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At least 1,117 records · Page 62Linked to original sources
PubMed1985

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.

Bacteroides↗
PubMed1992

Studies on the Mx transposable element system in maize recovered from X-irradiated stocks.

The unstable mutant bz-x3m arose in a plant subjected to X-irradiation. The element at the bronze locus is non-autonomous and recombination data indicate that an autonomous element is tightly linked. The autonomous element has been designated Mx (mobile element induced by X-rays) and the non-autonomous element, rMx (responder to Mx). Linkage data indicate that a second Mx lies near the end of the short arm of chromosome 9; in one plant, an Mx that is unlinked was detected. Distinguishing characteristics of bz-x3m are a large window of time in endosperm development during which somatic reversions can arise and a wide range in the frequency at which they occur; these features are heritable. With increasing doses of bz-x3m and Mx, the window expands and the frequency range increases. In kernels containing the bz-x3m allele and the tightly linked Mx, breakage occurs in chromosome 9 distal to the C locus, resulting in breakage-fusion-bridge patterns for endosperm markers that lie proximal to the break. The frequency of breaks and the developmental time at which they occur exhibit the same dosage effect as the somatic reversions of the bz-x3m allele. These observations suggest that an rMx (designated rMxBr) that causes chromosome breakage is positioned distal to the C locus. At the molecular level, the bz-x3m allele is associated with a 0.5 kb increase in fragment size in DNA samples digested with BglII, EcoRI, HindIII and PstI; in germinal revertants, the fragment size returns to that of the progenitor.

Alleles↗
PubMed2003

Gene and enhancer trap transposable elements reveal oxygen deprivation-regulated genes and their complex patterns of expression in Arabidopsis.

Transposon tagging with modified maize Ds-GUS constructs was used to isolate genes induced by oxygen deprivation in Arabidopsis thaliana. Seedlings of 800 gene-trap (DsG) and 600 enhancer-trap (DsE) lines were grown on vertically positioned plates for 1 week, oxygen deprived for up to 24 h and stained for GUS activity. Oxygen deprivation induced intricate patterns of gene expression in seedlings of 65 lines. The insertion site and phenotypes of 15 lines were examined. Surprisingly, none of the insertions were into genes that encode known anaerobic polypeptides. Insertions were identified within or adjacent to genes encoding proteins of regulatory, enzymatic, mitochondrial protein import and unknown function, as well as adjacent to genes encoding a putative receptor-like kinase and putative sensor-histidine kinase. Four lines had significantly lower ADH activity after 24 h of oxygen deprivation and three of these showed reduced stress tolerance. Two lines with wild-type levels of ADH were low-oxygen intolerant. Paradoxically, several lines had significantly higher ADH activity after 12 h of oxygen deprivation but reduced stress tolerance. Caffeine treatment, which increased ADH specific activity in wild-type seedlings under aerobic conditions, was sufficient to increase GUS staining in seven of the 15 lines, providing evidence that these genes may be regulated by cytosolic calcium levels. These results demonstrate the effectiveness of the Ds-GUS tagging system in the identification of genes that are regulated in response to oxygen deprivation and a calcium second messenger.

Arabidopsis↗
PubMed1983

Misregulation versus mutation in the alteration of gene expression by carcinogens through interactions with transposable elements in Drosophila melanogaster.

Carcinogens from different chemical series were tested on the wild-type allele of the complex white (w+) locus, which comprised some 5 recombinational subunits, the proximal part (w+4.5) exhibiting regulatory interactions with the neighbouring gene zeste (z). Three w+ loci were used, with different proximal regulatory sequences, including an unstable locus with a TE. Altered expression with each z w+ complex was assayed on the basis of the induction of aberrantly pigmented eye sectors known to be diagnostic of the interaction between z and the dosage of the functionally active w+4.5 subunits. All the tested carcinogens (DMN, DMBA and AFB1) were poorly active in the induction of the putative somatic deletions causing white (w-) eye sectors. In contrast, they were highly effective on the regulatory w+4.5 sequences in all test loci, as indicated by the significantly higher yield of red eye sectors (w-4.5) above the controls. However, this effect varied as a function of the chemical structure of the test compound and the genetic organisation of the regulatory targets. Germinal mutagenicity of the test compounds was assayed on X chromosomes carrying stable and unstable w+ loci, after the injection of adults and topical application on newly hatched larvae. Both techniques revealed that there was no association between the induction of somatic alterations in gene expression and the germinally induced mutations, including the TE w+4.5 deletions. Furthermore, the somatic events, unlike mutations, showed an association with the time of genetic determination during eye-disc cell differentiation. The present results were compatible with the concept of somatic gene misregulation by carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria.

To combine the features of the omega interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the omega interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (alph A) which is expressed well in many Gram-negative bacteria. The IS1 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain in which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.

Blotting, Southern↗
PubMed1986

A 2.6 kb DNA sequence of Streptomyces coelicolor A3(2) which functions as a transposable element.

Streptomyces coelicolor A3(2) contains CCC DNA molecules, 2.6 kb in size, with an average copy number of less than one per ten chromosomes. Southern hybridisation revealed, in addition, two linear, integrated copies (A and B) of this "mini-circle" sequence per chromosome. The two integrated copies have similar (if not identical) ends and are present in the same locations in various S. coelicolor A3(2) derivatives. The mini-circle sequence is absent from S. lividans 66 and S. violaceolatus ISP5438 and from several Streptomyces species less closely related to S. coelicolor A3(2). None of a variety of Streptomyces plasmids tested contained homology to the mini-circle sequence. When a 1.8 kb fragment of the mini-circle lacking the ends of the integrated copies was inserted into KC515 (a derivative of the temperate phage phi C31 which is unable to lysogenise host strains by the natural route because the phage attachment site has been deleted) the resulting phage lysogenized S. coelicolor A3(2) (integrating into the genome of this host by homologous recombination with resident minicircle sequences) but not S. lividans or a variety of other phi C31 hosts. In contrast, a KC515 derivative (KC591) carrying the entire 2.6 kb mini-circle sequence linearized at its single BclI site (and therefore containing the integration site of the free mini-circle) lysogenized not only S. coelicolor A3(2) but also S. lividans 66 and most other strains normally lysogenized by phi C31. The KC591 lysogens of the eight Streptomyces species tested contained a linear, integrated prophage with termini apparently identical to those of the linear mini-circle copies of S. coelicolor.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1993

Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants.

To develop an efficient gene tagging system in rice, a plasmid was constructed carrying a non-autonomous maize Ds element in the untranslated leader sequence of a hygromycin B resistance gene fused with the 35S promoter of cauliflower mosaic virus. This plasmid was cotransfected by electroporation into rice protoplasts together with a plasmid containing the maize Ac transposase gene transcribed from the 35S promoter. Five lines of evidence obtained from the analyses of hygromycin B-resistant calli, regenerated plants and their progeny showed that the introduced Ds was trans-activated by the Ac transposase gene in rice. (1) Cotransfection of the two plasmids is necessary for generation of hygromycin B resistant transformants. (2) Ds excision sites are detected by Southern blot hybridization. (3) Characteristic sequence alterations are found at Ds excision sites. (4) Newly integrated Ds is detected in the rice genome. (5) Generation of 8 bp target duplications is observed at the Ds integration sites on the rice chromosomes. Our results also show that Ds can be trans-activated by the transiently expressed Ac transposase at early stages of protoplast culture and integrated stably into the rice genome, while the cotransfected Ac transposase gene is not integrated. Segregation data from such a transgenic rice plant carrying no Ac transposase gene showed that four Ds copies were stably integrated into three different chromosomes, one of which also contained the functional hph gene restored by Ds excision. The results indicate that a dispersed distribution of Ds throughout genomes not bearing the active Ac transposase gene can be achieved by simultaneous transfection with Ds and the Ac transposase gene.

Base Sequence↗
PubMed1989

The transposable element Tn3 promotes general recombination at the neighboring regions.

Transposon Tn3 was inserted into a tRNA operon of the amber suppressor Su+2 on a transducing phage (lambda hcI857nin5pSu+2) by selecting phages with ampicillin resistance and Su- phenotypes. In a strain thus obtained, Tn3 was inserted between the promoter and the first tRNA gene of the operon, which was determined by DNA sequencing. The Su+2 tRNA operon on the transducing phage consisted of two tRNA genes for tRNA(Met) and Su+2 tRNA(2Gln), which was a deletion derivative of the supB-E tRNA operon of E. coli containing seven tRNA genes in the order of promoter-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2. Proliferating the lambda hcI857nin5pSu+2::Tn3 in E. coli cells, a number of phages which had lost Tn3 were isolated, and their tRNA gene compositions as well as the DNA structures of the tRNA operon were analyzed. In many cases the tRNA genes which had been deleted from the original transducing phage were regained from the chromosomal supB-E operon. Thus the loss of Tn3 from the phages was not due to excision of the transposon but due to the replacement of a portion of the tRNA operon, including Tn3, with the host homologous region that did not contain Tn3. This type of replacement takes place rather efficiently as a consequence of Tn3 insertion, owing to the general recombination occurring between homologous tRNA genes of phage and host chromosomes in the presence of either host recA or phage red. No such enhanced recombination in a similar cross between phage and host chromosomes was observed with the Tn3 present in the trans position on an independent plasmid. We conclude that inserting Tn3 in cis promotes general recombination in the neighboring regions. Possible mechanisms for this new type of genetic effect of Tn3 are discussed. During the course of this study, a natural defective mutation (T11) was also detected in one of the duplicated tRNA(2Gln) genes in an E. coli K12 strain we used.

Amino Acid Sequence↗
PubMed1988

Characterization of the maize transposable element Ac by internal deletions.

We have used the ability of Ac to transpose in tobacco to determine which Ac sequences are required for transposition, using a phenotypic assay for Ac excision from an NPTII gene in which excisions of Ac result in calli resistant to the antibiotic kanamycin (Baker et al., 1987). Here we show that deletion of the Ac DNA which encodes the untranslated leader of the Ac transcript does not prevent Ac excision. A deletion which removes 110 bp including the first 75 bp of long open reading frame prevents Ac excision in tobacco cells. However, it will excise in tobacco cells previously transformed with Ac indicating that deletion of the region prevents the synthesis of a product required for Ac excision. Deletion of the Ac sequences between bp 44 and bp 92 or from bp 75 to bp 181 abolishes, or strongly reduces, transposition in cells which are already transgenic for an active Ac element. This indicates that these deleted elements have lost sequences which are required for the transposon to respond to the transposase, when the enzyme is produced in trans. We also describe a tobacco strain transformed with a Ds element stably inserted wtihin an NPTII gene. This strain is Km and was retransformed with a construct containing the open reading frame (ORF) of the 3.5-kb Ac transcript expressed from a plant promoter. Expression of the cDNA construct promotes excision of the Ds element. These data suggest that the 3.5-kb transcript of Ac encodes the only Ac product required for transposition, i.e. the transposase function.

Journal Article↗
PubMed1987

Inactivation of the maize transposable element Activator (Ac) is associated with its DNA modification.

The Activator (Ac) element at the waxy locus (wx-m7 allele) has the ability to undergo changes in its genetic activity and cycles between an active and inactive phase. Comparison of active Ac elements at several loci and the inactive Ac at wx-m7 by Southern blot analysis revealed that the inactive Ac sequence was not susceptible to digestion by the methylation sensitive enzyme PvuII while active elements were susceptible to PvuII digestion. Restriction digest comparisons between the clones of the active and inactive Ac elements were indistinguishable. Further analyses with the enzymes SstII and the methylation sensitive and insensitive isoschizomers EcoRII and BstNI showed the inactive Ac sequence was methylated at these sites, whereas the active Ac was hypomethylated. Although the active Ac at the wx-m7 allele in different genetic backgrounds showed differences in the Ac DNA modification pattern, at least a fraction of genomic DNA contained Ac sequences that were unmethylated at all of the internal sites we assayed. These data may suggest a role for DNA modification in the ability of Ac to transpose from the waxy locus and to destabilize unlinked Ds elements.

Alleles↗
PubMed1997

Molecular domestication of mobile elements.

Transposable elements are ubiquitous in all organisms and represent a dynamic component of their genomes, causing mutations and thereby genetic variation. Because of their independent and expansive replication strategy, these elements are called selfish and were thought to have no impact on the adaptive evolution of their host organisms. Although most TE-induced mutations seem to exert only negative effects on the fitness of their carrier, recent evidence indicates that in the course of evolution at least some TE-mediated changes have become established features of the host genome. For example, the insertion of TEs may provide novel cis-regulatory regions to preexisting host genes or TE-derived trans-acting factors may undergo a molecular transition into novel host genes through a process described as molecular domestication. The stationary P element related gene clusters of D. guanche, D. madeirensis and D. subobscura provide an excellent model system to study the evolutionary impact of TEs on genome evolution. Each cluster unit consists of a cis-regulating section composed of different insertion sequences followed by the first three exons of a P element that are coding for a 66 kDa 'repressor-like' protein.

Animals↗
PubMed1986

Functional analysis of the transcriptional control regions of the copia transposable element.

The introduction of copia-based vectors in Drosophila hydei cells results in their high-level transient expression and the subsequent establishment of stably transformed cell lines containing multiple copies of vector integrated into host genomic DNA. Using transformation frequency and transient expression analysis as assays of promoter strength, we have defined the regions of copia essential for expression. We find that the essential sequences reside within the long terminal repeat, but 3' to the site of initiation of copia RNA. Deletion of the consensus enhancer-like sequences from copia appears to have no effect on vector expression.

Journal Article↗
PubMed2002

Tissue-culture enhanced transposition of the maize transposable element Dissociation in Brassica oleracea var. ' Italica'

To investigate the potential of heterologous transposons as a gene tagging system in broccoli ( Brassica oleracea var. Italica), we have introduced a Ds-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct ( Tpase). In three successive selfed generations of plants there was no evidence of germinal-excision events. To overcome this apparent inability to produce B. oleracea plants with germinal excisions, we performed a novel tissue-culture technique to select for fully green shoots from seed with somatic-excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%) and molecular analysis indicated that the plants genetically were like plants that contain a germinal-excision event. Further molecular analysis of these plants showed that 69% exhibited reinsertion of Ds back into the plant genome. Sequencing of donor-site footprints after Ds excision, revealed that there is an indication of more-severe deletions and rearrangements when higher concentrations of streptomycin are used in the tissue-culture selection process. Adapted versions of this regeneration technique have a high potential for providing germinal excision-like events in heterologous plants species which show low transposon activity. Alternatively, there is the potential to increase the proportion of 'germinal' plants in earlier generations of more-active plant species.

Journal Article↗
PubMed1997

Zebedee: a novel copia-Ty1 family of transposable elements in the genome of the medically important mosquito Aedes aegypti.

We have utilised PCR to directly identify a novel family of copia-Ty1 retrotransposable elements (RTPs) in the genome of the mosquito Aedes aegypti. Two members of the family have been sequenced in their entirety and their structural characteristics determined. ZebedeeI is 3505 bp long and appears to be flanked by 21bp direct repeat sequences. A single open reading frame (ORF) of 972 amino acids has the coding potential for a polyprotein with homology corresponding to the conserved amino acid motifs of Long Terminal Repeat (LTR) retrotransposon protease, integrase and reverse transcriptase. ZebedeeII likewise shares significant homology with these regions and also appears to be flanked by short direct terminal repeat sequences of 22 bp. Fifty copies of the 22 bp repeat sequence are present abutting the 5' end of ZebedeeII, with two (partial) representatives of this repeat sequence being present at the 3' end. The Zebedee family appears to have a low middle repetitive copy number in different strains of Ae. aegypti; and transcripts of the elements have been detected in cultured mosquito cells by RT-PCR. Despite the lack of a gag homologue or the LTR hallmarks of previously characterised copia-Ty1 RTPs, phylogenetic analyses place Zebedee within this group, showing considerable homology to copia from Drosophila melanogaster.

Aedes↗
PubMed2004

Transposable elements as a source of genetic innovation: expression and evolution of a family of retrotransposon-derived neogenes in mammals.

A family of functional neogenes called Mart, related to the gag gene of Sushi-like long terminal repeat retrotransposons from fish and amphibians, is present in the genome of human (11 genes) and other primates, as well as in mouse (11 genes), rat, dog (12 genes), cat, and cow. Mart genes have lost their capacity of retrotransposition through non-functionalizing rearrangements having principally affected long terminal repeats and pol open reading frame. Most Mart genes are located on the X chromosome in different mammals. Sequence database analysis suggested that Mart genes are present in opossum (marsupial), but absent from the genome of chicken. Hence, the Mart gene family might have been formed from Sushi-like retrotransposon(s) after the split of birds and mammals (310 myr ago), but before the divergence between placental mammals and marsupials (170 myr ago). RT-PCR analysis showed that at least six Mart genes are expressed during mouse embryonic development, with in situ hybridization analysis revealing rather ubiquitous expression patterns. Mart expression was also detected in adult mice, with some genes being expressed in all tissues tested, while others showed a much more restricted expression pattern. Although additional analysis will be required to establish the function of the retrotransposon-derived Mart neogenes, these observations support the evolutionary importance of retrotransposable elements as a source of genetic novelty.

Amino Acid Sequence↗