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PubMed1999

Precise excision and transposition of piggyBac in pink bollworm embryos.

Transposable elements such as P, hobo, Hermes, mariner and Minos have been successfully harnessed as gene vectors to achieve the transformation of several dipteran species including Drosophila melanogaster, Ceratitis capitata and Aedes aegypti. Plasmid-based excision and transposition assays have been useful indicators of an element's ability to be mobilized in vivo and thus potentially serve as a transforming vector. We report that the transposable element piggyBac is capable of precise excision and transposition in the pink bollworm (Pectinophora gossypiella), a worldwide pest of cultivated cotton. Combined with a suitable marker gene, the piggyBac element may serve as a vector for germline transformation in this and (potentially) other lepidopteran species.

Animals↗

PubMed2000

Transformation of Stomoxys calcitrans with a Hermes gene vector.

The ability of the Hermes transposable element to function as a germ line transformation vector was tested in the stable fly, Stomoxys calcitrans. Plasmid-based transposable element mobility assays indicated moderate mobility of Hermes in this species. Germline transformants were created using a Hermes element containing the enhanced green fluorescent protein (EGFP) under the regulatory control of the promoter from Actin5C gene of Drosophila melanogaster. Approximately 4% of the fifty-five adults that developed from the 1903 G(0) embryos injected with the vector produced transgenic progeny. In the four transgenic lines established, the EGFP expression pattern was distinctly nonuniform and levels of expression were low. Promoters other than the one from the Actin5C gene of D. melanogaster should be considered for widespread, constitutive expression. All transgenic lines contained multiple (2-4) integrated Hermes elements. Hermes integration events occurred through a canonical cut-and-paste mechanism.

Animals↗

PubMed1983

Transcription of the white locus in Drosophila melanogaster.

Genetic studies of the white locus have shown that it has a distal region where structural mutations occur and a proximal region where regulatory mutations occur. To better understand the molecular basis of this genetic organization we have analyzed white locus transcription. A 2.7-kilobase transcript comprising 0.0005% of poly(A)-RNA was detected in RNA prepared from pupae or adults. The structure of this transcript helps clarify some unusual genetic properties of the locus. There is a small 5' exon separated from the majority of the sequences found in the mature RNA by an intron of approximately 2.8 kilobases. This 5' exon is from the proximal region of the locus, whereas the main body of the RNA maps to the distal region. The mutationally silent region between the proximal and distal regions corresponds to the large intron. We have identified the family and determined the exact location of a number of transposable element insertions within the locus. These results show that transposable element insertions within introns can be without phenotypic effect. We have also investigated the effect on the white transcript of the zeste mutation, which represses white locus expression as judged by eye color phenotype. The RNA was unchanged in size or abundance in poly(A)-RNA from adult flies. This demonstrates that the zeste-white interaction does not occur by simply repressing transcription of the white locus in all tissues.

Journal Article↗

PubMed2006

Molecular structure and organization of the wheat genomic manganese superoxide dismutase gene.

The genomic structure of a manganese superoxide dismutase (MnSOD) gene in wheat was elucidated by sequencing a clone from a BAC library of a stripe rust resistant wheat line. The clone was identified by hybridization with a wheat MnSOD cDNA. The gene consisted of 6 exons interrupted by 5 introns with a total length of 4770 nucleotides from the start codon to the termination codon. The wheat MnSOD gene was the longest among those sequenced from plant species. The transcription initiation site was preceded by a G+C-rich promoter without a TATA or CAAT box. The promoter contained many putative cis-acting regulatory elements, including an abscisic acid (ABA)-responsive element, a stress-responsive element, and a GC-repeat, as well as several other structural features in common with the promoter of the rice MnSOD gene. A Stowaway-like transposable element was found in intron 5 of the wheat MnSOD gene, but further investigation revealed the transposable element was not present in all copies of the MnSOD genes.

Base Sequence↗

PubMed1982

FB elements are the common basis for the instability of the wDZL and wC Drosophila mutations.

The DNA insertions that cause the highly unstable mutations wC and wDZL share extensive homology with the FB family of transposable elements. FB elements carry long, internally repetitious, inverted terminal repeats and thus differ in structure from other transposable elements. Our results suggest that FB elements may excise and cause chromosomal rearrangements at unusually high frequencies. The wC insertion is a single FB element. The wDZL insertion differs in that it contains two FB elements, one at each terminus. The wC and wDZL insertions contain 4.0 and 6.5 kilobase nonhomologous segments between their terminal repeats. In contrast to the middle repetitive FB elements, the central segment of the wDZL insertion is single-copy and present at a fixed location in the wild-type genome. It has apparently been transposed by the action of flanking FB elements, causing the wDZL mutation at its new location.

Alleles↗

PubMed1987

Studies on the introduction and mobility of the maize Activator element in Arabidopsis thaliana and Daucus carota.

We have co-transformed carrot (Daucus carota) and Arabidopsis thaliana with an Agrobacterium tumefaciens non-tumorigenic T-DNA carrying the maize transposable element Activator (Ac) and an Agrobacterium rhizogenes Ri T-DNA. We present evidence that the Ac element transposes in transformed root or root-derived callus cultures of both species. We show that fertile plants can be regenerated from transformed, root-derived callus cultures of Arabidopsis, demonstrating the utility of the Ri plasmid for introducing the maize Ac element into plants. We also present evidence that Ac elements that excise from the transforming T-DNA early after transformation continue to be mobile in carrot root cultures.

Cells, Cultured↗

PubMed1997

What makes Grande1 retrotransposon different?

Grande1 elements constitute a family of Ty3 retrotransposons present in the Zea genus in more than 1000 copies in Zea diploperennis and maize. The sequences of three Grande1 flanking regions, two from Z. diploperennis and one from maize, reveal transposable elements as insertion targets, suggesting a preferential integration of Grande1 elements into other transposable elements. These retrotransposons are remarkable for their large size of around 14 kb, which is a consequence of a very large 3' region of more than 7 kb. Atypical entities within this region are two arrays of unrelated tandem repeats with potential stable stem-loop structures. A large portion of the same region is occupied by ORFs, although only ORF23, whose function is unknown, is presumably transcribed in antisense orientation to the reverse transcriptase ORF. Only ORF23 has a codon usage similar to the one tabulated for highly-expressed maize genes. Correspondingly, the transcript of 900 b that hybridizes with ORF23 probes is found in all the maize tissues explored. This is despite the high level of methylation in the DNA of Grande1. Genomic RNA has not been detected in any tissue or situation studied, probably reflecting a non-functional retrotransposon. The origin of ORF23 and the remainder 3' region might be due to a transduction event.

Base Sequence↗

PubMed1997

Insertion sequence IST3091 of Thiobacillus ferrooxidans.

An insertion sequence, designated as IST3091, was located adjacent to the putative origin of replication region of plasmid pTFI91 of Thiobacillus ferrooxidans TFI-91. The DNA sequence of the transposase gene of IST3091 revealed similarity with that of IS30, IS1086, IS4351, and the integrase gene of SpV1-R8A2 B (a bacteriophage of Spiroplasma citri). The sequence of IST3091 is 1063 bp long with partially matched 30-bp terminal inverted repeats. Several restriction fragments of plasmid pTFI91 of T. ferrooxidans containing the IST3091 element were cloned into the vector pHSG398. The hybrid plasmids (pBTL) were transformed into Escherichia coli NK7379 containing a miniF plasmid, which was devoid of transposable elements. The transposition function of the IST3091 element was confirmed by mobilizing hybrid plasmids via conjugation from transformed E. coli NK7379 (donor) to E. coli M8820 (recipient). The presence of the transposed element in transconjugants was detected by polymerase chain reaction amplification.

Amino Acid Sequence↗

PubMed1987

Rhizobium meliloti insertion element ISRm2 and its use for identification of the fixX gene.

Two of the three plasmids of the wild-type Rhizobium meliloti 41 (pRme41a and pRme41c) carry a copy of ISRm2, a 2.7-kilobase-long transposable element. ISRm2 is terminated by 22-base-pair (bp) inverted repeat sequences, exhibiting some homology to the inverted repeats of elements generating 9-bp target sequence duplication. Transposition of ISRm2 results in a duplication of 8 bp in length, rather rare among transposable elements. DNA sequences homologous to an internal fragment of ISRm2 were found in several Rhizobium species. Transposition of ISRm2 into fixation and nodulation genes located on the symbiotic plasmid pRme41b was detected at a high frequency. Exact locations of two copies of ISRm2 which transposed into the nod-nif region on the megaplasmid were determined. In one case, integration into the protein-coding region of the hsnD gene that determines a host specificity function of nodulation occurred. In the other mutant, ISRm2 was localized upstream of nifA, where a short open reading frame coding for a new fix gene (fixX) was identified. The product of fixX is a ferredoxin carrying a characteristic cluster of cysteine residues. On the basis of the observation that the arrangement of the ISRm2 copies is identical in the free-living wild-type cells and in nitrogen-fixing nodules, we concluded that the involvement of ISRm2 transposition in the development of nitrogen-fixing symbiosis is unlikely.

Cloning, Molecular↗

PubMed1989

The P-M and the 23.5 MRF (hobo) systems of hybrid dysgenesis in Drosophila melanogaster are independent of each other.

Strains of Drosophila melanogaster bearing the male recombination factor 23.5 MRF induce hybrid dysgenesis in a way which is highly reminiscent of the P-M system, and, most probably, causally related to the activity of the transposable element hobo. We have investigated potential interactions between the two systems of hybrid dysgenesis by studying mixed lines derived from bidirectional crosses between 23.5 MRF and P strains, and analyzed their potentials to induce or suppress the occurrence of dysgenesis. All new lines possess the P induction abilities, as determined by two different procedures, and have also acquired a P cytotype. In contrast, some of them lost their ability to induce the non-P-M dysgenesis, as well as to suppress the action of 23.5 MRF. This loss of the 23.5 MRF induction abilities parallels the selective loss of full-length hobo elements from the genome of these lines, providing further substantiation to the notion that the 23.5 MRF activity is directly linked to this transposable element.

Animals↗

PubMed1987

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

Amino Acid Sequence↗

PubMed2002

Homologous recombination between the inverted terminal repeats of defective transposon TCp3.2 causes an inversion in the genome of Cydia pomonella granulovirus.

In this study, a new mutant of the Cydia pomonella granulovirus (CpGV), which shows spontaneous inversion of a transposable element during in vivo replication, is described. CpGV-MCp4 is a natural mutant of CpGV-M, containing the transposable element TCp3.2, which originated from the genome of the host C. pomonella. During in vivo cloning studies of CpGV-MCp4, a mutant called CpGV-MCp4inv was isolated. CpGV-MCp4inv shows heterogeneity in the genome area of transposon insertion. Restriction mapping, PCR analysis and subsequent sequence analysis gave strong evidence that an inversion of TCp3.2 is caused by homologous recombination between the long inverted terminal repeats (ITRs) of the transposon. This finding demonstrated that extensive homologous repeat regions such as the ITRs of transposons cause inversions by homologous recombination during in vivo replication. The observed in vivo inversion between the ITRs can be considered as a model for the contribution of repeated sequences in the genome rearrangement of baculoviruses and a source for genetic heterogeneity among different baculoviruses and baculovirus genotypes.

Animals↗

PubMed1999

A novel pseudoautosomal human gene encodes a putative protein similar to Ac-like transposases.

We report the cloning of a novel gene, called Tramp, in the Xp/Yp PAR region that has a functional homologue on the Y chromosome and escapes X-inactivation. This gene encodes, within a single exon, a putative protein that has amino acid similarity with transposases of the Ac family. Flanking this gene we have identified putative terminal inverted repeats (TIRs) and a duplicate target site, suggesting that it may be an ancient transposable element. The nucleotide differences in these sites and the TIR-binding inactivity of the putative Tramp protein suggest that this element is not an autonomous transposon. In the human genome, the Tramp protein may be involved in the transposition of other transposable elements, like medium reiterated frequency repeats, or it could be specialized in the acquisition of a new cellular function.

Amino Acid Sequence↗

PubMed1985

How many processed pseudogenes are accumulated in a gene family?

A simple kinetic model is developed that describes the accumulation of processed pseudogenes in a functional gene family. Insertion of new pseudogenes occurs at rate v per gene and is countered by spontaneous deletion (at rate delta per DNA segment) of segments containing processed pseudogenes. If there are k functional genes in a gene family, the equilibrium number of processed pseudogenes is k(v/delta), and the percentage of functional genes in the gene family at equilibrium is 1/[1 + (v/delta)]. v/delta values estimated for five gene families ranged from 1.7 to 15. This fairly narrow range suggests that the rates of formation and deletion of processed pseudogenes may be positively correlated for these families. If delta is sufficiently large relative to the per nucleotide mutation rate mu (delta greater than 20 mu), processed pseudogenes will show high homology with each other, even in the absence of gene conversion between pseudogenes. We argue that formation of processed pseudogenes may share common pathways with transposable elements and retroviruses, creating the potential for correlated responses in the evolution of processed pseudogenes due to direct selection for control of transposable elements and/or retroviruses. Finally, we discuss the nature of the selective forces that may act directly or indirectly to influence the evolution of processed pseudogenes.

Animals↗

PubMed1983

An unusual transposon encoding heat shock inducible and developmentally regulated transcripts in Dictyostelium.

We have isolated and characterized a transposable element from Dictyostelium discoideum denoted Tdd-1. There are approximately 50 complete copies of the element per haploid genome and approximately 100-150 partial elements. Southern blots of DNA from different Dictyostelium discoideum strains show that Tdd-1 is a mobile element. Tdd-1 is 4.9 kb long, with 313 bp inverted repeats. These repeats lie near the termini, but unlike other transposable elements one end of Tdd-1 extends 36 bp past the repeat and the other extends 1 bp. Tdd-1 encodes a series of developmentally regulated transcripts, all with the same polarity, that increase dramatically in abundance after approximately 10 hr of development. We have also identified a heat shock inducible transcript of the opposite polarity.

Animals↗

PubMed1998

Transposition of the mariner element from Drosophila mauritiana in zebrafish.

With the increased popularity of zebrafish (Danio rerio) for mutagenesis studies, efficient methods for manipulation of its genome are needed. One approach is the use of a transposable element as a vector for gene transfer in this species. We report here the transformation of zebrafish and germ-line transmission of the mariner element from Drosophila mauritiana. The mariner element was selected because its transposition is independent of host-specific factors. One- to two-cell-stage zebrafish embryos were coinjected with a supercoiled plasmid carrying the nonautonomous mariner element peach and mRNA encoding the transposase. Surviving larvae were reared to adulthood, and the transmission of peach to the F1 generation was tested by PCR. Four of the 12 founders, following plasmid injections on 2 different days, transmitted the element to their progeny. Inheritance of the transgene from the F1 to the F2 generation showed a Mendelian pattern. No plasmid sequences were detected by PCR or Southern blot analysis, indicating transposition of peach rather than random integration of the plasmid DNA. These data provide evidence of transformation of a vertebrate with a transposable element and support the host-independent mechanism for transposition of the mariner element. We suggest this system could be used for insertional mutagenesis or for identifying active regions of the genome in the zebrafish.

Animals↗

PubMed1991

An indicator gene for detection of germline retrotransposition in transgenic Drosophila demonstrates RNA-mediated transposition of the LINE I element.

We have marked a cloned Drosophila transposable element--the I element--with an engineered neomycin-containing indicator gene, whose expression is conditioned by passage of the transposon through an RNA intermediate. Mobility of the marked element introduced into Drosophila as a transgene could be detected in vivo, upon in toto selection of developing embryos on G418-containing medium. For resistant individuals, Southern blot analysis and nucleotide sequencing after PCR amplification disclosed transposition of the marked element into new loci, with target site duplications and splicing out of the intron in the indicator gene. It demonstrates that the I element, which is closely related to the widespread mammalian LINEs, transposes through an RNA intermediate, as up to now only conjectured from sequence singularities of this class of 'non-viral retrotransposons'. The developed indicator gene provides a potent new genetic tool for detection and quantitative analysis of retrotransposon mobility and its regulation as it occurs in vivo.

Animals↗

PubMed2004

Mobile genetic elements as natural tools for genome evolution.

Transposable elements (TEs) are ubiquitous components of all living organisms, and in the course of their coexistence with their respective host genomes, these parasitic DNAs have played important roles in the evolution of complex genetic networks. The interaction between mobile DNAs and their host genomes are quite diverse, ranging from modifications of gene structure and regulation to alterations in general genome architecture. Thus over evolutionary time these elements can be regarded as natural molecular tools in shaping the organization, structure, and function of eukaryotic genes and genomes. Based on their intrinsic properties and features, mobile DNAs are widely applied at present as a technical "toolbox," essential for studying a diverse spectrum of biological questions. In this chapter we aim to review both the evolutionary impact of TEs on genome evolution and their valuable and diverse methodological applications as the molecular tools presented in this book.

Alu Elements↗