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PubMed1993

Characterization of the left 4 kb of conjugative transposon Tn916: determinants involved in excision.

The rate-limiting step in movement of the conjugative transposon Tn916, originally identified in Enterococcus faecalis, is believed to be an excision event that generates a non-replicative circular intermediate. When present on a plasmid vector in Escherichia coli, Tn916 generally excises at a high frequency. It was reported previously that insertion of Tn5 in a region near the left end of Tn916 eliminated the ability to excise; and the mutation could be complemented in trans. In this communication the nucleotide sequence of 4 kb of Tn916 DNA connecting the recently sequenced tet(M) determinant (Su et al., 1992; Burdett, 1990) with the left end of the transposon. Ten open reading frames (ORFs) were deduced, two of which (ORF3 and ORF4) were encoded in-frame within a third (ORF2). Mutants with Tn5 insertions in the ORF1 or ORF2 (ORF3 and ORF4) were defective in excision, but could be complemented in vivo by a co-resident plasmid containing the ORF1 or ORF2 determinant, respectively. The data support the view that both ORF1 and ORF2 are essential for excision. ORF1 and ORF2 are essentially identical to determinants designated xis-Tn and int-Tn, respectively, in the closely related Tn1545. A Tn5 insertion in ORF5 eliminated conjugative transfer between E. faecalis strains. Functions for the remaining ORFs (ORF6 through ORF10) remain unknown; however, nucleotide sequences within ORF6 and ORF9 had significant homology with sequence downstream of other tet(M) determinants.

Amino Acid Sequence↗
PubMed1994

Nucleolar introns from Physarum flavicomum contain insertion elements that may explain how mobile group I introns gained their open reading frames.

Comparison of two group I intron sequences in the nucleolar genome of the myxomycete Physarum flavicomum to their homologs in the closely related Physarum polycephalum revealed insertion-like elements. One of the insertion-like elements consists of two repetitive sequence motifs of 11 and 101 bp in five and three copies, respectively. The smaller motif, which flanks the larger, resembles a target duplication and indicates a relationship to transposons or retroelements. The insertion-like elements are found in the peripheral loops of the RNA structure; the positions occupied by the ORFs of mobile nucleolar group I introns. The P. flavicomum introns are 1184 and 637 bp in size, located in the large subunit ribosomal RNA gene, and can be folded into group I intron structures at the RNA level. However, the intron 2s from both P. flavicomum and P. polycephalum contain an unusual core region that lacks the P8 segment. None of the introns are able to self-splice in vitro. Southern analysis of different isolates indicates that the introns are not optional in myxomycetes.

Animals↗
PubMed1993

A new PCR based method for the generation of nested deletions.

We have developed a simple, PCR-based protocol, random primed/anchored-PCR (RPA-PCR), that allows the selective amplification and efficient cloning of segments that are adjacent to any known sequence. We demonstrate that RPA-PCR can be used to prepare a nested set of evenly spaced deletions suitable for DNA sequencing. However, it should also be possible to use this technique for a number of other purposes: generating deletions for the analysis of eukaryotic promoters, extending cDNA clones in the 5' direction, cloning the insertion sites of retroviral proviruses and transposons, and analyzing intron/exon boundaries.

Base Sequence↗
PubMed2005

The heat shock genes dnaK, dnaJ, and grpE are involved in regulation of putisolvin biosynthesis in Pseudomonas putida PCL1445.

Pseudomonas putida PCL1445 produces two cyclic lipopeptides, putisolvins I and II, which possess surfactant activity and play an important role in biofilm formation and degradation. In order to identify genes and traits that are involved in the regulation of putisolvin production of PCL1445, a Tn5luxAB library was generated and mutants were selected for the lack of biosurfactant production using a drop-collapsing assay. Sequence analysis of the Tn5luxAB flanking region of one biosurfactant mutant, strain PCL1627, showed that the transposon had inserted in a dnaK homologue which is located downstream of grpE and upstream of dnaJ. Analysis of putisolvin production and expression studies indicate that dnaK, together with the dnaJ and grpE heat shock genes, takes part in the positive regulation (directly or indirectly) of putisolvin biosynthesis at the transcriptional level. Growth of PCL1445 at low temperature resulted in an increased level of putisolvins, and mutant analyses showed that this requires dnaK and dnaJ but not grpE. In addition, putisolvin biosynthesis of PCL1445 was found to be dependent on the GacA/GacS two-component signaling system. Expression analysis indicated that dnaK is positively regulated by GacA/GacS.

Bacterial Proteins↗
PubMed1996

DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).

DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.

Animals↗
PubMed2004

In vitro shuttle mutagenesis using engineered mariner transposons.

Advances in our understanding of the protozoan parasite Leishmania have been facilitated by the development of molecular and genetic tools. One powerful approach for gene identification and analysis is transposon mutagenesis. This can be performed directly in vivo, but often it is more convenient to generate transpositions in vitro for subsequent analysis in vivo, in a process termed "shuttle mutagenesis." The Drosophila element mariner is well suited for application by either route. Minimal mariner elements containing cis-acting elements required for transposition have been generated, which can be further modified to suit the needs of the experimenter. Additional genetic markers and/or reporters can be introduced, which are useful for procedures such as insertional mutagenesis, shotgun sequencing, or the generation of protein and transcriptional fusions for subsequent analysis. Active transposase can readily be generated following expression in Escherichia coli, and efficiencies of 10-3/target can be obtained, allow-ing the generation of large transposon insertion libraries suitable for subsequent screening in vivo. This chapter explains the steps necessary to purify active Mos1 transposase and conduct an in vitro transposition reaction. We also discuss some of the considerations relevant to the design and application of functional mariner elements (donor plasmids) relevant to studies in Leishmania and other organisms.

Base Sequence↗
PubMed1988

The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936-kanamycin resistance element.

Tn1935, a 23.5-kb transposon mediating resistance to ampicillin, kanamycin, mercury, spectinomycin, and sulfonamide was isolated from pZM3, an IncFIme virulence plasmid from Salmonella wien. Tn1935 possesses the entire sequence of Tn21 and contains two additional DNA segments of 0.95 and 2.7 kb carrying the ampicillin and kanamycin resistance genes, respectively. The latter is part of a composite element since it is flanked by two IS15-like insertion sequences (IS1936) in direct orientation. IS1936 is about 800 bp long and is closely related to IS15 delta, IS26, IS46, IS140, and IS176. Functional analysis of IS1936-mediated cointegrates shows that both insertion sequences are active and able to form cointegrates at the same frequency. Resolution of the cointegrates requires the presence of the host Rec system. The presence of the composite IS1936-element within Tn1935 supports the hypothesis that multidrug resistance transposons evolved by insertion of antibiotic determinants which are themselves transposable.

Anti-Bacterial Agents↗
PubMed2006

Recent translational research: oncogene discovery by insertional mutagenesis gets a new boost.

Knowledge of the genes and genetic pathways involved in onco-genesis is essential if we are to identify novel targets for cancer therapy. Insertional mutagenesis in mouse models is among the most efficient tools to detect novel cancer genes. Retrovirus-mediated insertional mutagenesis received a tremendous boost by the availability of the mouse genome sequence and new PCR methods. Application of such advances were limited to lympho-magenesis but are now also being applied to mammary tumourigenesis. Novel transposons that allow insertional mutagenesis studies to be conducted in tumors of any mouse tissue may give cancer gene discovery a further boost.

Animals↗
PubMed2004

Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study.

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.

Animals↗
PubMed1988

The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component.

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.

ATP-Dependent Proteases↗
PubMed2002

Insertional polymorphisms of ETn retrotransposons include a disruption of the wiz gene in C57BL/6 mice.

ETn (early transposon) elements are moderate repetitive sequences present in hundreds of copies in the mouse genome. Their length ranges from 4.4 to 7.1 kb, and, like transposons, they contain long terminal repeats (LTRs) on both sides and are flanked by target site duplications (Kaghad et al. 1985). ETn-related elements can be grouped into three distinct families. Members of the ETn I and ETn II families mainly contain sequences of unknown origin in their core region. Only very short stretches of retrovirus-like sequences are present, and there are no ORFs. ETn I and ETn II elements differ primarily in the 3- half of both the 5- and 3- LTR, and in the 5- end of the core region (see Fig. 1). As a consequence, only ETn II elements contain a primer binding site for tRNALys. In contrast to ETn I and ETn II, members of the recently described MusD family (Mager and Freeman 2000) contain ORFs for (at least parts of) D-type virus Gag, Pro, and Pol proteins. However, in other regions they are structurally similar to ETn II elements and contain an intact primer binding site. It has been shown that MusD sequences are evolutionarily older than ETn II elements, suggesting that the latter might have arisen by recombinatory replacement of the MusD gene-coding sequences with sequences of unknown origin (Mager and Freeman 2000). ETn elements are still active as retrotransposons. In the past years, several germ line and somatic mutations caused by fresh ETn integrations have been found (Table 1). From 19 mutations, sufficient sequence is available in seven cases to show that the insertion was an ETn II element. In eight cases, the sequence data available indicate either an ETn II or a MusD element. ETn I has not been found to be the cause of any mutations, prompting the suggestion that ETn II is the "mobile" family, whereas ETn I elements have lost the capacity to retrotranspose.

Animals↗
PubMed2002

Seedling lethality in Nicotiana plumbaginifolia conferred by Ds transposable element insertion into a plant-specific gene.

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence and organization of an H2-uptake gene cluster from Rhizobium leguminosarum bv. viciae containing a rubredoxin-like gene and four additional open reading frames.

The nucleotide sequence of a 3.2 kb region following the hydrogenase structural operon (hupSLCDEF) in the H2-uptake gene cluster from Rhizobium leguminosarum by viciae strain 128C53 has been determined. Five closely linked genes encoding products of 16.3 (HupG), 30.5 (HupH), 8.0 (HupI), 18.4 (HupJ) and 38.7 (HupK) kDa were identified 166 bp downstream from hupF. Transposon insertions into hupG, hupH, hupJ and hupK suppress the H2-oxidizing capability of the wild-type strain. The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins. The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters. An involvement of the hupGHIJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin.

Amino Acid Sequence↗
PubMed1990

Primary sequence of the EcoRII endonuclease and properties of its fusions with beta-galactosidase.

The EcoRII endonuclease cleaves DNA containing the sequence CC(A/T)GG before the first cytosine. The methylation of the second cytosine in the sequence by either the EcoRII methylase or Dcm, a chromosomally coded protein in Escherichia coli, inhibits the cleavage. The gene for the EcoRII endonuclease was mapped by analysis of derivatives containing linker insertions, transposon insertions, and restriction fragment deletions. Surprisingly, plasmids carrying the wild-type endonuclease gene and the EcoRII methylase gene interrupted by transposon insertions appeared to be lethal to dcm+ strains of E. coli. We conclude that not all the EcoRII/Dcm recognition sites in the cellular DNA are methylated in dcm+ strains. The DNA sequence of a 1650-base pair fragment containing the endonuclease gene was determined. It revealed an open reading frame that could code for a 45.6-kDa protein. This predicted size is consistent with the known size of the endonuclease monomer (44 kDa). The endonuclease and methylase genes appear to be transcribed convergently from separate promoters. The reading frame of the endonuclease gene was confirmed at three points by generating random protein fusions between the endonuclease and beta-galactosidase, followed by an analysis of the sequence at the junctions. One of these fusions is missing 18 COOH-terminal amino acids of the endonuclease but still displays significant ability to restrict incoming phage in addition to beta-galactosidase activity. No striking similarity between the sequence of the endonuclease and any other protein in the PIR data base was found. The knowledge of the primary sequence of the endonuclease and the availability of the various constructs involving its gene should be helpful in the study of the interaction of the enzyme with its substrate DNA.

Amino Acid Sequence↗
PubMed1992

The 28S ribosomal RNA-encoding gene of Hymenoptera: inserted sequences in the retrotransposon-rich regions.

The genomes of two parasitoid wasps, Diadromus pulchellus and Eupelmus vuilleti, and the honey bee, Apis mellifera, contain few interspersed repeated sequences corresponding to transposons (Tn). This suggests that the genomic organisation of Hymenoptera could be due to the elimination of deleterious Tn in haploid males. We have used restriction-fragment length polymorphism analysis to show that nondeleterious Tn are present in the DNA (rDNA) encoding ribosomal RNA of twelve species of Hymenoptera. Sequence analysis of the 28S rDNA type-I and type-II insertion-rich regions of 80 species showed that this region is very highly conserved (95.8%). A consensus sequence and restriction map of the rDNA region were established. These sequence data were used to develop a strategy for detecting inserted elements in the rDNA fragments containing type-I or type-II insertion sites, and this strategy was used to screen twelve hymenopteran species and four non-Hymenoptera control species. The rDNA fragments from the Hymenoptera and control species contained inserted sequences in the area where type-I and type-II elements are inserted in the 28S rDNA retrotransposon-rich region of Diptera and Lepidoptera. The hymenopteran genomes therefore appear to contain repeated elements, the mobility and nature of which remain to be determined.

Animals↗
PubMed2005

Metabolic and evolutionary relationships among Pyrococcus Species: genetic exchange within a hydrothermal vent environment.

Pyrococcus furiosus and Pyrococcus woesei grow optimally at temperatures near 100 degrees C and were isolated from the same shallow marine volcanic vent system. Hybridization of genomic DNA from P. woesei to a DNA microarray containing all 2,065 open reading frames (ORFs) annotated in the P. furiosus genome, in combination with PCR analysis, indicated that homologs of 105 ORFs present in P. furiosus are absent from the uncharacterized genome of P. woesei. Pulsed-field electrophoresis indicated that the sizes of the two genomes are comparable, and the results were consistent with the hypothesis that P. woesei lacks the 105 ORFs found in P. furiosus. The missing ORFs are present in P. furiosus mainly in clusters. These clusters include one cluster (Mal I, PF1737 to PF1751) involved in maltose metabolism and another cluster (PF0691 to PF0695) whose products are thought to remove toxic reactive nitrogen species. Accordingly, it was found that P. woesei, in contrast to P. furiosus, is unable to utilize maltose as a carbon source for growth, and the growth of P. woesei on starch was inhibited by addition of a nitric oxide generator. In P. furiosus the ORF clusters not present in P. woesei are bracketed by or are in the vicinity of insertion sequences or long clusters of tandem repeats (LCTRs). While the role of LCTRs in lateral gene transfer is not known, the Mal I cluster in P. furiosus is a composite transposon that undergoes replicative transposition. The same locus in P. woesei lacks any evidence of insertion activity, indicating that P. woesei is a sister or even the parent of P. furiosus. P. woesei may have acquired by lateral gene transfer more than 100 ORFs from other organisms living in the same thermophilic environment to produce the type strain of P. furiosus.

DNA Transposable Elements↗
PubMed1988

Alcohol dehydrogenase gene from Alcaligenes eutrophus: subcloning, heterologous expression in Escherichia coli, sequencing, and location of Tn5 insertions.

The nucleotide sequence of the gene that encodes the fermentative, multifunctional alcohol dehydrogenase (ADH) in Alcaligenes eutrophus, and of adjacent regions on a 1.8-kilobase-pair PstI fragment was determined. From the deduced amino acid sequence, a molecular weight of 38,549 was calculated for the ADH subunit. The amino acid sequence reveals homologies from 22.3 to 26.3% with zinc-containing alcohol dehydrogenases from eucaryotic organisms (Schizosaccharomyces pombe, Zea mays, mouse, horse liver, and human liver). Most of the 22 amino acid residues, which are strictly conserved in this group of ADHs (H. Jörnvall, B. Persson, and J. Jeffery, Eur. J. Biochem. 167:195-201, 1987), either were present in the A. eutrophus enzyme or had been substituted by related amino acids. The A. eutrophus adh gene was transcribed in Escherichia coli only under the control of the lac promoter, but was not expressed by its own promoter. A sequence resembling the E. coli consensus promoter DNA sequence did not contain the invariant T, but a G, in the potential -10 region. In the transposon-induced mutants HC1409 and HC1421, which form ADH constitutively, the insertions of Tn5::mob were localized 56 and 66 base pairs, respectively, upstream of the presumptive translation initiation codon. In contrast to the promoter, the A. eutrophus ribosome-binding site with a GGAG Shine-Dalgarno sequence 6 base pairs upstream of the translation initiation codon was accepted by the E. coli translation apparatus. A stable hairpin structure, which may provide a transcription termination signal, is predicted to occur in the mRNA, with its starting point 21 base pairs downstream from the translation termination codon.

Alcaligenes↗