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M Chandler

Publications and source records attributed to M Chandler.

At least 55 records · Page 3Linked to original sources
PubMed1996

IS911-mediated transpositional recombination in vitro.

A cell-free system is described that accomplishes an unusual type of transposition/recombination involving the bacterial insertion sequence IS911. Using a plasmid substrate carrying a derivative of IS911, we show that bacterial cell extracts enriched for the IS911 transposase, OrfAB, carry out a single-strand cleavage and transfer reaction. This results in the formation of a figure-eight molecule in which a single strand of the element is circularized, faithfully reproducing an event previously detected in vivo. Moreover, when presented with a figure-eight substrate, OrfAB is capable of "reversing" strand transfer. This activity is equivalent to the "disintegration" reaction carried out by retroviral integrases. We demonstrate that the domain of OrfAB responsible for this catalytic activity is located in the carboxy-terminal region of the protein, since a peptide composed of this region retains disintegration activity. The OrfAB-mediated excision-circularization process previously observed in vivo was proposed to proceed via a figure-eight intermediate by circularization of the second transposon strand. The absence of transposon circles in cell-free reaction suggests either that the figure-eight form is not an intermediate or that additional host factors are required that are eliminated from the cell extract. Two types of model, replicative and non-replicative, are discussed to explain how the figure-eight molecule could be processed into the transposon circle.

Bacterial Proteins↗
PubMed1996

Interactions between the repressor and the early operator region of bacteriophage Mu.

The repressor of bacteriophage Mu, c, binds to three operator sites, O1, O2, and O3, overlapping two divergent promoters, which regulate the lytic and lysogenic pathways. Its binding to this operator region generates several complexes, which were analyzed by DNase I protection experiments. We demonstrate that c first binds to two 11-base pair partially repeated sequences in O2 that could represent "core" binding sites for the repressor. This initial interaction serves as an organizer of a more complex nucleoprotein structure in which O2, O1, and O3 become successively occupied. The quaternary structure of the repressor was also investigated. Size exclusion chromatography and protein-protein crosslinking experiments with chemicals that possess linking arms of various lengths indicate that the repressor oligomerizes in solution. A model is proposed describing the successive interactions of c with the operator sites O2, O1, and O3 leading to the elaboration of a higher order structure in which the early lytic functions are repressed.

Bacteriophage mu↗
PubMed1996

The role of strategic planning in accessing false-belief understanding.

Recently, Chandler and Hala found that actively involving 3-year-olds in planning a deception facilitated performance on false-belief questions. The methodology used, however, provided no basis for determining whether the good performance of these young subjects was the result of the deceptive intent of their planning efforts, or whether other sorts of planning would have been equally effective. The research reported here systematically varied both (a) subjects' responsibility for planning where to relocate an object and (b) whether the goal behind this relocation was a deceptive one. The present research demonstrated, first, when subjects simply watched the transfer take place, it made no difference whether the object was moved for deceptive or some more practical reason. In contrast, those subjects who had themselves strategically planned a deception were markedly better at answering questions about another's false beliefs than those who simply witnessed the transfer taking place. No comparable facilitating effect was found when subjects planned a transfer but without deceptive intent. We argue that strategic planning works to underscore the importance of the belief states of others and provide opportunities not afforded by "standard" unexpected change or transfer tasks for showcasing 3-year-olds' emerging understanding of the possibility of false belief.

Age Factors↗
PubMed1995

An in vivo transposase-catalyzed single-stranded DNA circularization reaction.

Expression of the bacterial insertion sequence IS911 transposase in vivo leads to excision and circularization of IS911-based transposons. We show here that transposase produces an unusual molecular form generated by single-strand cleavage, transfer, and ligation of one end of the element to the opposite end. When the transposon is carried by a circular plasmid, this results in the formation of a "figure-eight" molecule in which a single strand of the transposon is circularized while the corresponding strand of the vector backbone retains a single-strand interruption at this position. The results show that a 3' end of the transposon is transferred to the opposite target end. Transposase is therefore capable of introducing single-strand cleavages at the ends of the element, an activity similar to that of retroviral integrases with which it shares significant similarities in amino acid sequence. Kinetic studies demonstrate that the figure-eight accumulates earlier than transposon circles after transposase induction and disappears before circles after inhibition of transposase expression, raising the possibility that the figure-eight molecules are precursors to the circles. Therefore, IS911 excision as a circle may not occur by double-strand cleavage leading to its prior separation from the vector backbone in a linear form but could proceed by consecutive circularization of each strand.

Base Sequence↗
PubMed1995

IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.

Base Sequence↗
PubMed1995

Mutual stabilisation of bacteriophage Mu repressor and histone-like proteins in a nucleoprotein structure.

Integration host factor (IHF) binds in a sequence-specific manner to the bacteriophage Mu early operator. It participates with bound Mu repressor, c, in building stable, large molecular mass nucleoprotein complexes in vitro and enhances repression of early transcription in vivo. We demonstrate that, when the specific IHF binding site with the operator is mutated, the appearance of large molecular mass complexes still depends on IHF and c, but the efficiency of their formation is reduced. Moreover, the IHF-like HU protein, which binds DNA in a non-sequence-specific way, can substitute for IHF and participate in complex formation. Since the complexes require both c and a host factor (IHF or HU), the results imply that these proteins stabilise each other within the nucleoprotein structures. These results suggest that IHF and HU are directed to the repressor-operator complexes, even in the absence of detectable sequence-specific binding. This could be a consequence of their preferential recognition of DNA containing a distortion such as that introduced by repressor binding to the operator. The histone-like proteins could then stabilise the nucleoprotein complexes simply by their capacity to maintain a bend in DNA rather than by specific protein-protein interactions with c. This model is supported by the observation that the unrelated eukaryotic HMG-1 protein, which exhibits a similar marked preference for structurally deformed DNA, is also able to participate in the formation of higher-order complexes with c and the operator DNA.

Animals↗
PubMed1995

Bacterial transposases and retroviral integrases.

Transposable genetic elements have adopted two major strategies for their displacement from one site to another within and between genomes. One involves passage through an RNA intermediate prior to synthesis of a DNA copy while the other is limited uniquely to DNA intermediates. For both types of element, recombination reactions involved in integration are carried out by element-specific enzymes. These are called transposases in the case of DNA elements and integrases in the case of the best-characterized RNA elements, the retroviruses and retrotransposons. In spite of major differences between these two transposition strategies, one step in the process, that of insertion, appears to be chemically identical. Current evidence suggests that the similarities in integration mechanism are reflected in amino acid sequence similarities between the integrases and many transposases. These similarities are particularly marked in a region which is thought to form part of the active site, namely the DDE motif. In the light of these relationships, we attempt here to compare mechanistic aspects of retroviral integration with transposition of DNA elements and to summarize current understanding of the functional organization of integrases and transposases.

Amino Acid Sequence↗
PubMed1995

Mutagenesis of the IS1 transposase: importance of a His-Arg-Tyr triad for activity.

Inspection of the primary sequence of the IS1 transposase suggested that it carries residues which are characteristic of the active site of integrases of the bacteriophage lambda family (Int). In particular, these include a highly conserved triad: His-Arg-Tyr. The properties of mutants made at each of these positions were investigated in vivo. The results of several different assays confirm that each is important for transposase activity. Moreover, as in the case of members of the Int family, different mutations of the His residue exhibited different effects. In a particular, His-to-Leu mutation resulted in complete inactivation whereas the equivalent His-to-Gln mutation retained low but significant levels of activity.

Amino Acid Sequence↗
PubMed1994

Induction of the SOS response by IS1 transposase.

We find that IS1 transposase, like that of Tn10, can induce the SOS response when produced at high levels. Most of the activity (> 80%) requires IS1 ends in cis to the transposase gene and depends strictly on the presence of RecBCD function. This implies that processing of transposase-induced cleavages is responsible for generating the response. Induction of the SOS response during growth in a rich medium is seen only when cells approach stationary phase. The end-dependent induction is abolished by mutations in the ends of IS1 that eliminate transposition activity. IS1 ends in identical orientation on the same plasmid are inactive in transposition but stimulate SOS strongly. Even plasmids with a single end can stimulate SOS, probably as a consequence of plasmid dimer formation which places the ends in direct repeat orientation. These results imply that transposase-induced cleavages do not need inversely oriented ends. The system can therefore be used to dissociate cleavage activity from the other reactions of transposition. Induction of SOS by a series of short (67 to 114 bp) IS1-like elements was found to occur in a cyclical pattern as a function of length with a period of 10 to 11 bp. The frequency of cointegration promoted by these elements showed the same helix-phase dependence. These results suggest that transposase molecules bound to the ends of IS1 interact, and that this interaction is needed for the cleavages that initiate transposition.

Base Sequence↗
PubMed1994

Interaction of Fis protein with DNA: bending and specificity of binding.

The Escherichia coli Fis protein is a dimeric DNA-binding protein whose specific binding sites share a weak consensus sequence. Use of the gel retardation technique indicates that binding of Fis on a linear DNA fragment leads to the formation of a ladder of defined retarded complexes, independently of the presence of a specific site. This non-specific binding of Fis is consistent with a model where equivalent low-affinity sites on a given fragment would be bound randomly and independently of each other by consecutive Fis dimers. Evidence is presented that non-specific binding of Fis can, however, induce an apparent site-specific conformational change in the DNA. This observation is discussed in terms of a model in which each Fis:DNA complex detected in gel retardation experiments actually represents a dynamic equilibrium of a fixed number of Fis dimers distributed on the fragment.

Carrier Proteins↗
PubMed1994

Is the IS1 transposase, InsAB', the only IS1-encoded protein required for efficient transposition?

The transposase of the bacterial insertion sequence IS1 is normally expressed by inefficient translational frameshifting between an upstream reading frame which itself specifies a transposition inhibitor, InsA, and a second consecutive reading frame located immediately downstream. A fused-frame mutant which carries an additional base pair inserted at the point of frameshifting was constructed. This mutant exhibits high transposition activity and should express the transposase, InsAB', constitutively without frameshifting. Unexpectedly, a second protein species was observed to be expressed from this mutant. We demonstrate here that this protein, InsA*, results from continued frameshifting on the modified frameshift motif. The protein retains the activities of the repressor InsA. Its elimination, by further modification of the frameshift motif, results in a further increase in various transposition activities of IS1. These results support the hypothesis that a single IS1-encoded protein, InsAB', is necessary for transposition.

Bacterial Proteins↗
PubMed1994

One-ended insertion of IS911.

An apparently nonreplicative integration reaction mediated by the insertion sequence IS911 has been analyzed. It is shown to involve the right-end inverted repeat (IRR) of the element and sequences in the flanking vector DNA. The flanking sequences appear to behave as a surrogate IS911 end, since integration is greatly reduced when limited similarities with IRR are eliminated by site-directed mutagenesis. Data are presented which suggest that the activity of the IRR junction results from the proximity of the transposase gene and may therefore reflect preferential transposase recognition of IRR in cis.

Bacteriophage lambda↗
PubMed1993

Translational frameshifting in the control of transposition in bacteria.

The expression of an increasing number of genes of both prokaryotic and eukaryotic origin has been shown to be regulated at the translational level by programmed (sequence-specific) ribosomal frameshifting. Among these are the bacterial insertion sequences IS1 and two members of the widely distributed IS3-family, IS150 and IS911. Frameshifting provides a means of specifying several proteins with different functions using a minimum of genetic information. In this review, we survey present understanding of the way in which frameshifting is integrated into the overall control of transposition activity in these elements.

Bacteria↗
PubMed1993

Involvement of Escherichia coli FIS protein in maintenance of bacteriophage mu lysogeny by the repressor: control of early transcription and inhibition of transposition.

The Escherichia coli FIS (factor for inversion stimulation) protein has been implicated in assisting bacteriophage Mu repressor, c, in maintaining the lysogenic state under certain conditions. In a fis strain, a temperature-inducible Mucts62 prophage is induced at lower temperatures than in a wild-type host (M. Bétermier, V. Lefrère, C. Koch, R. Alazard, and M. Chandler, Mol. Microbiol. 3:459-468, 1989). Increasing the prophage copy number rendered Mucts62 less sensitive to this effect of the fis mutation, which thus seems to depend critically on the level of repressor activity. The present study also provides evidence that FIS affects the control of Mu gene expression and transposition. As judged by the use of lac transcriptional fusions, repression of early transcription was reduced three- to fourfold in a fis background, and this could be compensated by an increase in cts62 gene copy number. c was also shown to inhibit Mu transposition two- to fourfold less strongly in a fis host. These modulatory effects, however, could not be correlated to sequence-specific binding of FIS to the Mu genome, in particular to the strong site previously identified on the left end. We therefore speculate that a more general function of FIS is responsible for the observed modulation of Mu lysogeny.

Bacteriophage mu↗
PubMed1992

A simple and efficient system for the construction of phoA gene fusions in gram-negative bacteria.

We have developed a two-plasmid system for generating gene fusions between phoA and cloned genes encoding envelope proteins. The vector plasmid carries a temperature-sensitive replication system and can be rescued at high temperature by insertion of an IS1-based transposon carrying the ori region of pBR322 and a phoA gene lacking transcription and translation initiation signals. The vector plasmid also carries the transfer origin of the conjugative plasmid, F, permitting transfer into a suitable recipient strain. We have used this system in the analysis of the bla gene cloned from pBR322.

Alkaline Phosphatase↗
PubMed1992

Transposase-induced excision and circularization of the bacterial insertion sequence IS911.

We have investigated the role of three IS911-specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by -1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB-mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site-specific intramolecular transposition event. We present a model which explains both intra- and intermolecular transposition events in terms of a single reaction mechanism of the 'cut and paste' type.

Bacteria↗
PubMed1992

Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro.

Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator-binding sites in vitro. These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.

Bacterial Proteins↗