Alan Peter Lewis

Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence

A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.

Cloning and sequence analysis of κ and γ cynomolgus monkey immunoglobulin cDNAs

Abstract One γ heavy chain and 10 κ light chain cynomolgus monkey ( Macaca fasicularis ) immunoglobulin cDNAs have been cloned and sequenced. Comparisons of the variable (V) regions to human antibody sequences have revealed extensive identity, exhibiting 93% at the amino acid level for the V H framework regions, and 88–99% for the V κ frameworks. Identification of very few cynomolgus monkey-specific framework region residues suggests a role for cynomolgus monkey antibodies as donators of variable regions to chimeric monoclonal antibodies for utilisation in human therapy with human constant (C) regions. The cynomolgus monkey C κ region exhibited 83% amino acid identity to its human counterpart, and the C γ region was 95, 93, 95, and 95% similar to the human C γ1 , C γ2 , C γ3 , and C γ4 regions, respectively. Evolutionary analysis of the C γ genes, using the silent molecular clock, suggests that the divergence between cynomolgus monkey and human occurred before the time at which the ancestral γ gene diverged into the multiple isotypes observed in humans.

Differential Gene Expression Identifies Novel Markers of CD4+ and CD8+ T Cell Activation Following Stimulation by Mycobacterium tuberculosis

T cell activation in response to antigenic stimulation is a complex process, involving changes in the expression level of a large number of genes. We have used cDNA array technology to characterize the differences in gene expression between human CD4+ and CD8+ T cells. PBMC from six healthy donors were stimulated with live Mycobacterium tuberculosis, and the gene expression profiles of each donor’s CD4+ and CD8+ T cells were analyzed separately. ANOVA revealed 518 genes that were consistently differentially expressed between CD4+ and CD8+ T cells. These differentially expressed genes include a combination of well-known, previously characterized genes with a range of biological functions and unknown in silico predicted hypothetical genes. Where possible, the novel genes have been characterized using bioinformatics, and putative transcription factors, signaling molecules, transmembrane, and secreted factors have been identified. A subset of these differentially expressed genes could be exploited as markers of CD4+ and CD8+ T cell activation for use in vaccine trials. These observed differences in the gene expression profile of CD4+ and CD8+ T cells following activation by a human pathogen contribute to an increased understanding of T cell activation and differentiation and the roles these T cell subsets may play in immunity to infection.

SCALOP: sequence-based antibody canonical loop structure annotation

Abstract Motivation Canonical forms of the antibody complementarity-determining regions (CDRs) were first described in 1987 and have been redefined on multiple occasions since. The canonical forms are often used to approximate the antibody binding site shape as they can be predicted from sequence. A rapid predictor would facilitate the annotation of CDR structures in the large amounts of repertoire data now becoming available from next generation sequencing experiments. Results SCALOP annotates CDR canonical forms for antibody sequences, supported by an auto-updating database to capture the latest cluster information. Its accuracy is comparable to that of a standard structural predictor but it is 800 times faster. The auto-updating nature of SCALOP ensures that it always attains the best possible coverage. Availability and implementation SCALOP is available as a web application and for download under a GPLv3 license at opig.stats.ox.ac.uk/webapps/scalop. Supplementary information Supplementary data are available at Bioinformatics online.