Biomedical subjects

Michael Chandler

Publications and source records attributed to Michael Chandler.

10 recordsLinked to original sources

PubMed2006

Insertion sequences in prokaryotic genomes.

Insertion sequences (ISs) are small DNA segments that are often capable of moving neighbouring genes. Over 1500 different ISs have been identified to date. They can have large and spectacular effects in shaping and reshuffling the bacterial genome. Recent studies have provided dramatic examples of such IS activity, including massive IS expansion during the emergence of some pathogenic bacterial species and the intimate involvement of ISs in assembling genes into complex plasmid structures. However, a global understanding of their impact on bacterial genomes requires detailed knowledge of their distribution across the eubacterial and archaeal kingdoms, understanding their partition between chromosomes and extra-chromosomal elements (e.g. plasmids and viruses) and the factors which influence this, and appreciation of the different transposition mechanisms in action, the target preferences and the host factors that influence transposition. In addition, defective (non- autonomous) elements, which can be complemented by related active elements in the same cell, are often overlooked in genome annotations but also contribute to the evolution of genome organisation.

Bacteria↗

PubMed2006

Changing selves in changing worlds: youth suicide on the fault-lines of colliding cultures.

What does it mean to somehow override change and to count one's self as one and the same individual, continuous in time? What does "continuity" mean for whole cultural groups? How might disruptions to a sense of personal or cultural persistence deprive us of a past, and a connection to our as yet unrealized futures? Why is it that the bulk of us who succeed in knitting up our raveled sleeves of care choose for life, while those who loose the thread of their continuous existence so frequently make the opposite choice? The program of research outlined here--work that explores the relation between markers of self- and cultural continuity, and suicidal behaviors in both culturally mainstream and Canadian Aboriginal youth--provides evidence that personal persistence and persistent peoples have low or absent rates of youth suicide, while individuals and communities lacking a requisite sense of continuity regularly suffer suicides in epidemic numbers.

Adolescent↗

PubMed2005

Active site sharing and subterminal hairpin recognition in a new class of DNA transposases.

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg2+ and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalent intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.

Amino Acid Sequence↗

PubMed2005

Transposition of ISHp608, member of an unusual family of bacterial insertion sequences.

ISHp608 from Helicobacter pylori is active in Escherichia coli and represents a recently recognised group of insertion sequences. Its transposase and organisation suggest that it transposes using a different mechanism to that of other known transposons. The IS was shown to excise as a circular form, which is accompanied by the formation of a resealed donor plasmid backbone. We also demonstrate that TnpA, which is less than half the length of other transposases, is responsible for this and for ISHp608 transposition. Transposition was shown to be site specific: both insertion and transposon excision require a conserved target, 5'TTAC. Deletion analysis suggested that potential secondary structures at the left and right ends are important for transposition. In vitro TnpA bound both ends, showed a strong preference for a specific single-stranded DNA and introduced a single-strand break on the same strand at each end. Although many of the characteristics of ISHp608 appear similar to rolling-circle transposons, there are differences suggesting that, overall, transposition occurs by a different mechanism. The results have permitted the formulation of several related models.

Amino Acid Sequence↗

PubMed2004

Regulation of transposition in bacteria.

Mobile genetic elements (MGEs) play a central role in the evolution of bacterial genomes. Transposable elements (TE: transposons and insertion sequences) represent an important group of these elements. Comprehension of the dynamics of genome evolution requires an understanding of how the activity of TEs is regulated and how their activity responds to the physiology of the host cell. This article presents an overview of the large range of, often astute, regulatory mechanisms, which have been adopted by TEs. These include mechanisms intrinsic to the element at the level of gene expression, the presence of key checkpoints in the recombination pathway and the intervention of host proteins which provide a TE/host interface. The multiplicity and interaction of these mechanisms clearly illustrates the importance of limiting transposition activity and underlines the compromise that has been reached between TE activity and the host genome. Finally, we consider how TE activity can shape the host genome.

Bacteria↗

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↗

PubMed2004

IS911 partial transposition products and their processing by the Escherichia coli RecG helicase.

Insertion of bacterial insertion sequence IS911 can often be directed to sequences resembling its ends. We have investigated this type of transposition and shown that it can occur via cleavage of a single end and its targeted transfer next to another end. The single end transfer (SET) events generate branched DNA molecules that contain a nicked Holliday junction and can be considered as partial transposition products. Our results indicate that these can be processed by the Escherichia coli host independently of IS911-encoded proteins. Such resolution depends on the presence of homologous DNA regions neighbouring the cross-over point in the SET molecule. Processing is often accompanied by sequence conversion between donor and target sequences, suggesting that branch migration is involved. We show that resolution is greatly reduced in a recG host. Thus, the branched DNA-specific helicase, RecG, involved in processing of potentially lethal DNA structures such as stalled replication forks, also intervenes in the resolution of partial IS911 transposition products.

Base Sequence↗

PubMed2004

Functional domains of the IS1 transposase: analysis in vivo and in vitro.

The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB' transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB', but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB' using a series of site-directed InsAB' mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding.

Amino Acid Sequence↗

PubMed2004

Analysis of the N-terminal DNA binding domain of the IS30 transposase.

IS30 is the founding member of a large family of widely spread bacterial insertion sequences with closely related transposases. The N-terminal end of the IS30 transposase had been shown to retain sequence-specific DNA binding activity and to protect the IS30 terminal inverted repeats. Structural predictions revealed the presence of a helix-helix-turn-helix motif (H-HTH2) which, in the case of IS30, is preceded by an additional helix-turn-helix motif (HTH1). Analysis of deletion and point mutants in this region revealed that both motifs are important for IS30 transposition. IS30 exhibits two types of insertion specificity preferring either a 24 bp palindromic hot-spot (GOHS) or sequences resembling its ends [left and right terminal inverted repeat (IRL and IRR)]. Results are presented suggesting that the HTH1 region is required for GOHS targeting and interferes with the inverted repeat (IR) targeting. On the other hand, H-HTH2 appears to be required for both. The binding activities of the mutant proteins to the terminal IS30 IRs as measured by gel retardation correlated well with these results. Furthermore, close inspection of the H-HTH2 region revealed significant amino acid identity with a similar predicted secondary structure carried by the transcriptional regulator FixJ of Sinorhizobium meliloti and involved in FixJ binding to its target sequence. This suggests that FixJ and IS30 transposase share similar sequence-specific DNA binding mechanisms.

Amino Acid Sequence↗

PubMed2003

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Amino Acid Sequence↗