Whole-Genome Comparison of Two Campylobacter jejuni Isolates of the Same Sequence Type Reveals Multiple Loci of Different Ancestral Lineage

Campylobacter jejuni ST-474 is the most important human enteric pathogen in New Zealand, and yet this genotype is rarely found elsewhere in the world. Insight into the evolution of this organism was gained by a whole genome comparison of two ST-474, flaA SVR-14 isolates and other available C. jejuni isolates and genomes. The two isolates were collected from different sources, human (H22082) and retail poultry (P110b), at the same time and from the same geographical location. Solexa sequencing of each isolate resulted in 1.659 Mb (H22082) and 1.656 Mb (P110b) of assembled sequences within 28 (H22082) and 29 (P110b) contigs. We analysed 1502 genes for which we had sequences within both ST-474 isolates and within at least one of 11 C. jejuni reference genomes. Although 94.5% of genes were identical between the two ST-474 isolates, we identified 83 genes that differed by at least one nucleotide, including 55 genes with non-synonymous substitutions. These covered 101 kb and contained 672 point differences. We inferred that 22 (3.3%) of these differences were due to mutation and 650 (96.7%) were imported via recombination. Our analysis estimated 38 recombinant breakpoints within these 83 genes, which correspond to recombination events affecting at least 19 loci regions and gives a tract length estimate of 2 kb. This includes a 12 kb region displaying non-homologous recombination in one of the ST-474 genomes, with the insertion of two genes, including ykgC, a putative oxidoreductase, and a conserved hypothetical protein of unknown function. Furthermore, our analysis indicates that the source of this recombined DNA is more likely to have come from C. jejuni strains that are more closely related to ST-474. This suggests that the rates of recombination and mutation are similar in order of magnitude, but that recombination has been much more important for generating divergence between the two ST-474 isolates

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

The isolation and characterization of Rhizobium loti exopolysaccharide mutants : a thesis presented in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand

PN1 84, a streptomycin resistant derivative of the broad host range Rhizobium loti strain NZP2037, was shown to be Calcofluor-bright due to the production of a Calcofluor-binding exopolysaccharide (EPS) with both high and low molecular weight forms, the low molecular weight form being predominant Eight Calcofluor-dark EPS mutants (three smooth and five rough) were generated by Tn5 mutagenesis of PN1 84. Each mutant was shown to carry a single , independent Tn5 insertion. Cosmids that complemented the mutation carried by each of the rough, PN1 84-derived EPS mutants were isolated from a pLAFRl gene library to NZP2037 by complementation of the Calcofluor-dark phenotype. The genetic regions identified were shown to be located on the chromosome, and were not closely linked. The mutants were divided into three (complementation) groups. While the rough, PN1 84-derived EPS mutants failed to synthesize EPS, the smooth, PN1 84-derived EPS mutants were found to synthesize an EPS which failed to bind Calcofluor, and which was shown, by 1 H-NMR spectroscopy, to be significantly less acetylated than the EPS produced by PN1 84. Furthermore, PN1 177, one of the smooth, PN1 84-derived EPS mutants, was shown to produce only a small amount of high molecular weight EPS compared to PN1 84. All the PN1 84-derived EPS mutants induced the formation of fully effective (Nod+Fix+) nodules on Lotus pedunculatus, a determinate nodulating host legume, but, in contrast, induced the formation of ineffective (Nod+Fix-) nodules on Leucaena leucocephala, an indeterminate nodulating host legume. Each rough, PN1 84-derived EPS mutant, c arrying its complementing cosmid, was fully effective on L. leucocephala. PN4 1 15, a streptomycin resistant derivative of the restricted, effective host range R . loti strain NZP2213, was shown to be Calcofluor-dark. PN4 1 1 5 was shown to produce an EPS, which fails to bind Calcofluor, that is acetylated to approximately the same extent as the EPS produced by PN1 84. Like PN1 1 77, PN4 1 15 was shown to produce only a small amount of high molecular weight EPS. Examination of 1 H-NMR spectra of EPS from PN4 1 1 5 and the smooth, PN1 84-derived EPS mutants suggests that these strains produce an EPS of similar structure, with the exception of the degree of 0- acetylation. Three non-mucoid, Calcofluor-bright, EPS mutants were generated by Tn5 mutagenesis of PN4 1 15. Each mutant was shown to carry a single, independent Tn5 insertion. Cosmids could not be isolated which stably complemented the mutation carried by each mutant. None of the mutants produced EPS, but all three mutants produce a Calcofluor-binding EPS, possibly cellulose. All three PN4 1 15-derived EPS mutants induced the formation of fully effective nodules on Lotus corniculatus, a determinate nodulating host legume. On L. leucocephala, PN4 1 15 induced the formation of both small, ineffective, nodular swellings and large, ineffective, tumour-like structures. Occasionally, a low level of nitrogen fixation was observed. In contrast, the PN41 15-derived EPS mutants all induced the formation of only small, ineffective, nodular swellings. These results, obtained in isogenic Rhizobium backgrounds, support suggestions that EPS is required for effective nodulation of indeterminate nodulating legumes, but is not required for effective nodulation of determinate nodulating legumes

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter

Article on cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter

Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants

NMDA receptor gene variations as modifiers in Huntington disease: a replication study.

Several candidate modifier genes which, in addition to the pathogenic CAG repeat expansion, influence the age at onset (AO) in Huntington disease (HD) have already been described. The aim of this study was to replicate association of variations in the N-methyl D-aspartate receptor subtype genes GRIN2A and GRIN2B in the “REGISTRY” cohort from the European Huntington Disease Network (EHDN). The analyses did replicate the association reported between the GRIN2A rs2650427 variation and AO in the entire cohort. Yet, when subjects were stratified by AO subtypes, we found nominally significant evidence for an association of the GRIN2A rs1969060 variation and the GRIN2B rs1806201 variation. These findings further implicate the N-methyl D-aspartate receptor subtype genes as loci containing variation associated with AO in HD