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PubMed1994

IS1139 from Streptococcus salivarius: identification and characterization of an insertion sequence-like element related to mobile DNA elements from gram-negative bacteria.

An insertion sequence-like element, IS1139, was cloned and sequenced from Streptococcus salivarius ATCC 25975 chromosome. This insertion sequence-like element is 1168 bp long and is delimited by inverted repeats of 29 bp and by a duplicated sequence of 6 bp. This IS possesses an open reading frame that codes for a putative transposase of 339 amino acids which has, respectively, 94, 35, 33, and 30% amino-acid identity with the transposases of IS1161 from S. salivarius ATCC 25975, IS4351 from Bacteroides fragilis, IS30 from Escherichia coli, and IS1086 from Alcaligenes eutrophus. Sequence analysis revealed that these transposases may have evolved from a common ancestral gene. Southern hybridization of restriction endonuclease-digested genomic DNA from 21 strains of oral streptococci, using a probe specific to the transposase-encoding gene (tnpA), revealed that IS1139 is found in two strains of S. salivarius, ATCC 25975 and ATCC 13419, in eight and two copies, respectively.

Alcaligenes↗

PubMed1998

The tdcE gene in Escherichia coli strain W3110 is separated from the rest of the tdc operon by insertion of IS5 elements.

Unlike other Escherichia coli K-12 strains, W3110 contains multiple copies of the insertion sequence IS5. Some of these IS5 elements have been involved in tandem duplication of a portion of the chromosome which includes, amonst others, the tdcABC-DEFG operon genes. The nucleotide sequence and insertion site of one of these elements, IS5P, was determined. It was shown that IS5P has inserted within the coding sequence of the tdcA gene and is flanked, not by the remaining portion of the tdcA gene, but by the extreme 3' end of the tdcD gene. In other E. coli K-12 strains the tdcD gene and three other genes, tdcE, tdcF and tdcG, all form part of the tdc operon. Our results demonstrate that during the duplication event the tdcABCgenes have been amplified and separated from the remaining genes tdcE, tdcF and tdcG of the operon, which are each present in single copy.

Acyltransferases↗

PubMed1999

A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells.

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.

Fungal Proteins↗

PubMed1996

Characterization of an insertion sequence-like element identified in plasmid pCIT264 from Lactococcus lactis subsp. lactis biovar diacetylactis.

Plasmid pCIT264 from Lactococcus lactis subsp. lactis biovar diacetylactis (L. diacetylactis) contains an insertion sequence (IS)-like element located in the citrate utilization (citQRP) cluster. This 967-nucleotide long element is bounded by 17 bp perfect inverted repeats and contains an open reading frame (ORF1) composed of 296 codons, which could encode a transposase. Expression of the IS from pCIT264 generates two mRNAs of 2900 and 1900 nucleotides. The transcription is driven by the P3 promoter, composed of a -10 region located at the right end of the IS and of a -35 region positioned downstream of this element. The IS-like element (IS982) is present in seven copies in the L.diacetylactis genome. The copy present in pCIT264 is highly stable and does not promote rearrangements of the cit cluster. We suggest that the stable maintenance of the IS-like element in pCIT264 could be due to a translational control of the putative transposase by an antisense RNA.

Base Sequence↗

PubMed1984

Small fragments of herpesvirus DNA with transforming activity contain insertion sequence-like structures.

A 737-base-pair fragment of herpes simplex virus type 2 DNA with morphological-transforming ability was identified by transfecting into rodent cells deleted fragments of the left-hand end of the Bgl II N fragment region (map position 0.58-0.625), which were constructed in vitro. The transforming sequences lie within the coding region for a Mr 61,000 protein, but the fragment itself does not appear to specify a viral polypeptide. Contained within the transforming fragment are sequences that can be drawn as a stem-loop structure flanked by direct repeats, similar to an insertion sequence-like element. An insertion sequence-like structure was also found in a small fragment of human cytomegalovirus DNA that has transforming activity. Possible mechanisms of herpesvirus transformation are discussed.

Animals↗

PubMed2003

DNA ligase IV from a basidiomycete, Coprinus cinereus, and its expression during meiosis.

DNA ligase IV is thought to be involved in DNA double-strand break repair and DNA non-homologous end-joining pathways, but these mechanisms are still unclear. To investigate the roles of DNA ligase IV from a biologically functional viewpoint, the authors studied its relationship to meiosis in a basidiomycete, Coprinus cinereus, which shows a highly synchronous meiotic cell cycle. The C. cinereus cDNA homologue of DNA ligase IV (CcLIG4) was successfully cloned. The 3.2 kb clone including the ORF encoded a predicted product of 1025 amino acid residues with a molecular mass of 117 kDa. A specific inserted sequence composed of 95 amino acids rich in aspartic acid and glutamic acid could be detected between tandem BRCT domains. The inserted sequence had no sequence identity with other eukaryotic counterparts of DNA ligase IV or with another aspartic acid and glutamic acid rich sequence inserted in C. cinereus proliferating cell nuclear antigen (CcPCNA), although the length and the percentages of aspartic and glutamic acids were similar. In addition, the recombinant CcLIG4 protein not only showed ATP-dependent ligase activity, but also used (dT)(16)/poly(dA) and (dT)(16)/poly(rA) as substrates, and had double-strand ligation activity, like human DNA ligase IV. Northern hybridization analysis and in situ hybridization indicated that CcLIG4 was expressed not only at the pre-meiotic S phase but also at meiotic prophase I. Intense signals were observed in leptotene and zygotene. Based on these observations, the possible role(s) of C. cinereus DNA ligase IV during meiosis are discussed.

Alkylating Agents↗

PubMed1989

Analysis of separate isolates of Bordetella pertussis repeated DNA sequences.

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.

Base Sequence↗

PubMed1987

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris.

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.

Animals↗

PubMed1994

Molecular analysis of the rfb gene cluster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains.

The Salmonella enterica O antigen is a highly variable surface polysaccharide composed of a repeated oligosaccharide (the O unit). The O unit produced by serogroup D2 has structural features in common with those of groups D1 and E1, and hybridization studies had previously suggested that the D2 rfb gene cluster responsible for O-unit biosynthesis is indeed a hybrid of the two. In this study, the rfb gene cluster was cloned from a group D2 strain of S. enterica sv. Strasbourg. Mapping, hybridization, and DNA sequencing showed that the organization of the D2 rfb genes is similar to that of group D1, with the alpha-mannosyl transferase gene rfbU replaced by rfbO, the E1-specific beta-mannosyl transferase gene. The E1-specific polymerase gene (rfc) has also been acquired. Interestingly, the D1-like and E1-like rfb regions are separated by an additional sequence closely related to an element (Hinc repeat [H-rpt]) associated with the Rhs loci of Escherichia coli. The H-rpt resembles an insertion sequence and possibly mediated the intraspecific recombination events which produced the group D2 rfb gene organization.

Amino Acid Sequence↗

PubMed1991

Effect of RNA secondary structure on polyadenylation site selection.

Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. In agreement with previous results, random sequence insertions between the AAUAAA and G/U box signals were observed to inhibit poly(A) site function. However, sequence insertions of similar size that were predicted to form RNA stem-loop structures were found to have little effect on the efficiency of polyadenylation and instead induced a 3' shift in the site of polyadenylation that was equal to the length of the inserted stem-loop. The in vivo utilization of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by mutations that destabilized the predicted stem but was restored by compensatory mutations. These results strongly support the hypothesis that the appropriate spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. Sequence insertions that are able to form RNA secondary structures that maintain the correct spacing of these two RNA target sequences are well tolerated, whereas sequence insertions that disturb this spacing inhibit poly(A) site recognition. It is proposed that the effect of sequence insertions on poly(A) site function may be sufficiently predictable to allow the development of an assay for in vivo RNA secondary structure that uses poly(A) site selection as a readout.

Animals↗

PubMed1996

Transcriptional activation of the citrate permease P gene of Lactococcus lactis biovar diacetylactis by an insertion sequence-like element present in plasmid pCIT264.

The lactococcal plasmid pCIT264 contains a cluster of three genes (citQ, citR and citP) involved in the transport of citrate in Lactococcus lactis biovar diacetylactis. The cit cluster contains a copy of a newly discovered insertion sequence (IS)-like element located between its promoter P1 and the first gene of the cluster. In this report, we show that this IS-like element can act as a mobile switch for the downstream genes, creating two new transcriptional promoters named P2 and P2'. The P2 promoter is recognized by the lactococcal RNA polymerase in vivo. This is a hybrid promoter composed of a -35 region reading outwards 12bp from the right end of the IS-like element, and a nucleotide sequence from the recipient plasmid, adjacent to the element, which provides an appropriately spaced -10 region. Transcription of the citQRP cluster from this promoter takes place during the exponential and stationary phases of growth in L. lactis. Promoter P2' is included in the IS-like element and is the only promoter responsible for expression of citP in E. coli. Thus, it appears that the introduction of this element into pCIT264 allows expression of the citQRP cluster in E. coli, and increases its levels of expression in L. lactis.

Bacterial Proteins↗

PubMed1985

Bombyx mori 28S ribosomal genes contain insertion elements similar to the Type I and II elements of Drosophila melanogaster.

We have examined the 28S ribosomal genes of the silkmoth, Bombyx mori, for the presence of insertion sequences. Two types of insertion sequences were found, each approximately 5 kb in length, which do not share sequence homology. Comparison of the nucleotide sequences of the junction regions with the uninserted gene reveals that one type of insertion has resulted in a 14 bp duplication of the 28S coding region at the insertion site. The location of this insertion and the 14 bp duplication are identical to that found in the Type I ribosomal insertion element of Drosophila melanogaster. The second type of insertion element is located at a site corresponding to approximately 75 bp upstream of the first type. The location of this insertion, the variability detected at its 5' junction, and a short region of sequence homology at its 3' junction suggest that it is related to the Type II element of D. melanogaster. This is the first example of a Type II-like rDNA insertion outside of sibling species of D. melanogaster, and the first example of a Type I-like rDNA insertion outside of the higher Diptera.

Animals↗

PubMed1997

Identification and characterization of IS1476, an insertion sequence-like element that disrupts VanY function in a vancomycin-resistant Enterococcus faecium strain.

The vanY gene of vancomycin-resistant enterococci encodes a D,D-carboxypeptidase. By using a PCR detection strategy, a VanA Enterococcus faecium clinical isolate was found to have an insertion sequence (IS)-like element designated IS1476 in vanY. The activity of the VanY D,D-carboxypeptidase in this isolate was decreased in a fluorometric fluoraldehyde o-phthalaldehyde assay with diacetyl-L-Lys-D-Ala-D-Ala as the substrate. This, to our knowledge, is the first report of an IS-like element in a vancomycin resistance gene.

Anti-Bacterial Agents↗

PubMed1988

The human docking protein does not associate with the membrane of the rough endoplasmic reticulum via a signal or insertion sequence-mediated mechanism.

Docking protein (DP, or SRP receptor) is an essential component of the cellular machinery that mediates the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (ER). In this study we have investigated the nature of its own targeting to its site of function, the rough ER. Using an in vitro transcription-translation system we demonstrate that DP is not inserted into the membrane via a classical SRP/DP-mediated process (in contrast to human ribophorins), nor via hydrophobic insertion sequences (in contrast to cytochrome b5). Instead, we suggest that membrane assembly of DP is receptor-mediated; requiring the presence in the membrane of other proteins that mediate its targeting and insertion.

Animals↗

PubMed2006

Vibrio cholerae CTXPhi phage repressor gene is borne on an insertion sequence-like element and encodes a putative transposase.

Bioinformatics analyses show the presence of a novel insertion sequence-like element in the CTXPhi phage of Vibrio cholerae. The solitary open reading frame encompassed by the element is known to encode a repressor (RstR) of phage DNA replication and is responsible for phage heteroimmunity. Analysis of the nucleotide and protein sequence of the repressor and its flanking non-coding regions indicates that it resembles distinctly simple bacterial insertion elements in numerous aspects. Based on the results of extensive sequence analysis, we propose that the rstR gene is borne on an insertion sequence-like element and that the RstR protein encodes a putative transposase. We put forward the possibility that a bi-directional promoter could regulate the divergent transcription of the neighbouring genes of rstR and rstA. The possibility of overlapping reading frames yielding distinct repressor and transposase proteins is also expanded based on the analyses.

Amino Acid Sequence↗

PubMed2000

FK506 binding protein from a thermophilic archaeon, Methanococcus thermolithotrophicus, has chaperone-like activity in vitro.

The in vitro protein folding activity of an FKBP (FK506 binding protein, abbreviated to MTFK) from a thermophilic archaeon, Methanococcus thermolithotrophicus, was investigated. MTFK exhibited FK506 sensitive PPIase (peptidyl prolyl cis-trans isomerase) activity which accelerated the speed of ribonuclease T1 refolding, which is rate-limited by isomerization of two prolyl peptide bonds. In addition, MTFK suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. We called this activity of MTFK the chaperone activity. The chaperone activity of MTFK was also inhibited by FK506. Alignment of the amino acid sequences of MTFK with human FKBP12 showed that MTFK has two insertion sequences, consisting of 13 and 44 amino acids, at the N- and C-termini, respectively [Furutani, M., Iida, T., Yamano, S., Kamino, K., and Maruyama, T. (1998) J. Bacteriol. 180, 388-394]. To study the relationship between chaperone and PPIase activities of MTFK, mutant MTFKs with deletions of these insertion sequences or with amino acid substitutions were created. Their PPIase and chaperone activities were measured using a synthetic oligopeptide and denatured rhodanese as the substrates, respectively. The far-UV circular dichroism spectra of the wild type and the mutants were also analyzed. The results suggested that (1) the PPIase activity did not correlate with chaperone activity, (2) both insertion sequences were required for MTFK to take a proper conformation, and (3) the insertion sequence (44 amino acids) in the C-terminus was important for the chaperone activity.

Binding Sites↗

PubMed1982

A transposable element from Halobacterium halobium which inactivates the bacteriorhodopsin gene.

We describe the characterization of a transposable element from an archaebacterium. The bacteriorhodopsin genes from the wild-type and two mutant Halobacterium halobium strains have been cloned as BamHI fragments in pBR322. The cloned DNA fragments from the two mutants both contain a 1.1-kilobase-pair insertion sequence (ISH1) near the NH2 terminus of the bacteriorhodopsin coding sequence. ISH1 is present in the two mutants in an identical palindromic site but in opposite orientations. The complete sequence of ISH1 has been determined; it is 1,118 nucleotides long, it has 8-base-pair interrupted inverted repeats at the ends, and it duplicates an 8-base-pair (A-G-T-T-A-T-T-G) target sequence upon insertion. As for most eukaryotic and some prokaryotic transposable elements, the sequence of the ISH1 begins with T-G and ends in C-A. ISH1 contains an open reading frame 810 nucleotides long and codes for an RNA approximately 900 nucleotides long. The copy number of ISH1 ranges from one to five or more in different H. halobium strains. In at least one of the strains, one copy of ISH1 is present also on a plasmid DNA.

Bacteriorhodopsins↗

PubMed1994

Insertion sequence-related genetic variation in resting Escherichia coli K-12.

Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.

Biological Evolution↗