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PubMed1998

Recent B2 element insertions in the mouse genome.

B2 elements are a family of short interspersed repeats that have amplified within rodent genomes. Recent mobility of only two individual B2 elements has been reported to date. We identified an additional recent B2 insertion occurring within intron 4 of the murine beta-glucuronidase gene (Gus-s) of the BalbC strain of mouse by analyzing orthologous loci of a nonrandomly selected B2 element. The basis of selection for the B2 element was its high level of sequence identity to the B2 consensus. The selected B2 element was amplified by the polymerase chain reaction (PCR) using primers to the unique flanking sequences from genomic DNA of several species and laboratory strains of mice. Our results demonstrated the presence of the selected B2 element only in the genome of Mus musculus BalbC strain. Cloning and sequencing of a representative sample of the products obtained confirmed the absence of the B2 element within this intron in addition to other variations in the sequence. The detection of the B2 element only in the BalbC strain suggests that the element recently inserted within this mouse population when the initial laboratory colony was formed. Sequence comparison of the two previously identified recent B2 inserts also shows a low divergence in relation to the B2 type II consensus. The data presented confirms that recently inserted B2 elements closely match their consensus sequence, potentially allowing for their identification.

Animals↗
PubMed1999

Identification of the plasmid-mobilization potential of the strain Klebsiella pneumoniae ozenae KIIIA isolated from a polluted aquatic environment.

The Klebsiella pneumoniae ozenae KIIIA strain was isolated from the River Rhine soon after a serious mercury pollution episode and was selected for mercury resistance as well as for intergeneric DNA mobilization helper potential. This transfer helper capacity was shown to be related to the presence of a Tn3-like transposable element, Tn5403. Because transposon-mediated fusion was found to be involved in the mobilization potential of KIIIA, the visualization and the identification of the conjugative element, responsible for the transfer, were necessary. Our results show that, in addition to the four nonconjugative plasmids visualized in a previous study, K. pneumoniae ozenae KIIIA harbors two other plasmids, pK130 and pK45, of respective sizes of 130 and 45 kb, but none of these plasmids is involved in the mobilization mechanism. The presence of yet another extrachromosomal element pK225, with a size of 225 kb, was established by indirect methods, since yields of pK225 isolated from KIIIA were low and the plasmid was difficult to visualize directly. However, the integration of this plasmid into the chromosome was not detected. The present paper highlights the problem of detecting some plasmids in bacteria which have been isolated from the environment. For these plasmids, indirect approaches, that detect conjugative functions, constitute a feasible alternative for the investigation of the plasmid content of bacteria, if the direct approach fails. An analysis of the different types of transconjugants indicated that the mercury-resistance marker as well as the mobilization potentials, expressed by KIIIA, are linked to pK225. This plasmid could not be assigned to a described Inc group either by DNA hybridization or by PCR amplification.

Conjugation, Genetic↗
PubMed2005

Large-scale systematic study on stability of the Ds element and timing of transposition in rice.

Activator/Dissociation (Ac/Ds) transposon mutagenesis is a widely used tool for gene identification; however, several reports on silencing of the Ac/Ds element in starter lines and in stable transposants question the applicability of such an approach in later generations. We have performed a systematic analysis on various aspects of the silencing phenomenon in rice (Oryza sativa ssp. japonica cv. Nipponbare). High somatic and germinal transposition frequencies observed in earlier generations were maintained as late as T4 and T5 generations; thus the propagation of parental lines did not induce transposon silencing. Moreover, the stably transposed Ds element was active even at the F5 generation, since Ac could remobilize the Ds element as indicated by the footprint analysis of several revertants. Expression of the bar gene was monitored from F3 to F6 generations in >1,000 lines. Strikingly, substantial transgene silencing was not observed in any of the generations tested. We analyzed the timing of transposition during rice development and provide evidence that Ds is transposed late after tiller formation. The possibility, that the independent events could be the result of secondary transposition, was ruled out by analyzing potential footprints by reciprocal PCR. Our study validates the Ac/Ds system as a tool for large-scale mutagenesis in rice, since the Ds elements were active in the starter and insertion lines even in the later generations. We propose that harvesting rice seeds using their panicles is an alternative way to increase the number of independent transposants due to post-tillering transposition.

Base Sequence↗
PubMed2005

A transposon-based activation-tagging population in Arabidopsis thaliana (TAMARA) and its application in the identification of dominant developmental and metabolic mutations.

A population of 9471 stable activation-tagged lines was generated by transposable element mediated activation tagging mutagenesis in Arabidopsis (TAMARA) using the maize En/Spm transposon system. Based on DNA gel blot and flanking sequence analysis, this population contains approximately 6000 independent transposon insertions. A greenhouse-based screen identified six dominant or semi-dominant activation tagged mutants with obvious developmental alterations, among these a new pistillata mutant allele. In addition, a subset of 1500 lines was screened by a HPLC based high-throughput method for dominant activation tagged mutants with enhanced contents of phenolic compounds. One dominant activation tagged mutant (hpc1-1D) was isolated showing accumulation of a particular compound due to the upregulation of an R2R3-MYB transcription factor.

Amino Acid Sequence↗
PubMed2006

Identification, characterization and comparative genomics of chimpanzee endogenous retroviruses.

BACKGROUND: Retrotransposons, the most abundant and widespread class of eukaryotic transposable elements, are believed to play a significant role in mutation and disease and to have contributed significantly to the evolution of genome structure and function. The recent sequencing of the chimpanzee genome is providing an unprecedented opportunity to study the functional significance of these elements in two closely related primate species and to better evaluate their role in primate evolution. RESULTS: We report here that the chimpanzee genome contains at least 42 separate families of endogenous retroviruses, nine of which were not previously identified. All but two (CERV 1/PTERV1 and CERV 2) of the 42 families of chimpanzee endogenous retroviruses were found to have orthologs in humans. Molecular analysis (PCR and Southern hybridization) of CERV 2 elements demonstrates that this family is present in chimpanzee, bonobo, gorilla and old-world monkeys but absent in human, orangutan and new-world monkeys. A survey of endogenous retroviral positional variation between chimpanzees and humans determined that approximately 7% of all chimpanzee-human INDEL variation is associated with endogenous retroviral sequences. CONCLUSION: Nine families of chimpanzee endogenous retroviruses have been transpositionally active since chimpanzees and humans diverged from a common ancestor. Seven of these transpositionally active families have orthologs in humans, one of which has also been transpositionally active in humans since the human-chimpanzee divergence about six million years ago. Comparative analyses of orthologous regions of the human and chimpanzee genomes have revealed that a significant portion of INDEL variation between chimpanzees and humans is attributable to endogenous retroviruses and may be of evolutionary significance.

Animals↗
PubMed1990

Identification and molecular characterization of shrunken-2 cDNA clones of maize.

Mutation at the shrunken-2 (Sh2) locus of maize, a gene described more than 40 years ago, greatly reduces starch levels in the endosperm through its effect on the starch synthetic enzyme ADP-glucose pyrophosphorylase, an enzyme thought to be regulatory in this biosynthetic pathway. Although our previous work has suggested that Sh2 is a structural gene for this enzyme, we have also reported data compatible with Sh2 acting post-transcriptionally. In this study, we took advantage of a transposable element-induced Sh2 allele, its progenitor, and revertants to identify a clone for this locus. Although the cloning and identification were done independently of any knowledge concerning the product of this gene, examination of the deduced amino acid sequence revealed much similarity to known ADP-glucose pyrophosphorylase subunits of plants and bacteria, including regions involved in substrate binding and activator binding. Little sequence similarity, however, was found at the DNA level. These observations provide direct evidence that Sh2 encodes a subunit for endosperm ADP-glucose pyrophosphorylase. Analysis of several phenotypically wild-type alleles arising from a mutable sh2-Ds allele revealed one unexpected case in which DNA sequences of Sh2 were rearranged in comparison with the progenitor Sh2. In contrast to wild type, the Ds-induced sh2 allele conditions at least two transcripts in the endosperm.

Alleles↗
PubMed1996

A widespread transposable element masks expression of a yeast copper transport gene.

The trace element copper (Cu) is essential for cell growth. In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene. Ctr3p is a small intracellular cysteine-rich integral membrane protein that restores high-affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene. In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb. In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation. In such strains inactivation of both CTR1 and CTR3 is required to generate lethal copper-deficient phenotypes. Although Ctr1p and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions. These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.

Amino Acid Sequence↗
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↗
PubMed1996

Identification and cloning of a mobile transposon from Aspergillus niger var. awamori.

Aspergillus niger var. awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of the niaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in both A. niger var. awamori and A. niger. A single copy of Vader was detected from only one of the two laboratory strains of A. nidulans which were also examined. Insertion of the Vader element into the niaD gene of A. niger var. awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements at niaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. Mutants niaD392 and niaD436 reverted at a frequency of 9x10(-3) and 4x10(-2), respectively. Two of the mutants, niaD587 and niaD410, reverted at a lower frequency of 6x10(-4).

Aspergillus niger↗
PubMed2006

DNA methylation of fly genes and transposons.

The use of anti-5-methylcytosine antibodies in affinity columns allowed the identification of methylated sequences in the genome of Drosophila melanogaster adults. In view of the presence of transposable elements amongst the identified sequences, it has been suggested that DNA methylation is involved in transposon control in the fly genome. On the contrary, a reanalysis of these data furnishes several intriguing elements that could raise new questions about the role that DNA methylation plays in the fly genome. The aim of the present paper is to discuss some features that emerge from the analysis of the identified methylated sequences.

Animals↗
PubMed2002

A murine gene with circadian expression revealed by transposon insertion: self-sustained rhythmicity in the liver and the photoreceptors.

We have previously identified in some mouse strains (e.g. BALB/c, DBA/2) a murine Intracisternal A-particle (IAP) transposable element specifically expressed in the liver. This IAP sequence is inserted within a gene, mCCR4/m. nocturnin, the sequence of which is related to the circadian Xenopus nocturnin gene. Here we show, using real-time quantitative RT-PCR, that both the IAP sequence and the m. nocturnin gene display strong circadian expression in the liver, with peak abundance after dusk. Circadian oscillations of m. nocturnin RNA are maintained in mice without the IAP insertion (e.g. CBA/J, 129/sv), are free-running under constant light and dark conditions, and persist upon food and water privation, demonstrating that m. nocturnin is a circadian gene. In situ hybridization analyses (in 129/sv mice) further show circadian expression of m. nocturnin also in the retina, precisely at the level of the photoreceptors, a result consistent with the previously described circadian expression of the Xenopus gene. These results strengthen the strong conservation of the nocturnin gene with the identification of a functional mouse ortholog of the Xenopus gene, and demonstrate the reciprocal influence of nearby genes on the expression of transposable elements via "position effects".

Animals↗
PubMed1993

Linked and unlinked transposition of a genetically marked Dissociation element in transgenic tomato.

We have introduced a genetically marked Dissociation transposable element (Dsneo) into tomato. In the presence of Ac transposase, Dsneo excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Dsneo (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Dsneo, we examined the genomic distribution of Dsneo receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Dsneo elements was conducted in two tomato families, derived from independent primary transformants each bearing Dsneo within a T-DNA at a unique position in the genome. Transposition of Dsneo generated clusters of insertions that were positioned on several different tomato chromosomes. Dsneo insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Dsneo transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Dsneo. We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Dsneo, is an effective tool for transposon tagging experiments in tomato.

Alleles↗
PubMed1984

Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene.

Insertion mutations in yeast caused by the transposable element Ty have been identified at several genetic loci. In all cases so far, the site of Ty insertion has been in the 5' non-coding region of the affected gene. Experiments presented here demonstrate that the ura3-52 mutation, a non-reverting ura3 mutation, is caused by a Ty insertion mutation within the coding region of the URA3 gene. This Ty insertion within a gene has a similar structure to those in non-coding regions.

Base Sequence↗
PubMed2001

Transposition of autonomous and engineered impala transposons in Fusarium oxysporum and a related species.

The impala transposon of Fusarium oxysporum is an active element. We demonstrated that the imp160 copy, transposed into the gene encoding nitrate reductase, is an autonomous element, since it excises from this gene and reinserts at a new genomic position in backgrounds free of active elements. An element in which the transposase gene was replaced by a hygromycin B resistance gene was used (1) to demonstrate the absence of endogenous transposase in several F. oxysporum strains and (2) to check the ability of different genomic copies of impala to transactivate this defective element. This two-component system allowed the identification of autonomous elements in two impala subfamilies and revealed that transactivation can occur between highly divergent elements. We also demonstrate that the autonomous copy transposes in a closely related species complex, F. moniliforme, in a fashion similar to that observed in F. oxysporium. The ability of impala to function as a two-component system and to transpose in a heterologous host promises further advances in our understanding of the factors that modulate transposition efficiency and demonstrates the potential of impala as a means of establishing a transposon tagging system for a wide range of fungal species.

Anti-Bacterial Agents↗
PubMed2003

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.

Chromosome Mapping↗
PubMed2004

Establishment of an enhancer trap system with Ds and GUS for functional genomics in rice.

To develop an efficient means of enhancer trapping, a two-element system employing Ds and an Ac transposase (AcTPase) gene was tested in rice. We generated 263 transgenic rice plants, each of which harboured the maize transposable element Ds together with a GUS coding sequence under the control of a minimal promoter (Ds-GUS), and a gene that confers resistance to the herbicide chlorsulfuron. Among the 263 lines generated, 42 were shown to have a single copy of the Ds-GUS element. Four single-copy lines were crossed with each of six transgenic plants that carried the AcTPase gene. Excision of the Ds-GUS in leaves of F1 plants was detected in eight combinations out of seventeen examined. The frequency of transposition of Ds-GUS in germ cells in the F1 plants was examined using 10,524 F2 plants, and 675 (6%) were judged to be transposants. Their frequencies differed among F1 plants depending on the AcTPase x Ds-GUS cross considered, and also among panicles on the same F1 plant. This suggests that Ds-GUS tends to transpose during panicle development. Southern analysis with a GUS probe showed different band patterns among transposants derived from different panicles. Therefore, the transposants derived from different panicles must have arisen independently. Transposants showing tissue-specific GUS activities were obtained, and enhancers thus trapped by the Ds-GUS element were identified. These results demonstrate that the system is suitable for the isolation of large numbers of independent Ds-GUS transposants, and for the identification of various tissue-specific enhancers. The Ds-GUS lines generated in this study offer a potentially powerful tool for studies on the functional genomics of rice.

Blotting, Southern↗
PubMed1988

A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing.

A set of plasmid vectors was developed which allows fast sequencing by the chemical degradation method. These high-copy-number vectors are derivatives of the plasmid pUC8 containing different multiple-purpose cloning sites flanked by unique recognition sequences for the restriction enzymes BstEII, Tth111I and Eco81I as sites for end-labelling DNA. Due to their partially asymmetric recognition sequences, each of these three restriction sites can be singly end-labelled by a filling-in reaction with selected nucleotides. This allows easy single end-labelling of any cloned DNA fragment for sequencing by the chemical degradation method without any isolation and purification step after the labelling reaction. In addition, the nucleotide sequence of the complementary strand from the same end can be determined by the dideoxy chain termination procedure using the universal M13 primers. In most of the new vectors, the reading frame of the lacZ' gene is retained, allowing identification of cloned fragments.

Base Sequence↗
PubMed1982

Transposon-mediated site-specific recombination: identification of three binding sites for resolvase at the res sites of gamma delta and Tn3.

The tnpR gene product, resolvase, of the transposable element gamma delta mediates site-specific recombination between two copies of gamma delta directly repeated on the same replicon, and it negatively regulates transcription of the tnpA (transposase) gene and its own gene. The recombinational site, res, and the regulatory region both are located in the tnpA-tnpR intercistronic region. In studying the interaction of purified resolvase with DNA fragments derived from gamma delta and the related transposon, Tn3, that span this region, we have demonstrated that three sites specifically bind resolvase. Site I overlaps the recombinational crossover point and both transcriptional promoters. Sites II and III cover most of the DNA between the crossover point and the translational start of the tnpR gene. These are the only binding sites we have detected in a region of about 400 base pairs centered on the crossover point. Studies of cointegrates that contain only part of the region that binds resolvase indicate that site I is not sufficient for efficient site-specific recombination and suggest that all three sites are probably required.

Binding Sites↗