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M Chandler

Publications and source records attributed to M Chandler.

At least 37 records · Page 2Linked to original sources
PubMed2000

The role of tandem IS dimers in IS911 transposition.

Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR-IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination. This also creates an active IRR-IRL junction by generating a head-to-tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter-IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR-IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co-integrate structure. It is also shown here that the IRR-IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.

Bacterial Proteins↗
PubMed1999

Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region.

The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.

Africa, Eastern↗
PubMed1999

IS911 transposon circles give rise to linear forms that can undergo integration in vitro.

High levels of expression of the transposase OrfAB of bacterial insertion sequence IS911 leads to the formation of excised transposon circles, in which the two abutted ends are separated by 3 bp. Initially, OrfAB catalyses only single-strand cleavage at one 3' transposon end and strand transfer of that end to the other. It is believed that this molecule, in which both transposon ends are held together in a single-strand bridge, is then converted to the circular form by the action of host factors. The transposon circles can be integrated efficiently into an appropriate target in vivo and in vitro in the presence of OrfAB and a second IS911 protein OrfA. In the results reported here, we have identified linear transposon forms in vivo from a transposon present in a plasmid, raising the possibility that IS911 can also transpose using a cut-and-paste mechanism. However, the linear species appeared not to be derived directly from the plasmid-based copy by direct double-strand cleavages at both ends, but from preformed excised transposon circles. This was confirmed further by the observation that OrfAB can cleave a cloned circle junction both in vivo and in vitro by two single-strand cleavages at the 3' transposon ends to generate a linear transposon form with a 3'-OH and a three-nucleotide 5' overhang at the ends. Moreover, while significantly less efficient than the transposon circle, a precleaved linear transposon underwent detectable levels of integration in vitro. The possible role of such molecules in the IS911 transposition pathway is discussed.

Bacterial Proteins↗
PubMed1999

Progressive visual field defects from experimental glaucoma: measurements with white and colored stimuli.

PURPOSE: Our purpose was to study the effects of using monochromatic test stimuli to measure the relative rate of progression of visual field defects caused by experimental glaucoma. METHODS: Visual field measurements were obtained by static perimetry from trained macaque monkeys with laser-induced, unilateral glaucoma. The visual field defects were assessed by perimetric (global) indices derived from comparisons of experimental visual fields to the expected normal fields of monkeys. Three types of perimetry stimuli were used, the conventional white Goldmann III and two monochromatic (460 and 620 nm) Goldmann V test stimuli. The relationships between field defects with white and monochromatic stimuli were investigated by linear regression of the Z-scores for the perimetric indices. RESULTS: The correlations between the mean deviation global indices for chromatic vs. white stimuli were high (r > 0.9) and linear throughout the period of progression of field defects. The slopes of the regression lines typically were greater than unity, indicating that statistical significance was higher for visual field defects measured with chromatic stimuli than with white light stimuli. The higher significance level for defects measured with chromatic stimuli was not explained by a difference in visual thresholds, because the thresholds with chromatic and white light were highly correlated across the full range of visual field defects, from initial-onset to end-state. This result also suggests that the early detection of glaucomatous visual defects with monochromatic stimuli does not reflect a selective loss of retinal ganglion cells. CONCLUSIONS: Although these experiments do not suggest an alternative neural mechanism for the clinical utility of perimetry with chromatic light for the early detection of glaucoma, it is very likely that the combinations of neural and/or analytical factors that explain the utility of perimetry with chromatic stimuli will also provide an explanation for the higher sensitivities in identifying early glaucoma reported for other prototype stimuli.

Animals↗
PubMed1999

X linked severe mental retardation, craniofacial dysmorphology, epilepsy, ophthalmoplegia, and cerebellar atrophy in a large South African kindred is localised to Xq24-q27.

To date over 150 X linked mental retardation (XLMR) conditions have been documented. We describe a five generation South African family with XLMR, comprising 16 affected males and 10 carrier females. The clinical features common to the 16 males included profound mental retardation (100%), mutism despite apparently normal hearing (100%), grand mal epilepsy (87.5%), and limited life expectancy (68.8%). Of the four affected males examined, all had mild craniofacial dysmorphology and three were noted to have bilateral ophthalmoplegia and truncal ataxia. Three of 10 obligate female carriers had mild mental retardation. Cerebellar and brain stem atrophy was shown by cranial imaging and postmortem examination. Linkage analysis shows the gene to be located between markers DXS424 (Xq24) and DXS548 (Xq27.3), with a maximum two point lod score of 3.10.

Abnormalities, Multiple↗
PubMed1999

Integrating DNA: transposases and retroviral integrases.

Transposable elements appear quite disparate in their organization and in the types of genetic rearrangements they promote. In spite of this diversity, retroviruses and many transposons of both prokaryotes and eukaryotes show clear similarities in the chemical reactions involved in their transposition. This is reflected in the enzymes, integrases and transposases, that catalyze these reactions and that are essential for the mobility of the elements. In this chapter, we examine the structure-function relationships between these enzymes and the different ways in which the individual steps are assembled to produce a complete transposition cycle.

Catalytic Domain↗
PubMed1998

Functional effects of FGF-13 on human lung fibroblasts, dermal microvascular endothelial cells, and aortic smooth muscle cells.

We studied the effects of FGF-13 and FGF-2 on human lung fibroblasts, dermal microvascular endothelial cells, and aortic smooth muscle cells. FGF-13 induced cell growth of lung fibroblasts and aortic smooth muscle cells but had no effect on dermal vascular endothelial cells. FGF-2 induced cell growth in all the three cell types. FGF-13 and FGF-2 had little effect on IL-6 production by lung fibroblasts and aortic smooth muscle cells and substantially enhanced that induced by IL-1alpha. In contrast, FGF-13 and FGF-2 had little effect on IL-6 production by dermal vascular endothelial cells, either alone or in synergy with IL-1alpha.

Aorta↗
PubMed1998

Oligomeric structure of the repressor of the bacteriophage Mu early operon.

The regulation of the lytic and lysogenic development in the life cycle of bacteriophage Mu is regulated in part by its repressor, c, which binds to three operator sites, O1, O2 and O3, overlapping two divergent promoters. The oligomeric structure of this repressor protein was investigated by hydrodynamic and biochemical methods. Size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, crosslinking and direct electron microscopy observations suggest that c exists primarily as a hexamer with a molecular mass of 120-140 kDa at low concentrations, i.e. in the 10-microM range. This molecule undergoes a self-assembly process leading to dodecamers and higher order species as the concentration is further increased in a manner depending on the nature of the solvent. Our results also suggest that these species have an elongated structure, and a possible arrangement of the subunits within the hexamer is proposed. The implication of this unusual quaternary structure for a repressor in its interaction with the operator sites O1 and O2 remains to be elucidated.

Bacteriophage mu↗
PubMed1998

Efficient transposition of IS911 circles in vitro.

An in vitro system has been developed which supports efficient integration of transposon circles derived from the bacterial insertion sequence IS911. Using relatively pure preparations of IS911-encoded proteins it has been demonstrated that integration into a suitable target required both the transposase, OrfAB, a fusion protein produced by translational frameshifting between two consecutive open reading frames, orfA and orfB, and OrfA, a protein synthesized independently from the upstream orfA. Intermolecular reaction products were identified in which one or both transposon ends were used. The reaction also generated various intramolecular transposition products including adjacent deletions and inversions. The circle junction, composed of abutted left and right IS ends, retained efficient integration activity when carried on a linear donor molecule, demonstrating that supercoiling in the donor molecule is not necessary for the reaction. Both two-ended integration and a lower level of single-ended insertions were observed under these conditions. The frequency of these events depended on the spacing between the transposon ends. Two-ended insertion was most efficient with a natural spacing of 3 bp. These results demonstrate that transposon circles can act as intermediates in IS911 transposition and provide evidence for collaboration between the two major IS911-encoded proteins, OrfA and OrfAB.

DNA Transposable Elements↗
PubMed1998

Multiple oligomerisation domains in the IS911 transposase: a leucine zipper motif is essential for activity.

Structure-function relationships involved in oligomerisation of the transposase OrfAB of the bacterial insertion sequence IS911 have been investigated. Site-directed mutagenesis and sequential deletion coupled with immunoprecipitation have led to the definition of three regions of the protein capable of promoting multimerisation. These include a region predicted to assume a coiled-coil conformation, which is shown to be essential for activity, promoting correct multimerisation of the N-terminal domain of OrfAB and sequence-specific binding to the IS911 terminal inverted repeats mediated by this domain. This region presents the structural and functional characteristics of the leucine zipper motif described in eukaryotic proteins. The two other regions are located further towards the C-terminal end of the protein, adjacent to the leucine zipper and in the region that carries the conserved catalytic DD(35)E motif.

Amino Acid Sequence↗
PubMed1998

Prenatal screening and women's perception of infant disability: a Sophie's Choice for every mother.

Prenatal screening can significantly benefit parents and the community. However, it has created a dilemma for women as it requires them to quickly decide whether to continue a pregnancy or terminate it should the test indicate a foetal abnormality. This can be psychologically traumatic for women torn between their connection to an unborn child with all its possible imperfections, and a desire to prevent its suffering as a disabled child in later life. A woman must also consider her own and her family's future welfare. Extensive research into the physical aspects of prenatal screening has not explored the meaning of the experience for women or whether termination is the most appropriate option. This article examines recent qualitative studies, concluding that women who terminate a pregnancy following prenatal screening may experience an acute grief reaction or be plagued by guilt and fear that can precipitate marital breakdown. Additionally, there is a risk that through striving to eradicate congenital disability, a community risks promoting a cult of perfectionism that may have discriminatory effects on disabled people.

Abortion, Induced↗
PubMed1998

Insertion sequences.

Insertion sequences (ISs) constitute an important component of most bacterial genomes. Over 500 individual ISs have been described in the literature to date, and many more are being discovered in the ongoing prokaryotic and eukaryotic genome-sequencing projects. The last 10 years have also seen some striking advances in our understanding of the transposition process itself. Not least of these has been the development of various in vitro transposition systems for both prokaryotic and eukaryotic elements and, for several of these, a detailed understanding of the transposition process at the chemical level. This review presents a general overview of the organization and function of insertion sequences of eubacterial, archaebacterial, and eukaryotic origins with particular emphasis on bacterial elements and on different aspects of the transposition mechanism. It also attempts to provide a framework for classification of these elements by assigning them to various families or groups. A total of 443 members of the collection have been grouped in 17 families based on combinations of the following criteria: (i) similarities in genetic organization (arrangement of open reading frames); (ii) marked identities or similarities in the enzymes which mediate the transposition reactions, the recombinases/transposases (Tpases); (iii) similar features of their ends (terminal IRs); and (iv) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length). A brief description of the mechanism(s) involved in the mobility of individual ISs in each family and of the structure-function relationships of the individual Tpases is included where available.

Animals↗
PubMed1997

Assembly of a strong promoter following IS911 circularization and the role of circles in transposition.

When supplied with high levels of the IS911-encoded transposase, IS911-based transposons can excise as circles in which the right and left terminal inverted repeats are abutted. Formation of the circle junction is shown here to create a promoter, p(junc), which is significantly stronger than the indigenous promoter, pIRL, and is also capable of driving expression of the IS911 transposition proteins. High transposase expression from the circular transposon may promote use of the circle as an integration substrate. The results demonstrate that IS911 circles are highly efficient substrates for insertion into a target molecule in vivo. Insertion leads to the disassembly of p(junc) and thus to a lower level of synthesis of the transposition proteins. The observation that normal levels of IS911 transposition proteins supplied by wild-type copies of IS911 are also capable of generating transposon circles, albeit at a low level, reinforces the idea that the transposon circles might form part of the natural transposition cycle of IS911. These observations form the elements of a feedback control mechanism and have been incorporated into a model describing one possible pathway of IS911 transposition.

Base Sequence↗
PubMed1997

IS911-mediated intramolecular transposition is naturally temperature sensitive.

It is shown here that the bacterial insertion sequence IS911 exhibits a temperature-sensitive transposition phenotype. Previous results have demonstrated that elevated levels of the IS911 transposase OrfAB generate significant quantities of a figure-eight form, created by cleavage and circularization of one of the transposon strands, and of an excised circular form, in which both transposon strands have been circularized. We show here that the level of both types of molecule observed in vivo was greatly reduced at 42 degrees C compared with 37 degrees C. On the other hand, reducing the temperature to 30 degrees C resulted in a significant increase in production. Transposition activity at this temperature was sufficiently high to permit detection in vivo of an excised circular form of a defective single IS911 chromosomal copy when OrfAB is supplied in trans. A similar temperature-activity profile is observed for a cell-free reaction that uses partially purified OrfAB and generates the figure-eight form uniquely. Moreover, two point mutants of OrfAB were obtained, which render the reactions partially temperature resistant both in vivo and in vitro. These results suggest that some property of transposase itself is sensitive to elevated temperatures.

Bacterial Proteins↗
PubMed1997

Rescuing magical thinking from the jaws of social determinism.

Although there is otherwise much to recommend it, by riveting attention too narrowly on the contents of magical thought, and by recasting what is left of process in exclusively substantiative terms, this target article works to create the unwarranted impression that the magical thoughts of children and adults are all of a common piece. This commentary reads these oversights and omissions as symptoms of an unspoken new-situationalism working behind the back of Woolley's review.

Adult↗