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Biomedical subjects

M Chandler

Publications and source records attributed to M Chandler.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1992

Use of gel retardation to analyze protein-nucleic acid interactions.

Protein-nucleic acid interactions are crucial in the regulation of many fundamental cellular processes. The nature of these interactions is susceptible to analysis by a variety of methods, but the combination of high analytical power and technical simplicity offered by the gel retardation (band shift) technique has made this perhaps the most widely used such method over the last decade. This procedure is based on the observation that the formation of protein-nucleic complexes generally reduces the electrophoretic mobility of the nucleic acid component in the gel matrix. This review attempts to give a simplified account of the physical basis of the behavior of protein-nucleic acid complexes in gels and an overview of many of the applications in which the technique has proved especially useful. The factors which contribute most to the resolution of the complex from the naked nucleic acid are the gel pore size, the relative mass of protein compared with nucleic acid, and changes in nucleic acid conformation (bending) induced by binding. The consequences of induced bending on the mobility of double-strand DNA fragments are similar to those arising from sequence-directed bends, and the latter can be used to help characterize the angle and direction of protein-induced bends. Whether a complex formed in solution is actually detected as a retarded band on a gel depends not only on resolution but also on complex stability within the gel. This is strongly influenced by the composition and, particularly, the ionic strength of the gel buffer. We discuss the applications of the technique to analyzing complex formation and stability, including characterizing cooperative binding, defining binding sites on nucleic acids, analyzing DNA conformation in complexes, assessing binding to supercoiled DNA, defining protein complexes by using cell extracts, and analyzing biological processes such as transcription and splicing.

Base Sequence↗
PubMed1991

Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911.

The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase.

Amino Acid Sequence↗
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↗
PubMed1991

Clinical and neuropsychological effects of desipramine in children with attention deficit hyperactivity disorder.

Desipramine is a tricyclic antidepressant with demonstrated efficacy for some children with attention deficit hyperactivity disorder (ADHD). In this controlled study, clinical improvement was noted in a group of 12 ADHD children. There also were neuropsychological effects associated with desipramine treatment: a small but significant decline in motor performance and an improvement in long-term verbal memory. The decline in motor performance may be of only limited clinical significance, but it is an effect that desipramine seems to share with other tricyclic antidepressants. The improvement in memory performance is an effect it shares with the psychostimulants.

Affect↗
PubMed1990

Overo lethal white foal syndrome: equine model of aganglionic megacolon (Hirschsprung disease).

The lethal white foal syndrome (LWFS) is a congenital abnormality of overo spotted horses which is a model for human aganglionic megacolon or Hirschsprung disease. Foals with LWFS have an all white, or nearly all white, coat. They also present clinically with an intestinal obstruction that proves fatal within the first few days of life. The LWFS involves both melanocytes and intestinal ganglion cells, and appears to result from a genetic defect involving neural crest cells. This report describes pathologic studies of two recent cases of LWFS. Two different hypothetical models of inheritance of LWFS are presented and discussed.

Animals↗
PubMed1990

Relativism and stations of epistemic doubt.

This sequence of studies examined the role that relativistic thinking plays in the cognitive and social-emotional lives of adolescents. Study 1 introduces an assessment strategy and associated descriptive model employed in evaluating how 70 concrete and formal operational adolescents differently interpret and resolve problems involving competing knowledge claims. A second study explored the relations between the epistemic orientations evidenced by 61 of these subjects and their current level of identity development. Study 3 compared the epistemic assumptions of a group of 29 psychiatrically hospitalized adolescents with those of a matched group of normal controls. Results from these studies indicate that relativistic approaches to problems of belief entitlement are: a) routinely characteristic of most normal adolescents; b) available to formal operational, but not concrete operational individuals; c) associated with more mature ego-identity statuses; and d) typically absent in groups of psychiatrically hospitalized youth.

Adolescent↗
PubMed1990

Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences?

Members of the IS3 family of insertion sequences are found in a wide range of bacteria. At least 10 members of this family carry two major open reading frames: a small upstream frame (0 phase), and a longer downstream frame in the -1 phase. The downstream frame shows significant similarity at the amino acid level. A highly conserved region of this frame also exhibits notable similarity with a region of the integrase (endonuclease) domain of retroviruses. Although the overall transposition mechanism of the insertion sequence and retroviral elements is certainly different, the two groups may share additional common features, including a -1 frameshift resulting in the production of a fusion protein.

Amino Acid Sequence↗