse I footprinting. Only mariner elements that were very closely related to each other (ca. 84% identity) cross-mobilized, and then inefficiently. Because of the dramatic suppression of transposition between closely related elements, we propose that to isolate elements functionally, only minor changes might be necessary between elements, in both inverted terminal repeat and amino acid sequence. We further propose a mechanism to explain mariner diversification based on this phenomenon.

Animals↗
PubMed1989

Eukaryotic transposable elements and genome evolution.

The changes in DNA sequence that have taken place during the evolution of eukaryotic genomes cannot be accounted for simply by base substitutions; some more complex mutations must have taken place as well. Transposable elements can affect gene structure and expression in several ways that suggest that they may have contributed to these evolutionary events.

Base Sequence↗