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PubMed1999

The first detection of the insertion sequence ISW1 in the intracellular reproductive parasite Wolbachia.

Wolbachia are maternally inherited intracellular rickettsia-like bacteria known to infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis, and feminization. We report on a novel insertion sequence (IS), ISW1, which was identified in the region downstream of groEL of a Wolbachia strain, wTai. The 573-bp-long ISW1 sequence is the first IS element observed in this organism, displays significant similarity to IS200, and lacks terminal inverted repeats. There were more than 20 copies of ISW1 on the chromosome of wTai. Sequence analysis of nine distinct ISW1 copies and their flanking regions showed that the copies were identical and suggested that ISW1 has no preference for its insertion sites. Possible roles of ISW1 in the adaptation of Wolbachia to intracellular environments and in various reproductive alterations caused by this bacterium are discussed.

Amino Acid Sequence↗

PubMed2001

Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2.

The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3' untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

3' Untranslated Regions↗

PubMed2002

Frequency of the insertion sequence IS4Bsu1 among Bacillus subtilis strains isolated from fermented soybean foods in Southeast Asia.

Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome. In contrast, none of 49 B. subtilis strains of non-food origin contained IS4Bsu1. Frequent occurrence of this mobile DNA element in the soybean-fermenting B. subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.

Asia, Southeastern↗

PubMed1998

The nature of the minimal 'selenocysteine insertion sequence' (SECIS) in Escherichia coli.

The UGA codon, usually a stop codon, can also direct the incorporation into a protein of the modified amino acid selenocysteine. This UGA decoding process requires a cis -acting mRNA element called 'selenocysteine insertion sequence' (SECIS) that can form a stem-loop structure. In Escherichia coli the SECIS of the selenoprotein formate dehydrogenase (FdhH) mRNA has been previously described to consist of at least 40 nucleotides following the UGA codon. Here we determined the nature of the minimal SECIS required for the in vivo UGA-directed selenocysteine incorporation in E.coli . Our study is based on extensive mutational analysis of the fdhF SECIS DNA located in a lac' Z fusion. We found that the whole stem-loop RNA structure of the E.coli fdhF SECIS previously described is not required for the UGA-directed selenocysteine incorporation in vivo . Rather, only its upper stem-loop structure of 17 nucleotides is necessary on the condition that it is located in a proper distance (11 nucleotides) from the UGA codon. Based on these observations, we present a new model for the minimal E.coli SECIS.

Base Sequence↗

PubMed1995

The Corynebacterium xerosis composite transposon Tn5432 consists of two identical insertion sequences, designated IS1249, flanking the erythromycin resistance gene ermCX.

Analysis of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium xerosis M82B revealed that the erythromycin resistance gene, ermCX, is located on a 4524-bp composite transposable element, Tn5432. The ends of Tn5432 are identical, direct repeats of an insertion sequence, designated IS1249, encoding a putative transposase of the IS256 family. IS1249 consists of 1385 bp with 45/42 imperfect terminal inverted repeats. The nucleotide sequence of the 1754-bp Tn5432 central region is 99% identical to the previously sequenced erythromycin resistance region of the Corynebacterium diphtheriae plasmid pNG2. It encodes the erythromycin resistance gene, ermCX, and an ORF homologous to the amino-terminal end of the transposase of IS31831 from Corynebacterium glutamicum. Transposons with regions flanking the insertion sites were recovered from the C. glutamicum chromosome by a plasmid rescue technique. Insertion of Tn5432 created 8-bp target site duplications. A Tn5432-induced isoleucine/valine-auxotrophic mutant was found to carry the transposon in the 5' region of the ilvBNC cluster; in pTP10 the transposon is inserted in a region similar to replication and partitioning functions of the Enterococcus faecalis plasmid pAD1 and the Agrobacterium tumefaciens plasmid pTAR.

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↗

PubMed1991

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors.

On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.

Blotting, Southern↗

PubMed2004

A novel mitochondrial DNA-like sequence insertion polymorphism in Intron I of the FOXO1A gene.

The human forkhead box O1A (FOXO1A) gene belongs to the human forkhead gene family and acts downstream of the human insulin signalling pathway. In this study, polymorphisms of the Intron I of FOXO1A gene were studied in Italian healthy people and insulin resistant subjects. No significant association between the germ-line variability in the Intron I of FOXO1A and insulin resistance was observed. Interestingly, during the study, a new 39-bp sequence insertion polymorphism in Intron I of FOXO1A gene was described. The polymorphism was found to co-segregate in a co-dominant Mendelian fashion and to be present in an ethnically distinct population (Greeks). A BLAST search showed that the sequence shares 100% identity with a mtDNA (mitochondrial DNA) sequence coding for the ATP synthase 8 (ATPase8) and ATP synthase 6 (ATPase6) genes. Hence, FOXO1A Intron I is a polymorphic nuclear region involved in the exchange of DNA material between mitochondrial and genomic DNA, which is a well-established mechanism of evolutionary change in eukaryotes.

Adenosine Triphosphatases↗

PubMed2006

Insertion sequence ISEcp1-like element connected with a novel aph(2'') allele [aph(2'')-Ie] conferring high-level gentamicin resistance and a novel streptomycin adenylyltransferase gene in Enterococcus.

Enterococcus casseliflavus HZ95 is an enterococcus with high-level resistance to aminoglycosides. Nine genes responsible for high-level aminoglycoside resistance, including aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id, aph(3')-IIIa, aac(6')-Ii, ant(3')-Ia, ant(4')-Ia and ant(6')-Ia, were not detected in HZ95. An 8 kb fragment from unconjugative plasmids of HZ95 was cloned, and expressed gentamicin resistance in Escherichia coli DH5alpha. The genetic structures ( approximately 8 kb DNA fragment) containing these aminoglycoside-modifying enzyme genes in Ent. casseliflavus HZ95 were determined. The deduced amino acid sequence of the novel aph(2'') allele, aph(2'')-Ie, had 93.7 % amino acid identity with APH(2'')-Id. The aph(2'')-Ie gene was bracketed upstream by an insertion sequence (IS)Ecp1-like element and downstream by a streptomycin adenylyltransferase gene (str). The streptomycin adenylyltransferase encoded by the str gene had 80.3 % amino acid identity with the protein encoded by aadE. The plasmid of approximately 16 kb could hybridize with a PCR-generated aph(2'')-Ie intragenic probe. The ISEcp1-like element had 91 % identity with ISEcp1. ISEcp1, which commonly acts as a key factor in the dissemination of CTX-M-type beta-lactamase genes in Gram-negative bacteria, has not been reported in Enterococcus.

Alleles↗

PubMed1997

The Deinococcus radiodurans uvr A gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria.

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.

Adenosine Triphosphatases↗

PubMed2004

Molecular basis of the indole-negative reaction in Shigella strains: extensive damages to the tna operon by insertion sequences.

The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of Shigella strains, in effect clones of Escherichia coli, was investigated. Analysis of the tna operon of 23 Shigella strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the tna operon, leading to inability to convert tryptophan to indole. These events differ for cluster 1, cluster 3, and the outlier Shigella strains, confirming our previous observation of independent origins of these lineages from within E. coli. Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in Shigella strains and advantages those without it.

DNA Transposable Elements↗

PubMed2000

The selenocysteine insertion sequence binding protein SBP is different from the Y-box protein dbpB.

In eukaryotes, translation of internal UGA selenocysteine codons requires the SECIS stem-loop structure in the 3'UTR of selenoprotein mRNAs. In an earlier work, we identified SBP as a selenocysteine insertion sequence (SECIS)-binding protein. Here, the yeast three-hybrid screen was employed to capture the cDNA of SBP. One candidate, satisfying the genetic screens, was identified as the already known dbpB protein. Although it was also found by another group, but with a different strategy, to carry SECIS-binding activity, further experiments enabled us to show that dbpB was unable to bind the SECIS element in vitro. Altogether, our findings led us to conclude that, under our conditions, dbpB and SBP are two distinct proteins.

Animals↗

PubMed2000

The bulged nucleotide in the Escherichia coli minimal selenocysteine insertion sequence participates in interaction with SelB: a genetic approach.

The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB.

Bacterial Proteins↗

PubMed1994

Possible role of insertion sequence IS257 in dissemination and expression of high- and low-level trimethoprim resistance in staphylococci.

The transposon-like structure Tn4003 and related elements were found to encode high- and low-level trimethoprim resistance (Tpr) in Staphylococcus aureus and coagulase-negative staphylococci. By using transcriptional fusions in Escherichia coli, the variation in resistance levels was found to correlate with the transcriptional activity of the region presumed to carry the promoter for the operon containing the Tpr dihydrofolate reductase gene, dfrA, encoded by these elements. The reduced transcriptional activities exhibited by elements encoding low-level Tpr appear to be a consequence of deletions adjacent to the copy of IS257 which normally encodes the -35 sequences of these promoters. The data obtained not only support the involvement of IS257 in the transcription of the proposed thyE-dfrA-orf-140 operon of Tn4003 but may also implicate this insertion sequence in the mechanisms resulting in the variation in Tpr levels observed in staphylococci.

Base Sequence↗

PubMed2004

Distribution of insertion sequences associated with Tn1546-like elements among Enterococcus faecium isolates from patients in Korea.

The vanA gene cluster is carried as a part of Tn1546-like elements. The genetic diversity in Tn1546-like elements has been documented previously. The differences described thus far have included the integration of insertion sequence (IS) elements IS1216V, IS1251, IS1476, and IS1542. Among these, IS1216V has been reported to be widespread in VanA enterococci of diverse geographic areas, whereas IS1542 and IS1476 have been reported only in the United Kingdom and Canada, respectively. We investigated the distribution of ISs among 20 vanA-containing Enterococcus faecium isolates from human patients in nine different university hospitals in Korea. Pulsed-field gel electrophoresis (PFGE) was performed to identify the clonality of the isolates. Moreover, PCR amplification of the internal regions of Tn1546 was performed for structural analysis of the van gene, and both DNA strands of the PCR amplicons were directly sequenced by the dideoxy termination method. The PFGE patterns revealed a high degree of clonal diversity. Structural analyses of the van gene detected IS1542 and IS1216V in the genomes of all 20 isolates, whereas it did not detect IS1476 or IS1251 in the genomes of any of the isolates. In addition, IS19 was detected in the vanS-vanH intergenic region of one isolate. These data indicate that identification of the IS within a vanA gene cluster could be a useful tool in epidemiological investigations. In addition, the distribution of ISs associated with Tn1546-like elements among the Korean isolates is therefore similar to that among European vancomycin-resistant enterococci.

Bacterial Proteins↗

PubMed2001

Characterization of the common nodulation genes of the photosynthetic Bradyrhizobium sp. ORS285 reveals the presence of a new insertion sequence upstream of nodA.

We isolated and characterized nodA genes from photosynthetic and non-photosynthetic rhizobia nodulating the legume genus Aeschynomene, and found that the nodA sequence from photosynthetic stem-nodulating bacteria was phylogenetically distant from the other already described nodA genes. Characterization of the photosynthetic strain ORS285 common nod gene cluster (nodABC) showed, upstream of nodA, the presence of a new insertion sequence element belonging to the IS3 family and specific to a group of photosynthetic strains from Aeschynomene.

Acyltransferases↗

PubMed1990

Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus.

Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients [Lo et al., Am. J. Trop. Med. Hyg. 41 (1989) 364-376; 601-616], contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements. This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini. Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element. Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3. In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M. incognitus. This potentially transposable element has many characteristic features in common with bacterial IS elements.

Amino Acid Sequence↗

PubMed1977

Heteroduplex mapping of small plasmids derived from R-factor R12: in vivo recombination occurs at IS1 insertion sequences.

Small, autonomously replicating plasmids derived by in vivo recombination from R-factor R12 (= R100) have been structurally mapped by heteroduplex formation between the plasmids and an R-factor which is structurally closely related to R6-5. Recombination resulting in generation of the small resistance-free plasmids occurs between the (IS1)b insertion sequence and various other sites on the opposite side of an origin of replication. A larger R12-derived plasmid pSM17, carrying streptomycin (Sm), sulfadiazole (Sa), and chloramphenicol (Cm) resistances, has recombined in a similar manner but at the (IS1)a sequence. A new structural coordinate origin for R100 and for partially homologous R-factors is proposed based upon the location of the (IS1)b sequence.

DNA, Bacterial↗