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PubMed1995

IS1222: analysis and distribution of a new insertion sequence in Enterobacter agglomerans 339.

With a length of 1221 bp and 44-bp inverted repeats with ten mismatches, IS1222 was identified as an endogenous insertion sequence in Enterobacter agglomerans 339. In this host strain, four copies were located, three on the nif plasmid pEA9 and one at the chromosome. Sequence analysis showed two consecutive open reading frames, orfA and orfB, encoding putative polypeptides of 87 and 276 amino acids. In-between both reading frames, a potential frameshift window of the homonucleotide type was postulated, followed by a pseudoknot structure and a ribosome-binding site. Based on significant homology at the sequence level and similarity of the features discussed, IS1222 was placed among the group of IS3 elements with IS407, IS476 and ISR1 being the most closely related IS. Hybridization experiments suggest that the distribution of IS1222 is limited to a group of related bacterial strains among Enterobacteriaceae.

Amino Acid Sequence↗

PubMed1989

Specificity of insertion of IS91, an insertion sequence present in alpha-haemolysin plasmids of Escherichia coli.

We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5'-CGAGTAGG...CCTATCGAT. IS91 inserts specifically 5' to either one of the tetranucleotides 5'-GAAC or 5'-CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.

Bacterial Proteins↗

PubMed1990

Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction.

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

Animals↗

PubMed1995

Virulence-associated fyuA/irp2 gene cluster of Yersinia enterocolitica biotype 1B carries a novel insertion sequence IS1328.

The fyuA/irp2 gene cluster, which is part of the Yersinia pestis pigmentation (pgm) locus encoding genes involved in iron uptake and virulence, is present in all pesticin-sensitive bacteria. In Y. enterocolitica biotype 1B strains (serotypes O8, O20, O21), the fyuA/irp2 gene cluster carries an insertion of a novel repetitive sequence, IS1328. It was also found in the genome of Y. enterocolitica O5 (biotype 1A) and O13 (biotype 1B), but not in pesticin-sensitive Y. pseudotuberculosis O1 and Escherichia coli Phi. The 1353-bp repetitive element has 12-bp perfect inverted terminal repeats. A single open reading frame is capable of encoding a 334-amino acid polypeptide. IS1328 DNA has high homology with the DNA sequences located downstream of the aggR gene from the enteroaggregative E. coli (EAggEC), to the region of the R751 plasmid flanking Tn501, to the sequence following the merR gene of S. marcescens pDU1358 plasmid, and to the sequences of K. pneumoniae plasmid pCFF04. The putative polypeptide has 36.4% identity with the transposase encoded by the Coxiella burnetii IS1111a insertion sequence. The IS1328 insertion sequence could be responsible for the deletions of the fyuA/irp2 gene cluster observed in Y. enterocolitica O8 and could represent a member of a new group of widely distributed repetitive elements.

Amino Acid Sequence↗

PubMed1988

Direct repeats flanking the Bacteroides transposon Tn4351 are insertion sequence elements.

The clindamycin-erythromycin resistance (Ccr Emr) region of the Bacteroides transposon Tn4351 is flanked by direct repeats. This study showed that the direct repeats are insertion sequence (IS) elements. Although both IS elements can mediate transfer of the chloramphenicol (Cmr) marker on pBR328 by cointegrate formation with the conjugal IncW plasmid R388, IS4351R-mediated transfer of Cmr occurred at a consistently lower frequency than did the transfer mediated by IS4351L. Analysis of plasmids from the resultant transconjugants revealed IS-mediated activities such as deletions, tandem duplication of IS4351L, and excision of IS4351R.

Bacteroides↗

PubMed1986

An extrachromosomal plasmid is the etiological precursor of kalDNA insertion sequences in the mitochondrial chromosome of senescent neurospora.

In the kalilo strains of N. intermedia, senescence is initiated by insertion of a 9.0 kb foreign nucleotide sequence, kalDNA, into mitochondrial DNA. A 9.0 kb linear DNA plasmid that is structurally homologous to the mitochondrial kalDNA insertion sequences exists in high copy numbers in close association with the nuclei of presenescent and senescent kalilo cells, but is not present in cells of long-lived normal strains. The free kalilo plasmid has not been detected in mitochondria, suggesting that the element does not contain a mitochondrial origin of replication. Unexpectedly, the nuclear plasmid, like the mitochondrial insertion element, follows a strict pattern of maternal inheritance. We surmise that the extramitochondrial plasmid is the etiological precursor of the kalDNA insertion sequences that appear in the mtDNAs of senescent cell lines and conclude that the kalilo element induces senescence because it is a mutator of mitochondrial genes.

DNA Replication↗

PubMed1989

Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA.

Transposition of insertion sequence IS10 is regulated by an anti-sense RNA which inhibits transposase expression when IS10 is present in multiple copies per cell. The anti-sense RNA (RNA-OUT) consists of a stem domain topped by a flexibly paired loop; the 5' end of the target molecule, RNA-IN, is complementary to the top of the loop, and complementarity extends for 35 base-pairs down one side of RNA-OUT. We present here genetic evidence that anti-sense pairing, both in vitro and in vivo, initiates by interaction of the 5' end of RNA-IN and the loop domain of RNA-OUT; other features of the reaction are discussed. In the context of this model, we discuss features of this anti-sense system which are important for its biological effectiveness, and suggest that IS10 provides a convenient model for design of efficient artificial anti-sense RNA molecules.

DNA Transposable Elements↗

PubMed1990

Role of instability in the cis action of the insertion sequence IS903 transposase.

An unusual subset of DNA-binding proteins, termed cis-acting proteins, has been shown to act preferentially at their site of synthesis; the transposases of several bacterial insertion sequences (ISs) fall into this class. The transposase of IS903 exhibits a strong preference for action in cis: complementation of defective transposons in trans occurs at less than 1%. Furthermore, transposition mediated by transposase acting in cis is extremely sensitive to the distance between the 3' end of the transposase gene and the nearest transposon inverted repeat; we find that an insertion of 1 kilobase of DNA reduces transposition to 1-2% of control levels. Here we show that there is a strong correlation between the stability of transposase and its ability to act in trans. We found that the wild-type transposase is a very unstable protein with a physical half-life of about 3 min. However, a transposase-beta-galactosidase fusion protein has a much greater half-life and can act equally well in cis or in trans. In addition, the native transposase is stabilized in lon- strains of Escherichia coli, and, in these protease-deficient strains, trans action of transposase is increased 10- to 100-fold. These results suggest that instability of the IS903 transposase is a major determinant of its cis action and that the La protease, product of the lon gene, is an important determinant of transposase instability.

DNA Transposable Elements↗

PubMed1988

Specific insertion and deletion of insertion sequence 1-like DNA element causes the reversible expression of the virulence capsular antigen Vi of Citrobacter freundii in Escherichia coli.

Citrobacter freundii strain WR7004 reversibly expresses the virulence capsular antigen Vi, whose production is controlled by two distinct chromosomal loci, viaA and viaB. The rate of oscillation between the Vi-producing (Vi+) state and the Vi-nonproducing (Vi-) state in strain WR7004 ranges from 2 X 10(-4) to 7 X 10(-3) transitions per bacterium per generation. A similarly high conversion rate from Vi+ to Vi- occurs in Escherichia coli HB101 harboring pWR127, a plasmid that contains the 18-kilobase-pair (kb) viaB region cloned from WR7004. However, the Vi- state in HB101 harboring pWR127 was so stable that transition to the Vi+ state was not detected (less than 10(-10) per bacterium per generation). When pWR127 DNA derived from Vi- strains of HB101 harboring pWR127 was transformed in HB101 recipient, a small number of Vi+ transformants were seen among the transformants, which were predominantly Vi-. The viaB region consists of two EcoRI digestion fragments, A (8.6 kb) and B (9.4 kb). A discrete 700- to 800-base-pair DNA element was found to be inserted in the EcoRI B fragment of the viaB region of pWR127 when derived from Vi- strains. However, no such DNA element was found in the pWR127 DNA isolated from Vi+ strains. This discrete DNA element inserts into a recombinational hot spot 1.1 kb from the end of the EcoRI B fragment and behaves as an insertion sequence 1 (IS1)-like element. The insertion of this IS1-like element in the viaB region thus disrupts expression of the Vi antigen. Restoration of Vi expression results when this element is excised.

Antigens, Bacterial↗

PubMed1988

IST2: an insertion sequence from Thiobacillus ferrooxidans.

The genome of Thiobacillus ferrooxidans (strain ATCC 19859) contains at least two families of repeated sequences, termed family 1 and 2. The nucleotide sequence of a family 2 member was determined. It is 1408 base pairs long and has structural features similar to those of insertion sequences (IS elements). Terminal inverted repeats 25 base pairs in length are present. These inverted repeats are imperfect and adjacent to target-site duplications 9 base pairs in length. Several open reading frames were detected (the longest was 888 base pairs). We have named this IS element-like sequence IST2. The ends of a second example of IST2 were analyzed and compared to those of the first. The DNA sequences are identical and similarly sized target-site duplications are present.

Base Sequence↗

PubMed1979

Inserted sequence in the mitochondrial 23S ribosomal RNA gene of the yeast Saccharomyces cerevisiae.

The sequence organization of the yeast mit-DNA region carrying the large ribosomal RNA gene and the polar locus omega was examined. Hybridization studies using rho- deletion mutants and electron microscopy of the heteroduplexes formed between 23S rRNA and the appropriate restriction fragments, lead to the conclusion that the 23S rRNA1 gene of the omega+ strains is split by an insertion sequence of 1,000-1,100 bp. In contrast, no detactable insertion was found in the 23S rRNA gene of the omega- strains. The size and the location of the insert found in the 23S rRNA gene of the omega+ strains appear to be identical to those of the sequence delta which had previously been found to characterize the difference (at the omega locus) between the mitDNA of the wild type strains carrying the omega+ or omega- alleles (Jacq et al., 1977).

Base Sequence↗

PubMed1990

A second isoform of chicken brush border myosin I contains a 29-residue inserted sequence that binds calmodulin.

Chicken brush border myosin I (CBB-MI) is a single-headed, nonfilamentous, myosin-like mechanoenzyme which, as isolated, has 3 mol of calmodulin (CAM) 'light chains' bound per mole of 119 kDa heavy chain. We have isolated a partial cDNA clone for CBB-MI that encodes the C-terminal approximately 35 kDa of the heavy chain. The sequence of this clone is identical to that of an authentic, near-full-length CBB-MI cDNA clone reported recently, except for an 87-bp/29-residue insertion occurring approximately 32 kDa from the C-terminus. This insert, which is probably generated by an alternate splicing event, is expressed in brush border as part of a message of the size predicted for the CBB-MI heavy chain, although the steady state level of this transcript is approximately 8-fold lower than for transcripts lacking the insert. 125I-CAM overlays of this cDNA clone (expressed as a trpE fusion protein in E. coli) indicate that it binds one more calmodulin than does a second cDNA clone that lacks the 29-residue insert. A synthetic peptide corresponding to the insert sequence binds tightly to CAM-Sepharose, demonstrates a shift and enhancement of fluorescence in the presence of CAM, and binds CAM in solution with a KD of 190 nM (in 100 mM KCl). We conclude that a second, low-abundance isoform of CBB-MI contains an additional (and possibly fourth) CAM binding site as a result of a 29-residue peptide that is inserted into the tail domain by an apparent alternate splicing event.

Amino Acid Sequence↗

PubMed1981

pED100, a conjugative F plasmid derivative without insertion sequences.

The largest HindIII fragment of F includes the entire replication and transfer regions, and its circularisation with ligase gave the conjugative plasmid pED100. This plasmid, which contains none of the F insertion sequences, was essentially unable to mobilise the E. coli chromosome or to give integrative suppression of a dnaA strain.

Chromosome Mapping↗

PubMed1985

Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains.

Bacillus subtilis↗

PubMed1992

The effects of retrotransposon-derived sequence insertions on gene expression in a model system.

Retrotransposition has been well documented as a significant source of mutagenesis in diverse eukaryotic organisms, including humans. Insertions of retrotransposons within or in close proximity to transcription units cause many spontaneous mutations, and have been implicated as a source of heritable genetic defects and as a cause of carcinogenesis. The mechanisms by which retrotransposon insertions produce mutant phenotypes are diverse and, in many cases, not fully understood. A model transcriptional system was utilized to test the effects of copia retrotransposon-derived sequence insertions upon gene expression in different cellular environments. The results of these experiments indicate that retrotransposon insertions within nontranslated regions of a transcription unit inhibit gene expression by at least two concurrently acting mechanisms: 1) transcriptional interference due to an active internal promoter and, 2) trans RNA hybridization interactions between transcripts containing complementary retrotransposon sequences.

3T3 Cells↗

PubMed1995

Isolation and characterization of a novel insertion sequence element, IS1248, in Paracoccus denitrificans.

A new suicide vector, pRVS3, was constructed to facilitate gene replacements in the genome of Paracoccus denitrificans. In control experiments, incorporation of this suicide vector into the genome did not depend on the presence of homologous DNA. Using appropriate restriction enzymes, the suicide vector and flanking DNA were recovered from the genomic DNA. Sequence analysis demonstrated that both up- and downstream of the ex-integrant vector there was an element that showed high homology with bacterial insertion sequences (IS). Southern blot analysis of wild-type and integrant strains revealed that at least four copies of this IS element reside in the P. denitrificans genome, one of which, designated IS1248, had been involved in the transpositional event described here. IS1248 is 830 bp long, has 13-bp imperfect inverted repeats at the borders, and contains five open reading frames. With respect to the organization and primary sequences of the open reading frames, IS1248 closely resembles IS869 and IS427 of Agrobacterium tumefaciens, IS402 of Pseudomonas cepacia, and ISmyco found in Mycobacterium tuberculosis.

Amino Acid Sequence↗

PubMed1991

Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120.

A family of repetitive elements from the cyanobacterium Anabaena sp. strain PCC 7120 was identified through the proximity of one element to the psbAI gene. Four members of this seven-member family were isolated and shown to have structures characteristic of bacterial insertion sequences. Each element is approximately 1,200 bp in length, is delimited by a 30-bp inverted repeat, and contains two open reading frames in tandem on the same DNA strand. The four copies differ from each other by small insertions or deletions, some of which alter the open reading frames. By using a system designed to trap insertion elements, one of the elements, denoted IS895, was shown to be mobile. The target site was not duplicated upon insertion of the element. Two other filamentous cyanobacterial strains were also found to contain sequences homologous to IS895.

Amino Acid Sequence↗

PubMed1991

Molecular typing of Shigella strains using pulsed field gel electrophoresis and genome hybridization with insertion sequences.

The genomes of 18 independent Shigella isolates (9 Shigella sonnei, 5 Shigella dysenteriae and 4 Shigella flexneri) as well as of 4 epidemic S. flexneri strains were analysed by pulsed field gel electrophoresis (PFGE) and by the distribution of insertion sequences (IS1, IS2 and IS911). Despite the close relatedness observed among the 9 independent S. sonnei, all of them could be differentiated from each other. The 4 independent S. flexneri isolates showed clearly distinguishable DNA profiles. Nearly complete genetic identity was detected within the 4 epidemic S. flexneri when analysed by PFGE or for IS1 and IS2 patterns. However, IS911 was found to be too mobile in these epidemic S. flexneri to be used as a typing probe. The 5 S. dysenteriae isolates could also be distinguished by the techniques used. The diversity found within this species is striking: of the 5 investigated isolates, 3 completely different DNA profiles were revealed. In conclusion, both PFGE and IS probing demonstrated their potential usefulness in molecular epidemiology and in typing of Shigella strains. The degree of differentiation given by these two methods was generally comparable, although IS probes showed better discrimination of the isolates.

Bacterial Typing Techniques↗