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PubMed1995

A novel insertion sequence (IS)-like element of the thermophilic bacterium PS3 promotes expression of the alanine carrier protein-encoding gene.

A novel insertion sequence (IS)-like element was found in the 5'-upstream region of the alanine carrier protein-encoding gene (acp) in the thermophilic bacterium PS3 chromosomal DNA. The sequence contained an open reading frame (ORF) encoding a polypeptide of 369 amino acids which revealed high similarity with ORFs from IS891 from the cyanobacterium Anabaena and IS1136 from Saccharopolyspora erythraea. The direction of transcription was the same as that of acp, and typical inverted and direct repeats characteristic of IS were found in both the 5' and 3' region of the ORF. Southern hybridization analysis of the chromosomal DNA revealed that multiple copies of the ORF sequence were contained in the PS3 genome. This element might well be a member of a new IS family including IS891 and IS1136, and we have designated this element IS1341. The analysis of acp expression in Escherichia coli cells indicated that IS1341 promotes the expression of acp.

Amino Acid Sequence↗

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↗

PubMed1994

Isolation and characterization of IS1181, an insertion sequence from Staphylococcus aureus.

The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.

Amino Acid Sequence↗

PubMed1994

The pigmentation locus of Yersinia pestis KIM6+ is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2.

The pigmentation (Pgm+) phenotype of Yersinia pestis includes a number of different characteristics which appear to be associated with a 102 kb segment of chromosomal DNA known as the pgm locus. In Y. pestis KIM6+, the pgm locus is flanked by direct copies of a repeated element that probably plays a role in the spontaneous deletion of this region. We have sequenced the ends of these elements and shown that they have features in common with bacterial insertion sequences. In addition we show that a clone, pSDR498, from the pgm locus of KIM6+ restores pesticin sensitivity and the iron-regulated expression of three polypeptides, 240 kDa, 190 kDa, and 68 kDa in size, to Pgm- cells. In vitro transcription/translation assays and Escherichia coli minicells were used to analyse the products encoded by various subclones of pSDR498. Pesticin sensitivity mapped to a 5.9 kb fragment that encodes a 68 kDa protein derived from a 72 kDa precursor. Synthesis of the 190 kDa protein was restored by a 19.2 kb clone, indicating that the structural gene for this protein also resides within the pgm locus of Y. pestis KIM6+. Finally, a survey of our Pgm- strains indicates that 97% have also deleted the sequences encoding the 190 kDa protein and pesticin sensitivity.

Bacterial Proteins↗

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↗

PubMed1994

Molecular characterization of catalase from Bordetella pertussis: identification of the katA promoter in an upstream insertion sequence.

In this report we evaluate the role of catalase in the survival of Bordetella pertussis within human polymorphonuclear leukocytes (PMNs). Crude extracts of B. pertussis exhibited a single catalase activity when subjected to non-denaturing polyacrylamide gel electrophoresis and assayed for catalase activity. A plasmid containing B. pertussis katA was identified by complementation of UM255, a catalase-deficient strain of Escherichia coli. The nucleotide sequence of katA predicts a 55 kDa protein that shares homology with a class of haem-containing catalases found in both eubacteria and eukaryotes. Analysis of the nucleotide sequence upstream of katA revealed the presence of a copy of IS481, a B. pertussis-specific insertion sequence. The start site of transcription of katA was mapped to a T residue in IS481 by primer extension analysis performed with B. pertussis RNA and a katA-specific primer. A catalase-deficient strain of B. pertussis, DD900, was constructed by gene replacement. DD900 was more sensitive to killing by 1 and 5 mM H2O2 than the parental strain, BP339. However, there was no difference in the ability of DD900 and BP339 to survive for 2 h in human PMNs. This suggests that catalase plays no significant role in the survival of B. pertussis within PMNs.

Base Sequence↗

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↗