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PubMed2004

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis.

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.

Animals↗
PubMed1994

Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements.

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.

Amino Acid Sequence↗
PubMed2005

Generation of biologically active linear and cyclic peptides has revealed a unique fine specificity of rituximab and its possible cross-reactivity with acid sphingomyelinase-like phosphodiesterase 3b precursor.

Heterogeneity of the effector functions displayed by rituximab and other anti-CD20 monoclonal antibodies (mAbs) apparently recognizing the same CD20 epitope suggests that additional mechanisms, probably related to mAb fine specificity, are responsible for B-cell depletion. To improve our understanding of rituximab's function, its fine specificity was investigated by means of phage display peptide library (PDPL)-expressing 7-mer cyclic (c7c) or 7-/12-mer linear peptides. Rituximab-specific c7c PDPL-derived clone insert sequences expressed the motif A(S)NPS overlapping the human CD20 170ANPS173. P172 was the most critical for rituximab binding, since its replacement with S172 (of mouse CD20) abolished the reactivity. The WPXWLE motif expressed by the linear PDPL-derived clone insert sequences could only be aligned to the reverse-oriented 161WPXWLE156 of acid sphingomyelinase-like phosphodiesterase 3b precursor (ASMLPD), though linear peptides bearing WPXWLE competed with cyclic ones for rituximab-paratope binding. Anti-CD20 mAb 1F5 only displayed a reactivity profile similar to that of rituximab, which also reacted with ASMLPD-derived peptides. Peptides induced antibodies with specificity and effector functions similar to those of rituximab. Our results show a unique fine specificity of rituximab, define the molecular basis for the lack of rituximab reactivity with mouse CD20 (mCD20), and the potential of targeting CD20 in an active immunotherapy setting. A possible rituximab interaction with ASMLPD is suggested.

Amino Acid Motifs↗
PubMed1994

Isolation and characterization of IS31831, a transposable element from Corynebacterium glutamicum.

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS1096 from Mycobacterium smegmatis. Both IS31831 and IS1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.

Amino Acid Sequence↗
PubMed2000

Restriction fragment length polymorphism analysis of Mycobacterium avium isolates from animal and human sources.

Restriction fragment length polymorphism (RFLP) analysis using probes derived from the insertion sequences IS901, IS1245 and IS1311, was carried out on Mycobacterium avium isolates obtained from 18 human patients, 44 deer, 14 pigs and five cattle in the Republic of Ireland. Forty-two of the cervine isolates and two of the bovine isolates contained IS901, while this insertion sequence was absent from all of the human and porcine isolates. RFLP analysis with IS901 probe differentiated the 44 field isolates which contained this element into three types. All of the IS901-positive isolates had a characteristic three-band IS1245 hybridisation pattern and a characteristic single-band IS1311 hybridisation pattern. The IS901-negative isolates exhibited highly polymorphic IS1245 and IS1311 hybridisation patterns which differentiated the human and porcine isolates into a wide diversity of strain types.

Animals↗
PubMed1994

Differential distribution of amyloid protein precursor immunoreactivity in the rat brain studied by using five different antibodies.

The beta-amyloid or A4 protein is found deposited in neuritic plaques and neurofibrillary tangles in Alzheimer's disease (AD) affected brains and in the brains of adults with Down's Syndrome. The precursor to this 42 amino acid protein is the 695 amino acid long amyloid protein precursor (APP-695). Two additional APP species, APP-751 and APP-770, each contain a 56-amino-acid insert sequence that is analogous to Kunitz protease inhibitors. APP mRNA is widely distributed in both the human and rat brain, although the adult rat does not develop mature amyloid pathology. In this study we used antibodies against the N-terminus, junction site (unique to APP-695) insert sequence (unique to APP-751,-770), A4 region, and C-terminus of APP to immunolabel sections from throughout the young adult rat brain. From these results we constructed maps of the staining pattern of each antibody. We found that APP is widely distributed throughout the brain, that labelling is predominantly neuronal in character, and that there is marked variation among the antibodies in the extent of labelling, the particular cell populations stained, and the structures labelled within individual cells. The differential staining patterns observed with the five different antibodies suggest that the way APP is processed differs from one region to another and within different compartments in the cell. The specificity of the antibodies was established by Western blot analysis, in which APP species of approximately 95 and 110 kD were found. Our findings on the distribution of APP provide a foundation for further investigations into the normal role of APP and the pathogenesis of AD.

Amyloid beta-Protein Precursor↗
PubMed1982

Control of transposon Tn5 transposition in Escherichia coli.

Tn5 is a composite transposable element in which the insertion sequences IS50R and IS50L bracket a central region encoding kanamycin resistance (kanr). IS50R encodes a functional transposase, whereas IS50L contains the promoter of the kanr gene. To determine the relative activities of IS50R and IS50L in transposition we examined the structures of chimeric DNA molecules generated by insertion of segments of pBR322::Tn5 dimeric plasmids into red- lambda phage in recA- Escherichia coli. Restriction endonuclease analyses showed that the inserted sequences contained direct terminal repeats of pairs of IS50R or of IS50L elements and that the frequencies of usage of IS50R vs. IS50L depended on the position and orientation of Tn5 in the plasmid vector: IS50R was used preferentially when Tn5 was in transcriptionally quiescent regions of the vector (in either orientation) or when IS50L was immediately downstream from a strong promoter in the vector. In contrast, IS50L was used preferentially when IS50R was downstream from a strong promoter. We conclude IS50R tends to be used preferentially but that when transcription impinges on the end of an IS50 element the participation of that element in transposition is inhibited.

Base Sequence↗
PubMed2005

Intervening sequence acquired by lateral gene transfer in Tropheryma whipplei results in 23S rRNA fragmentation.

Completion of Tropheryma whipplei genome sequencing may provide insights into the evolution of the molecular mechanisms underlying the pathogenicity of this microorganism. The first postgenomic application was the successful design of a comprehensive culture medium that allows axenic growth of this bacterium, which is particularly recalcitrant to cultivation. This achievement in turn permitted analysis of T. whipplei RNA without contaminating eukaryotic nucleic acids. To obtain high-quality RNA, several extraction methods were compared, but under all conditions tested an atypical profile was observed. By using a Northern blot assay we demonstrated that an insertion sequence previously described in T. whipplei 23S rRNA is in fact an intervening sequence excised during maturation. This cleavage could involve an RNase III identified in the genome of this microorganism. Among the bacteria with a 23S rRNA insertion sequence, T. whipplei is the only gram-positive microorganism. We present phylogenetic evidence that this mobile genetic element was acquired by lateral gene transfer from another enteric bacterium.

Actinomycetales↗
PubMed2004

Host processing of branched DNA intermediates is involved in targeted transposition of IS911.

A simplified system using bacterial insertion sequence IS911 has been developed to investigate targeted insertion next to DNA sequences resembling IS ends. We show here that these IR-targeted events occur by an unusual mechanism. In the circular IS911 transposition intermediate the two IRs are abutted to form an IR/IR junction. IR-targeted insertion involves transfer of a single end of the junction to the target IR to generate a branched DNA structure. The single-end transfer (SET) intermediate, but not the final insertion product, can be detected in an in vitro reaction. SET intermediates must be processed by the bacterial host to obtain the final insertion products. Sequence analysis of these IR-targeted insertion products and of those obtained in vivo revealed high levels of DNA sequence conversion in which mutations from one IR were transferred to another. These sequence changes cannot be explained by the classic transposition pathway. A model is presented in which the four-way Holliday-like junction created by SET is processed by host-mediated branch migration, resolution, repair and replication. This pathway resembles those described for processing other branched DNA structures such as stalled replication forks.

DNA Transposable Elements↗
PubMed2000

Application of four molecular techniques for typing outbreak-associated Mycobacterium tuberculosis strains.

We applied four molecular techniques for the typing of strains of Mycobacterium tuberculosis associated with outbreaks: RFLP of the IS6110 insertion sequence, spoligotyping, RAPD, and PCR-IS6110. All 4 techniques were applied to 18 strains which were shown by epidemiological data to be involved in 6 outbreaks. All the methods classified the strains into the same groups as the classical epidemiological data did, but RFLP of the IS6110 insertion sequence and spoligotyping are laborious techniques requiring more than a full day's work, whilst RAPD and PCR IS6110 are simple methods easily incorporated into the daily routine. Nevertheless, a large-scale process of standardization and evaluation is necessary in order to be able to establish the true value of the latter two methods as intraspecific characterization markers for M. tuberculosis isolates.

Bacterial Typing Techniques↗
PubMed1990

Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression.

The yopE gene of Yersinia pseudotuberculosis was recently sequenced, and YopE was identified as an indispensable virulence determinant when tested in a mouse model (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). In the study described here, the DNA sequences of the yopE genes of Yersinia pestis EV76 and Yersinia enterocolitica 8081 were determined and compared with that of the Y. pseudotuberculosis gene. Only two codons were found to differ, both leading to amino acid replacements, when the gene from Y. pestis was compared. These two replacements were also present in the gene from Y. enterocolitica; in addition, 18 other codons were found to differ. Thirteen of these substitutions led to amino acid replacements. Downstream of the yopE gene, the plasmid partition locus par was found to be conserved in all three species. In Y. enterocolitica 8081, the sequence homology was interrupted by a putative insertion sequence element inserted between the yopE gene and the par region at a position only 5 base pairs downstream of the yopE stop codon. Upstream of the yopE gene, 620 base pairs were conserved in the three species. This region contained a 130-amino-acid-long open reading frame reading in the opposite direction to the yopE gene and expressed a 14-kilodalton protein in minicells. An insertion mutation in this region constructed in Y. pseudotuberculosis expressed significantly lower amounts of YopE protein in vitro than did the corresponding wild type. The expression level could be restored by transcomplementation. This new locus was designated yerA, for yopE-regulating gene A. The yerA mutant was avirulent when mice were challenged by oral infection.

Amino Acid Sequence↗
PubMed1998

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA.

At a time when optimal case ascertainment for meningococcal infection is a high priority, the need for non-culture case confirmation, in particular by DNA amplification, is seen as being of vital importance to assist contact management and cluster recognition. A solution hybridisation assay with colorimetric microtitre plate detection (polymerase chain reaction-enzyme-linked immunosorbent assay (PCR ELISA)¿ has been developed using the multicopy insertion sequence IS1106 which had reportedly achieved a specificity of 100% and was described as being meningococcal specific. This PCR ELISA assay was evaluated on specimens from over 5000 patients at the national Meningococcal Reference Unit (MRU) between late 1995 and early 1997 and was found to be highly sensitive. Insertion sequences, however, are genetically mobile with the ability to spread between species and even genera. During the evaluation period of the IS1106 PCR ELISA a number of false positives proved to be caused by organisms other than N. meningitidis were recorded resulting in the withdrawal of this assay as a front line screening assay for routine confirmation of meningococcal infection.

Bacteriological Techniques↗
PubMed1981

The nucleotide sequence and protein-coding capability of the transposable element IS5.

The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined. It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch. One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids. Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp. Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand). The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim.

Bacteriophage mu↗
PubMed1980

Plasmid cloning vectors that can be nicked at a unique site.

We describe ColEl-type plasmids, with relaxed DNA replication, based on pMB9, and carrying the CmR determinant of R1, in addition to the TcR determinant of pMB9. One of the plasmids, pPH207, has unique sites for EcoRI, HindIII, BamI, SalI and HpaI. Insertion of foreign DNA into all but the last of these inactivates either the CmR or the TcR determinant. The original CmR TcR plasmid (pCM2) contains a copy of IS1 which produces deletions to left and to right. Most of these inactivate either the CmR or the TcR determinant. An internal 280 bp deletion of IS1 DNA in pPH207 greatly reduces the frequency at which deletions are observed. The main feature of these plasmids is a site that is cleaved by some preparations of EcoRI in only one strand of the DNA duplex (the EcoRIn site). This site facilitates strand separation of sequences inserted at the HindIII, BamI and SalI sites of the TcR gene, and also of any inserted at the true EcoRI site by a method that destroys that site. Since the orientation of the EcoRIn site is known, the orientation of sequences inserted at the neighbouring sites can be easily determined. Plasmid pPH207 is not mobilised by a Hfr, but its mobilisation is promoted by ColEl. It is therefore Mob- bom+. Experiments with minicells show that it directs the copious synthesis of chloramphenicol transacetylase.

DNA Replication↗
PubMed1998

UGA codon position affects the efficiency of selenocysteine incorporation into glutathione peroxidase-1.

A UGA codon and a selenocysteine insertion sequence in the 3'-untranslated region are the only established mRNA elements necessary for selenocysteine (Sec or U) incorporation during translation. These two elements, however, do not universally confer efficient Sec incorporation. The objective of this study was to systematically examine the effect of UGA codon position on efficiency of Sec insertion. In a glutathione peroxidase-1 (F-GPX1) expression vector, the UGA at the native position (U47) was mutated to a cysteine codon, and codons for Ser-7, Ser-12, Ser-18, Ser-29, Ser-45, Ser-93, Cys-154, Val-172, Ser-178, and Ser-195 were individually mutated to UGA and transiently expressed in COS-7 cells. 75Se incorporation at the 11 positions was 31, 72, 54, 105, 90, 100, 146, 135, 13, 11, and 43%, respectively, of 75Se incorporation at U47, suggesting that Sec is more efficiently incorporated at UGA codons positioned in the middle of the coding region rather than close to the 5' or 3' ends. Ribonuclease protection showed that these differences were not due to differences in mRNA level. When the green fluorescence protein (GFP) coding region was placed in-frame at the 5' or 3' ends of the coding region in F-GPX1 to produce chimeric 50-51-kDa GFP/GPX1 proteins, Sec incorporation at UGA codons, formerly close to the 5' or 3' ends, was increased to levels comparable to the UGA at U47. Insertion of GFP after the UAA-stop was just as effective in increasing Sec insertion efficiency as GFP inserted before the stop. These studies used a recombinant expression model that incorporated Sec at non-native UGA codons at rates equal to those of endogenous glutathione peroxidase-1 and showed that the efficiency of Sec incorporation can be modulated by UGA position; Sec incorporation at high efficiency appears to require that the UGA be >21 nucleotides from the AUG-start and >204 nucleotides from the selenocysteine insertion sequence element.

3' Untranslated Regions↗
PubMedDate not supplied

Sequence-specific insertion of the Drosophila transposable genetic element 17.6.

As in the case of retrovirus proviruses, most of the Drosophila copia-like transposable elements so far examined are bounded by 5'TG...CA3' and inserted into the chromosome without obvious site-specificity. In the other copia-like elements, 297, HMS Beagle and 17.6 (refs 5-7), terminal dinucleotides (5'TG...CA3') are completely absent and, instead, 5'(A)GT is present at least at one of the termini. One important feature of these three elements may be frequent insertion into 'TATA' boxes, since the three of four insertion sites so far examined were TATA boxes, two for H3 histone genes and one for a cuticle gene. Because of the importance of this type of insertion, we extensively analysed site-specificity using 17.6 as a model. Our results, described here, suggest that insertion of 17.6 takes place in a site-specific fashion, using a target 5'ATAT corresponding to the major portion of the consensus TATA box, TATAATAAT.

Animals↗
PubMed2004

The control of copy number of IS6110 in Mycobacterium tuberculosis.

Insertion sequence (IS) elements are bacterial genes that are able to transpose to different locations in the genome. These elements are often used in molecular epidemiology as genetic markers that track the spread of pathogens. Transposable elements have frequently been described as "selfish DNA" because they facilitate their own transposition, causing damage when they insert into coding regions, while contributing little if anything to the bacterial host. According to this hypothesis, the expansion of copy number of insertion sequences is opposed by negative selection against high copy numbers. From an alternative point of view, we might expect IS elements to intrinsically regulate transposition within cells, thereby limiting damage to their bacterial host. Here, we report evidence that the copy number of IS6110 in Mycobacterium tuberculosis is controlled by selection against the element. We first construct 12 different models of marker change resulting from a combination of possible transposition functions and selective regimes. We then compute the Akaike Information Criterion for each model to identify the models that best explain data consisting of serial isolates of M. tuberculosis genotyped with IS6110. We find that the best performing models all include selection against the accumulation of copies. Specifically, our analysis points to the interaction of separate copies of the element causing lethal effects. We discuss the implications of these findings for genome evolution and molecular epidemiology.

DNA Transposable Elements↗