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PubMed1983

Topological modifications and template activation are induced in chimaeric plasmids by inserted sequences.

The effect of the insertion of foreign genes or gene systems in closed DNA domains has been investigated in vitro in purified systems. We observe that in chimaeric plasmids two apparently independent classes of modifications, (1) functional and (2) topological, do take place in defined instances. (1) Among the screened yeast gene systems, examples have been found of DNA sequences that upon insertion cause activation of in vitro transcription of distant genes. (2) Foreign DNA sequences may lead to new topological features of the harbouring plasmids; it is shown that more than one S1-sensitive secondary structure may be contemporaneously present on the same chimaeric plasmid. DNA superhelicity is a prerequisite of these modifications. The two classes of effects (1) functional and (2) topological are not a priori directly related one to the other but appear to be two independent consequences of the same cause: the insertion of foreign DNA sequences into closed DNA domains. These observations suggest a regulatory model of gene expression based on alternative topologies of closed DNA domains.

Chimera↗

PubMed1979

The occurrence of long transcription units among the X and Y ribosomal genes of Drosophila melanogaster: transcription of insertion sequences.

Most of the ribosomal transcription units (rTUs) in Drosophila melanogaster observed by electron microscopy measure about 8 kb; a length which corresponds to the size of the 38S precursor to ribosomal RNA in D. melanogaster. However, interspersed among these rTUs are transcription units that are much longer (up to 14.6 kb) than the 8 kb expected for rTUs. Some of these larger length estimates can be attributed to stretching but an important fraction is significantly larger and has up to 60 more fibers per gene.--The following evidence suggests that these larger transcription units are ribosomal genes consisting of insertion sequences. The long transcription units are within the sizes expected for rTUs containing insertion sequences as reported by other workers. Their RNP fibers cross-react with antibodies raised against ribosomal proteins in a manner similar to that observed for ribosomal RNP. They are interspersed among rTUs in the X chromosome.--These putative ribosomal genes carrying insertions are present both in the X and, although to a lesser extent, in the Y ribosomal chromatin as is indicated by their existence in nurse cells of both Oregon R females and females of the genotype sc4sc8/sc4sc8/y+ Y. Analysis of the fiber patterns of "long TUs" supports the hypothesis that the insertion region is being transcribed.--"Long TUs" are found in tandem with non-transcribed spacer regions which are heterogeneous in length with a mean of 1.53+/-0.61 micrometers (or 8.5+/-3.4 kb).

Animals↗

PubMed1991

Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli.

Transposition events mediated by plasmidborne copies of the insertion sequence IS3 of Escherichia coli are difficult to detect because of a low frequency of cointegrate formation. We found that cointegration activity could be strongly enhanced by using plasmid constructions in which a second IS3 element, disabled by a large deletion, was placed adjacent to an intact IS3 copy. Attempts to construct plasmids containing two adjacent intact IS3 copies were unsuccessful, probably because of instability. Transpositional hyperactivity of tandemly duplicated IS sequences was previously described for spontaneous duplications of IS21 and IS30 and may well be a more general phenomenon. The frequency of cointegration events was also strongly increased in an E. coli strain deficient in Dam methylation, suggesting that IS3, like some other Dam site-containing IS elements, is regulated by the Dam methylation system. Insertion sites were strongly clustered within the target lambda repressor gene: however no sequence specificity determinants could be identified. All insertions analyzed carried the IS element in the same orientation; target sequence duplications were mostly 3 bp, but in some cases 4 bp long. To obtain information about the roles of the open reading frames (ORFs) in IS3, we constructed plasmid-borne mutant elements in which potentially functional reading frames were inactivated by site-directed mutations; the mutants were introduced into partial tandem constructions and tested in cointegration assays. Mutations inactivating the putative initiation condons of ORF I and II in the intact element reduced insertion activity to less than 4% of the wild type, whereas the introduction of a termination codon into ORF IV had no effect on cointegration frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1986

Genetic analysis of promoters on the insertion sequence IS21 of plasmid R68.45.

Tandem duplication of a 2.1-kb DNA sequence on R68 leads to the active insertion element IS21 on the enhanced chromosome mobilizing plasmid R68.45. The HindIII/SalI fragment which carries the single copy or the tandem duplication of IS21 was cloned from R68 and R68.45, respectively, into the multicopy plasmid pED815. Promoters on the two HindIII/SalI fragments were subsequently identified by cloning Sau3A fragments into the BglII site of the promoter cloning vector pGA46. Three promoters were identified on the HindIII/SalI fragment derived from R68 or R68.45, two of them mapped on Sau3A fragments of 214 bp and 82 bp, respectively, on IS21. The promoter on the 82 bp Sau3A fragment which maps at the SmaI site close to the left end of IS21 reads inward. The Sau3A fragment of 214 bp contains the left end of IS21 and transcription from its promoter proceeds outward. In R68.45, readthrough from this preexisting promoter located near the junction of the tandem copies of IS21 proceeds from the right-hand copy into the left, opposing the reading direction of the promoter mapped at the SmaI site of IS21. The expression of genes on one copy of IS21 by readthrough from a promoter on the other one is a possible explanation for the transpositional activity of the tandem configuration of IS21. The similarity of IS21 to other insertion sequences and especially to "mobile promoters" is discussed.

Cloning, Molecular↗

PubMed1992

The evolution of insertion sequences within enteric bacteria.

To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.

Base Sequence↗

PubMed1992

p43, the protein product of the atypical insertion sequence IS900, is expressed in Mycobacterium paratuberculosis.

The novel mycobacterial insertion sequence IS900 was analysed by coupled transcription-translation, of both strands independently, in a cell-free E. coli extract using an exogenous promoter. This revealed only one protein product, p43, as predicted from the nucleotide sequence. The protein was readily translated in recombinant E. coli, using the tac promoter, though it did not appear as a major product by SDS-PAGE analysis. A synthetic peptide was used to generate and affinity-purify a specific anti-p43 antibody, which clearly identified the protein in recombinant E. coli. p43 was relatively stable in exponential phase and stationery phase bacteria, though a 28 kDa processed form was seen to accumulate over a period of hours. Both forms appeared in the soluble fraction of the bacterial lysate. The anti-p43 antibody also identified p43, as a 28 kDa processed product, in Western blots of protein extracts from Mycobacterium paratuberculosis, indicating a level of expression which would be unusually high for a classical transposase. These data have important implications for the relationship between IS900 and its host.

Amino Acid Sequence↗

PubMed1986

Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6.

Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47 degrees C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome. Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401. The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements.

DNA Restriction Enzymes↗

PubMed1982

Factors determining frequency of plasmid cointegration mediated by insertion sequence IS1.

We demonstrate that mutants with deletions at either end of the insertion sequence IS1 lose the ability to mediate cointegration of two plasmids, whereas mutants with deletions or an insertion within IS1 can mediate cointegration at a reduced frequency. These results, together with the nucleotide sequence analysis of the IS1 mutants, indicate that the two ends of IS1 (insL and insR) and two genes (insA and insB) that are encoded by IS1 are required for cointegration. Using a plasmid carrying two copies of IS1, we found that the individual IS1s mediate cointegration at different characteristic frequencies, and that each of two parts of plasmid DNA segments flanked by the two IS1s is a transposon, mediating plasmid cointegration at a unique frequency. When one IS1 was replaced with a mutant IS1, the remaining wild-type IS1 complemented the cointegration ability of the mutant IS1 as well as a resulting mutant transposon that was then flanked by a wild-type IS1 and a mutant IS1. The efficiency of this complementation reflected the characteristic ability of an individual IS1 present on the plasmid to promote cointegration. The results suggest that the IS1-encoded proteins are produced in different amounts, depending on the location of IS1 in the plasmid, and that these amounts determine the efficiency of complementation of the cointegration ability of a mutant IS1 as well as a mutant transposon. However, the location of an individual IS1 itself can also determine the frequency of cointegration in the presence of a given amount of the IS1 proteins. On the basis of the observation that the cointegration ability of a mutant IS1 is less efficiently complemented than is the ability of a mutant transposon, we also suggest that the IS1-encoded proteins can function in trans, but act preferentially on the IS1 or transposon sequence from which they are produced in promoting cointegration.

Codon↗

PubMed1989

Analysis of the chromosomal location of two copies of a Bordetella pertussis insertion sequence.

IS481v1 and IS481v2 are two copies of a Bordetella pertussis insertion sequence element. We have shown that IS481v1 is located within 3 kbp of the start of the adenylate cyclase gene whilst IS481v2 is immediately adjacent to the end of the agglutinogen 2 gene and provides the stop codon for that gene. In addition, IS481v1 and IS481v2 were present at these two specific sites in nine strains of B. pertussis, including two Phase IV strains which expressed neither adenylate cyclase nor agglutinogen 2 and three Phase I strains which did not express agglutinogen 2. The loss of expression in these strains is not the result of DNA rearrangements at the sites of IS481v1 or IS481v2.

Adenylate Cyclase Toxin↗

PubMed1991

IS406 and IS407, two gene-activating insertion sequences for Pseudomonas cepacia.

We have determined the nucleotide sequences of IS406 (1368 bp) and IS407 (1236 bp), two insertion sequence (IS) elements isolated from Pseudomonas cepacia 249 on the basis of their abilities to activate the expression of the lac genes of Tn951. IS406 and IS407 when inserted into the lac promoter/operator region of Tn951 generated, respectively, duplications of 8 and 4 bp of target DNA. IS406 had 41-bp terminal inverted repeat (IR) sequences with eleven mismatches. IR-L (left) contained a 12-bp motif present at the ends of Tn2501. In other respects, IS406 was distinct from previously described bacterial IS elements listed in the GenBank and EMBL databases. IS407 had 49-bp terminal IRs with 18 mismatches. IR-R (right) contained an outwardly directed sigma 70-like promoter. IS407 was closely related to IS476 and ISR1 from Xanthomonas and Rhizobium sp., respectively.

Amino Acid Sequence↗

PubMed1992

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains.

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.

DNA Transposable Elements↗

PubMed1992

Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.

Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.

BCG Vaccine↗

PubMed1990

The istA gene of insertion sequence IS21 is essential for cleavage at the inner 3' ends of tandemly repeated IS21 elements in vitro.

The bacterial 2.1 kb insertion sequence IS21 occurs as a tandem repeat [=(IS21)2] on the broad host range plasmid R68.45. In (IS21)2, the two IS21 elements are separated by 3 bp termed junction sequence. Plasmids carrying (IS21)2 form cointegrates with other replicons at high frequencies. The two IS21 genes, istA and istB, were found to be necessary for cointegrate formation in vivo. Since the outer ends of (IS21)2 are dispensable for cointegrate formation, we favor a transposition model according to which a plasmid carrying (IS21)2 is cleaved at the junction sequence; the opened plasmid is then inserted into a target replicon. Here we show that Escherichia coli cell extracts, which contained over-produced IstA protein, nicked a supercoiled (IS21)2 plasmid precisely at the inner 3' termini of IS21; the resulting staggered cut generated 5' protrusions. The istA gene, but not the istB gene, was required for in vitro cleavage of an IS21-IS21 junction. Because of this cleavage and our previous findings (generation of 4 bp target duplications and loss of the junction sequence after cointegrate formation in vivo) we propose that plasmids with (IS21)2 produce cointegrates by a mechanism which involves joining of the inner 3' ends of IS21 to the 5' ends of the target.

Base Sequence↗

PubMed1990

Discovery of an insertion sequence, IS116, from Streptomyces clavuligerus and its relatedness to other transposable elements from actinomycetes.

We have identified an insertion sequence, IS116, present in Streptomyces clavuligerus at one copy per genome. The element was discovered as a 1.4 kb insertion into the multicopy plasmid pIJ702 after propagation in S. clavuligerus. The nucleotide sequence of IS116 and the flanking sequences from pIJ702 have been determined. The junctions with pIJ702 show no target site duplication and there are no inverted repeats at the ends of the element. One putative coding open reading frame of 1197 bp was identified which would code for a protein product of 399 amino acids. This protein resembles deduced integrase/transposase proteins specified by three other transposable elements of actinomycetes: IS110 and the mini-circle from Streptomyces coelicolor A3(2), and--most particularly--IS900 of Mycobacterium paratuberculosis. Two regions that are relatively conserved among these gene products show features found in similar positions in many reverse transcriptases. IS116 and IS900 are also closely similar in their general organization and (apparently) in their insertion site specificity, whereas IS110 and the mini-circle are quite different in these features.

Actinomycetales↗

PubMed1992

IS231D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family.

IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231F). Like the previously described IS231A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478-amino-acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso-IS231 terminal inverted repeats are strongly conserved 20 bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231F is bordered by a 12 bp direct repeat. No direct repeats were found flanking IS231D or IS231E.

Amino Acid Sequence↗

PubMed1991

Characterization of insertion sequence IS892 and related elements from the cyanobacterium Anabaena sp. strain PCC 7120.

IS892, one of the several insertion sequence (IS) elements discovered in Anabaena sp. strain PCC 7120 (Y. Cai and C. P. Wolk, J. Bacteriol. 172:3138-3145, 1990), is 1,675 bp with 24-bp near-perfect inverted terminal repeats and has two open reading frames (ORFs) that could code for proteins of 233 and 137 amino acids. Upon insertion into target sites, this IS generates an 8-bp directly repeated target duplication. A 32-bp sequence in the region between ORF1 and ORF2 is similar to the sequence of the inverted termini. Similar inverted repeats are found within each of those three segments, and the sequences of these repeats bear some similarity to the 11-bp direct repeats flanking the 11-kb insertion interrupting the nifD gene of this strain (J. W. Golden, S. J. Robinson, and R. Haselkorn, Nature [London] 314:419-423, 1985). A sequence similar to that of a binding site for the Escherichia coli integration host factor is found about 120 bp from the left end of IS892. Partial nucleotide sequences of active IS elements IS892N and IS892T, members of the IS892 family from the same Anabaena strain, were shown to be very similar to the sequence of IS892.

Amino Acid Sequence↗

PubMed1988

Insertion sequence IS5 contains a sharply curved DNA structure at its terminus.

It was demonstrated that insertion sequence IS5 contains a sequence-directed bent (sharply curved) DNA structure at its terminus, close to one of its 16 bp terminal repeats. The minimal number of copies of IS5 related sequences and the locations of the latter on the Escherichia coli K12 W3110 chromosome were determined. Evidence is presented of the occurrence of IS5 mediated translocation and duplication of a large DNA segment on the E. coli chromosome.

Bacteriophage lambda↗

PubMed1991

Translational control of transposition activity of the bacterial insertion sequence IS1.

The experiments reported here provide strong evidence indicating that the transposition frequency of the bacterial insertion sequence IS1 is determined principally by two IS1-specified proteins. The first, InsA, was previously shown to bind to the ends of the element and to act as a repressor. We present both physical and genetic evidence which reveals that the second, the InsAB' transposase, is a fusion of InsA with the product of a downstream reading frame, InsB'. Synthesis of this protein occurs by a -1 frameshift between the insA and insB' frames. It requires the presence of an intact retroviral-like frameshift signal composed of an A6C motif and a downstream region able to form several alternative secondary structures. In vivo studies show that IS1 transposition activity depends on the relative rather than on the absolute levels of InsA and InsAB'. The ratio is determined primarily at the translational level by frameshifting and appears to be relatively insensitive to large variations in levels of transcription. This novel homeostatic control could therefore protect IS1 from activation as a consequence of insertion into active transcription units.

Base Composition↗