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PubMed1989

Method for selection of transposable DNA and characterization of a new insertion sequence, IS493, from Streptomyces lividans.

A method to select for transposable elements from Streptomyces spp. by using insertional inactivation of a repressor gene that functions in Escherichia coli was developed. Plasmid pCZA126, which can replicate in Streptomyces spp. or E. coli, contains a gene coding for the lambda cI857 repressor and a gene, under repressor control, coding for apramycin resistance. E. coli cells containing the plasmid are apramycin sensitive but become apramycin resistant if the cI857 repressor gene is disrupted. Plasmids propagated in Streptomyces spp. can be screened for transposable elements that have disrupted the cI857 gene by transforming E. coli cells to apramycin resistance. This method was used to isolate a new 1.6-kilobase insertion sequence, IS493, from Streptomyces lividans CT2. IS493 duplicated host DNA at the target site, had inverted repeats at its ends, and contained two tandem open reading frames on each strand. IS493 was present in three copies in the same genomic locations in several S. lividans strains. Two of the copies appeared to be present in regions of similar DNA context that extended at least 11.5 kilobases. Several other Streptomyces spp. did not appear to contain copies of IS493.

Amino Acid Sequence↗

PubMed1990

The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element.

The gene for the insertion sequence (IS) 30 transposase is placed under the control of the tac promoter, and large quantities of transposase are expressed upon induction. The resulting protein precipitates inside the Escherichia coli cells in the form of inclusion bodies which, upon cell lysis, cannot be dissolved under nondenaturing conditions. In contrast, the N-terminal third of the transposase, a 17-kDa protein produced by a truncated gene, can be purified and is able to interact site specifically with the ends of the IS30 element. In DNase I footprint experiments, regions of 26 nucleotides on one DNA strand and 19 nucleotides on the other strand at either end of the element are protected from nuclease digestion. It is concluded that a functional DNA-binding domain can be formed by expression of only one-third of the complete IS30 transposase. Sequence comparison shows a homology of the IS30 ends to the ends of IS4351 and to the L1 end of bacteriophage Mu.

Amino Acid Sequence↗

PubMed1983

An insertion sequence blocks the expression of a soybean lectin gene.

We investigated soybean lectin gene structure and expression in a genetic line that accumulates normal levels of seed lectin (Le+ line) and in a mutant line that produces no detectable seed lectin (Le- line). We found that the nuclear and cytoplasmic lectin RNA levels in Le- embryos are approximately 0.01% of those in Le+ embryos. Runoff transcription experiments with isolated nuclei showed that the reduced RNA levels are caused by decreased lectin gene transcription. Each genetic line contains two lectin genes, L1 and L2. L1 encodes seed lectin, while the function of L2 is unknown. We found that a 3.4 kb DNA segment, consisting of repeated sequences, has been inserted into the L1 gene of the Le- line. We conclude that this insertion results in reduced transcription of the mutant L1 gene, and leads to the lectinless phenotype.

DNA↗

PubMed1992

Identification and characterization of a novel insertion sequence, IS1106, downstream of the porA gene in B15 Neisseria meningitidis.

Examination of Neisseria meningitidis strains associated with endemic meningococcal disease demonstrated differences in the number of copies of a repetitive sequence. Characterization of a copy of this repetitive sequence present in B15 strains has revealed the presence of a novel insertion sequence (IS1106) located within a complex repetitive region downstream of the gene for the major surface antigen (porA). IS1106 has a length of 1137 bp and is flanked by 36bp inverted repeats. Two open reading frames (ORF1 and ORF2) are present in opposite strands in codon-codon register with ORF2 entirely located within ORF1. The predicted protein from ORF1 demonstrates homology with the 5A protein of IS5 (Kroger and Hobom, 1982). Strains from two independent outbreaks of B15 meningococcal disease in the UK were found to contain the same genomic deletion removing a copy of IS1106 downstream of the porA gene.

Alleles↗

PubMed1987

Detection and characterization of IS256, an insertion sequence in Staphylococcus aureus.

Resistance to the aminoglycosides gentamicin (Gmr), tobramycin (Tmr) and kanamycin (Kmr) in strains of Staphylococcus aureus isolated in Australia is mediated by the transposon Tn4001. The 1.35 kb inverted repeat of this transposon exhibits many of the characteristics of an insertion sequence and has consequently been designated IS256. Tandem duplication of IS256 contiguous with Tn4001 results in an increase in the level of GmrTmrKmr, thereby implying that the element possesses strong promoter sequences. Both contiguous and independent insertions of IS256 into the staphylococcal chromosome have been observed, the latter suggesting that the element may play a role in molecular rearrangements of the genome.

Chromosome Mapping↗

PubMed1981

Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points.

A pBR322-derived plasmid pKEN221 carrying a Serratia marcescens lpp gene overproduces the outer membrane lipoprotein in an Escherichia coli lpp- cell. However, when this strain was continuously cultured in a rich medium for about thirty generations, many Lpp- mutants were accumulated. Out of six mutants analyzed, three were found to carry insertion mutation in the lpp gene in pKEN221. From resistance enzyme mapping and hybridization analysis of the mutant plasmid DNA, it was found that two mutants were caused by insertion sequence IS1 and one by IS5. Nucleotide sequence analysis of these mutant DNAs revealed that both IS1 and IS5 insertions occurred in the A-T rich 5' untranslated region of the lpp gene. While the IS1 insertion resulted in a direct duplication of a nine-base-pair sequence in the original pKEN221 DNA at the junction with IS1, the IS5 insertion resulted in a direct duplication of a four-base-pair sequence. IS5 was found to contain inverted-repeat sequences of twelve nucleotides at its exact ends. This is the first example of the nucleotide sequence analysis of an IS5 insertion mutation. By Southern blot hybridization, the E. coli chromosomal DNA was found to contain about ten copies of IS5.

Bacterial Proteins↗

PubMed1976

Structural evolution of bacterial plasmids: role of translocating genetic elements and DNA sequence insertions.

Recent evidence suggests that plasmids have evolved by site-specific recombinational events involving translocation and insertion of discretely defined DNA segments. The role of translocating genetic elements and repeated DNA sequences in the formation and structural evolution of bacterial plasmids, and in the control of plasmid gene expression, is the subject of this brief review. Insertion sequence (IS) regions are discrete segments of DNA that are known to cause strongly polar mutations in the genes of Escherichia coli and several bacteriophages as a consequence of their insertion into bacterial or phage genomes. Recent investigations have identified three separate kinds of IS segments on plasmids, and have indicated that such regions may have a role in 1) site-specific reversible dissociation of antibiotic resistance plasmids into their component segments, 2) recombination of certain plasmids with the bacterial chromosome, and 3) translocation of segments of plasmid DNA onto other replicons, or onto different sites of the same replicon. In addition, such DNA sequences, which may be repeated on plasmid genomes in either direct or reverse orientation, are involved in the control of plasmid gene expression. Inverted repeats other than the genetically characterized IS segments also appear to be involved in recA-independent, recombination and translocation of plasmid DNA segments. These inverted repeats contain palindromic nucleotide sequences on each strand of DNA and are detectable as hairpin-loop structures by electron microscope heteroduplex analysis. Such palindromes resemble the recognition sites for restriction endonucleases, some of which are encoded by plasmids, suggesting that similar endonucleolytic enzymes may be involved in the translocation of plasmid DNA segments.

Base Sequence↗

PubMed1992

A sequence insertion targeting vector for Leishmania enriettii.

We have demonstrated previously that Leishmania enriettii contains the enzymatic machinery to mediate efficient interplasmidic homologous recombination. In this report we show that a sequence insertion targeting vector, pALT-Neo-Tub, can be inserted into the genome of L. enriettii by homologous recombination between alpha-tubulin sequences found in the plasmid and their homologs in the genome. pALT-Neo-Tub, a pBluescript-derived vector containing the neor gene flanked by the alpha-tubulin intergenic and alpha-tubulin coding sequences, was used to transfect cells to G418 resistance. Analysis of the DNA from the drug resistant clones indicates that all of the insertion events are restricted to the alpha-tubulin gene repeats. As little as 200 base pairs of sequence homology between the plasmid and the genome is required for integration. Nonhomologous recombination events are not detected. These results indicate that exogenous DNA sequences can be integrated into the L. enriettii genome provided that they are flanked by homologous DNA sequences.

Animals↗

PubMed1989

Identification of insertion sequence element IS427 in pTiT37 plasmid DNA of an Agrobacterium tumefaciens T37 isolate.

The isolation and characterization of an insertion sequence (IS) element, IS427, from Agrobacterium tumefaciens T37 is described. IS427 is present in three nonidentical copies on the pTiT37 plasmid. The copy that was isolated through transposition on the entrapment vector pUCD800 contains at its ends a 16-bp imperfect inverted repeat and generates a 2-bp duplication of the target DNA. IS427 does not show homology with previously characterized IS elements of A. tumefaciens, based on hybridization experiments and/or sequence comparison.

Bacillus subtilis↗

PubMed1991

Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability.

Acetobacter pasteurianus NCI1380, a thermophilic strain isolated from the surface culture of acetic acid fermentation, showed genetic instability to produce at high frequency spontaneous mutants which were deficient in ethanol oxidation because of the loss of alcohol dehydrogenase activity. Southern hybridization experiments with the cloned alcohol dehydrogenase-cytochrome c gene cluster as the probe showed insertion of an unknown DNA fragment into a specific position in the cytochrome c gene in most of the mutant strains. Cloning and sequencing analyses revealed that the inserted sequence was 1,665 bp in length and had a terminal inverted repeat of 15 bp. In addition, this inserted sequence was found to generate a 4-bp duplication at the inserted site upon transposition. The target site specificity was not very strict, but a TCGA sequence appeared to be preferentially used. The inserted sequence contains two long open reading frames of 461 and 222 amino acids which are overlapped and encoded by different strands. Although these open reading frames showed no homology to any protein registered in the DNA data bases, the longer open reading frame contained many basic amino acids (87 of 461), as was observed with transposases of so-called insertion sequence (IS) elements. All of these characteristics are typical of IS elements, and the sequence was named IS1380. The copy number of IS1380 in a cell of A. pasteurianus NCI1380 was estimated to be about 100. Several strains of acetic acid bacteria also contained IS1380 at high copy numbers. These results suggest that IS1380 is associated with the genetic loss of ethanol-oxidizing ability as well as the genetic instability of acetic acid bacteria in general.

Acetobacter↗

PubMed1989

Characterization of an insertion sequence (IS891) of novel structure from the cyanobacterium Anabaena sp. strain M-131.

When recombinant plasmids that were transferred to the cyanobacterium Anabaena sp. strain M-131 were transferred back to Escherichia coli, some of the transformants contained inserts. One of the insertion sequences (ISs) was characterized by sequencing. This 1,351-base-pair IS contained an open reading frame that was capable of encoding a peptide of 310 amino acids and had terminal sequences with distinctive structures, but it lacked terminal inverted repeats and did not duplicate target DNA upon insertion. The element bore no significant sequence homology to any sequence stored in the GenBank data base. Restriction analysis of the genomes of Anabaena sp. strain M-131 and Anabaena sp. strain PCC 7120 showed those strains to be closely related. Sequences homologous to the IS element were also present in the DNA of Anabaena strain PCC 7120, but the copy numbers and chromosomal locations of such sequences differed in the two strains. The largest visualized plasmid was 425 kilobases (kb) in M-131 and 410 kb in PCC 7120; at least the former plasmid contained multiple copies of the element, as did a 115-kb plasmid in M-131.

Amino Acid Sequence↗

PubMed1989

A Mycoplasma genetic element resembling prokaryotic insertion sequences.

Nucleotide sequence analysis of two Mycoplasma hyopneumoniae-derived copies of a repetitive genetic element revealed structural similarities to typical prokaryotic insertion sequences. This is the first such sequence identified in the class Mollicutes. The element spans approximately 1550bp, with 28bp inverted terminal repeats. Two open reading frames occur within the sequence, one potentially encoding a protein with a size-variant alpha-helical domain containing heptameric leucine periodicity. Hybridization data with several strains from each of two mycoplasma species showed that the repetitive sequence is variably distributed within the M. hyopneumoniae and Mycoplasma hyorhinis chromosomes and indicated that in some cases the repeated sequence is contained within a larger genetic element which may be the result of phage or plasmid insertion.

Amino Acid Sequence↗

PubMed1989

Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli.

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.

Blotting, Southern↗

PubMed1976

Insertion sequence IS2 near the gene for prophage lambda excision.

In this study we characterize a variant of the lambdacI857S7 prophage, designated lambdabi2cI857S7, which carries a DNA insertion. The insertion sequence is IS2, and it resides in the antipolar orientation II just upstream from the gene for prophage excision (xis) at 61.6%lambda. This bi2 insertion mutant could prove valuable for studies on possible recombination functions of IS2 DNA and of its effect on the lambda integration and excision functions.

Chromosome Mapping↗

PubMed1983

A new insertion sequence, IS121, is found on the Mu dI1 (Ap lac) bacteriophage and the Escherichia coli K-12 chromosome.

We have discovered a new insertion sequence, now designated IS121, as a component of the Mu dI1 (Ap lac) phage. This sequence is 1.2 kilobases long and contains single recognition sites for the HincII, Bg1II, and HindIII restriction endonucleases. IS121 is present in at least three copies in the chromosome of several Escherichia coli K-12 strains. When present in the nonconjugative plasmid pBR322, IS121 can mediate cointegrate formation with an F' lac plasmid and transfer of pBR322 sequences to suitable recipients. IS121 is also capable of precise or nearly precise excision. As part of the study of IS121, we have determined the physical structure of the Mu dI1 (Ap lac) phage and established an extensive restriction endonuclease map of this phage. A revised schema for the formation of the Mu dI1 (Ap lac) phage is presented.

Chromosomes, Bacterial↗

PubMed1982

ISRm1: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes.

After transposon Tn5 mutagenesis, a high proportion of Rhizobium meliloti symbiotic mutants do not contain Tn5 insertions in symbiotic genes. Instead, the mutations in these strains are correlated with the presence of an endogenous insertion sequence (ISRm1) in nitrogen fixation (nif) or symbiotic genes which are adjacent to the nif genes. ISRm1 is 1.4 kb and transposes to at least three restriction fragments in the nif region at a frequency between 10(-2) and 10(-3). A nif region restriction fragment containing ISRm1 was cloned from one of the mutant strains unable to fix nitrogen symbiotically (Fix-) and the resulting plasmid was used as a hybridization probe. ISRm1 is present at least ten times in the R. meliloti genome but is not present in any other R. meliloti strains, E. coli strains, or Rhizobium species tested. We demonstrated that the Fix- phenotype correlated with ISRm1 transposition is indeed caused by ISRm1 insertion by conjugating a cloned fragment containing ISRm1 into a wild type Fix+ R. meliloti host and replacing the normal genomic nif fragment with the nif::ISRm1 fragment. The resulting strain was Fix-.

DNA Transposable Elements↗

PubMed1988

Synthesis and overproduction of the 5A protein of insertion sequence IS5.

We have demonstrated both the synthesis and overproduction of the 5A protein encoded by the longest open reading frame of the bacterial insertion sequence IS5. Expression was obtained in vitro and in Escherichia coli maxicells from plasmids containing IS5 in either orientation, as well as in vitro from a restriction fragment containing exclusively IS5 DNA. When IS5 was cloned in the appropriate orientation downstream of a strong tac promoter, production of the 5A protein was increased to 10 to 20% of the total protein synthesized in vitro.

Carrier Proteins↗

PubMed1991

Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12.

In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.

Amino Acid Sequence↗