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Item Mutations in the riboflavin biosynthesis pathway confer resistance to furazolidone and abolish the synergistic interaction between furazolidone and vancomycin in Escherichia coli.(Microbiology Society, England, 2025-02-11) Wykes H; Le VVH; Rakonjac JThe combined application of furazolidone and vancomycin has previously been shown to be synergistic against Gram-negative pathogens, with great therapeutic promise. However, the emergence and mechanism of resistance to this antibiotic combination have not been characterized. To fill this gap, we here selected Escherichia coli progeny for growth on the furazolidone-vancomycin combination at the concentration where the parent was sensitive. We show that selected clones were associated with increased resistance to neither, only one drug, or both furazolidone and vancomycin, but in all cases were associated with a decrease in the growth inhibition synergy. Using whole-genome sequencing, we identified various gene mutations in the resistant mutants. We further investigated the mechanism behind the most frequently arising mutations, those in the riboflavin biosynthesis genes ribB and ribE, that represent novel mutations causing furazolidone resistance and diminished vancomycin-furazolidone synergy. It was found that these ribB/ribE mutations act predominantly by decreasing the activity of the NfsA and NfsB nitroreductases. The emergence of the ribB/ribE mutations imposes a significant fitness cost on bacterial growth. Surprisingly, supplementing the medium with riboflavin, which compensates for the affected riboflavin biosynthesis pathway, could restore the normal growth of the ribB/ribE mutants while having no effects on the furazolidone resistance phenotype. Searching the ribB/ribE mutations in the public sequencing database detects the presence of the furazolidone-resistance-conferring ribE mutations (TKAG131-134 deletion or duplication) in clinical isolates from different countries. Hypotheses explaining why these ribE mutations were found in clinical isolates despite having poor fitness were further discussed.Item Prevalence of the ABCB1-1Δ gene mutation in a sample of New Zealand Huntaway dogs(Informa UK Limited, trading as Taylor & Francis Group, 2023-03-13) Gedye K; Poole-Crowe E; Shepherd M; Wilding A; Parton K; Lopez-Villalobos N; Cave NAIMS: To determine the prevalence of the ATP Binding Cassette Subfamily B Member 1-1Δ mutation (ABCB1-1Δ; previously Multidrug Resistance 1 (MDR1) mutation) in a cohort of New Zealand Huntaway dogs. MATERIALS AND METHODS: Samples were opportunistically collected from Huntaway dogs (n = 189) from throughout New Zealand. Buccal swabs were collected from 42 Huntaways from the Wairarapa region and 147 blood samples from Huntaways from the Gisborne, Waikato, Manawatū/Whanganui, Hawkes Bay, Canterbury and Otago regions. DNA was extracted from all samples and tested for the presence of the ABCB1-1Δ allele. RESULTS: Of 189 Huntaway dogs that were tested, two were found to be heterozygous carriers of the ABCB1-1Δ allele and the remaining 187/189 dogs were homozygous for the wild type allele. No dogs homozygous for the mutation were identified. CONCLUSIONS AND CLINICAL RELEVANCE: The results of this study show that the ABCB1-1Δ allele is present in Huntaway dogs. The low prevalence in this convenience sample suggests that the prevalence of this allele in the Huntaway population is likely to be low. We recommend that veterinary clinicians discuss the potential for this mutation in Huntaways with dog owners including the clinical implications for dogs that are homozygous for the mutated allele and the potential for testing for the mutation, as they would do for other known mutations.Item A novel mutation in IAA16 is associated with dicamba resistance in Chenopodium album(John Wiley and Sons Ltd on behalf of Society of Chemical Industry, 2024-07) Ghanizadeh H; He L; Griffiths AG; Harrington KC; Carbone V; Wu H; Tian K; Bo H; Xinhui DBACKGROUND: Resistance to dicamba in Chenopodium album was first documented over a decade ago, however, the molecular basis of dicamba resistance in this species has not been elucidated. In this research, the resistance mechanism in a dicamba-resistant C. album phenotype was investigated using a transcriptomics (RNA-sequence) approach. RESULTS: The dose-response assay showed that the resistant (R) phenotype was nearly 25-fold more resistant to dicamba than a susceptible (S) phenotype of C. album. Also, dicamba treatment significantly induced transcription of the known auxin-responsive genes, Gretchen Hagen 3 (GH3), small auxin-up RNAs (SAURs), and 1-aminocyclopropane-1-carboxylate synthase (ACS) genes in the susceptible phenotype. Comparing the transcripts of auxin TIR/AFB receptors and auxin/indole-3-acetic acid (AUX/IAA) proteins identified from C. album transcriptomic analysis revealed that the R phenotype contained a novel mutation at the first codon of the GWPPV degron motif of IAA16, resulting in an amino acid substitution of glycine (G) with aspartic acid (D). Sequencing the IAA16 gene in other R and S individuals further confirmed that all the R individuals contained the mutation. CONCLUSION: In this research, we describe the dicamba resistance mechanism in the only case of dicamba-resistant C. album reported to date. Prior work has shown that the dicamba resistance allele confers significant growth defects to the R phenotype investigated here, suggesting that dicamba-resistant C. album carrying this novel mutation in the IAA16 gene may not persist at high frequencies upon removal of dicamba application.Item A new mechanism for a familiar mutation - bovine DGAT1 K232A modulates gene expression through multi-junction exon splice enhancement(BioMed Central Ltd, 2020-08-26) Fink T; Lopdell TJ; Tiplady K; Handley R; Johnson TJJ; Spelman RJ; Davis SR; Snell RG; Littlejohn MDBACKGROUND: The DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Since first described over 15 years ago, a protein-coding variant K232A has been assumed as the causative variant underlying these effects, following in-vitro studies that demonstrated differing levels of triglyceride synthesis between the two protein isoforms. RESULTS: We used a large RNAseq dataset to re-examine the underlying mechanisms of this large milk production QTL, and hereby report novel expression-based functions of the chr14 g.1802265AA > GC variant that encodes the DGAT1 K232A substitution. Using expression QTL (eQTL) mapping, we demonstrate a highly-significant mammary eQTL for DGAT1, where the K232A mutation appears as one of the top associated variants for this effect. By conducting in vitro expression and splicing experiments in bovine mammary cell culture, we further show modulation of splicing efficiency by this mutation, likely through disruption of an exon splice enhancer as a consequence of the allele encoding the 232A variant. CONCLUSIONS: The relative contributions of the enzymatic and transcription-based mechanisms now attributed to K232A remain unclear; however, these results suggest that transcriptional impacts contribute to the diversity of lactation effects observed at the DGAT1 locus.Item A frameshift-deletion mutation in Reelin causes cerebellar hypoplasia in White Swiss Shepherd dogs(John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 2023-10) Littlejohn MD; Sneddon N; Dittmer K; Keehan M; Stephen M; Drögemüller M; Garrick DCerebellar hypoplasia is a heterogeneous neurological condition in which the cerebellum is smaller than usual or not completely developed. The condition can have genetic origins, with Mendelian-effect mutations described in several mammalian species. Here, we describe a genetic investigation of cerebellar hypoplasia in White Swiss Shepherd dogs, where two affected puppies were identified from a litter with a recent common ancestor on both sides of their pedigree. Whole genome sequencing was conducted for 10 dogs in this family, and filtering of these data based on a recessive transmission hypothesis highlighted five protein-altering candidate variants - including a frameshift-deletion of the Reelin (RELN) gene (p.Val947*). Given the status of RELN as a gene responsible for cerebellar hypoplasia in humans, sheep and mice, these data strongly suggest the loss-of-function variant as underlying these effects. This variant has not been found in other dog breeds nor in a cohort of European White Swiss Shepherds, suggesting a recent mutation event. This finding will support the genotyping of a more diverse sample of dogs, and should aid future management of the harmful allele through optimised mating schemes.Item The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis(BioMed Central Ltd, 2021-06-17) Jivanji S; Harland C; Cole S; Brophy B; Garrick D; Snell R; Littlejohn M; Laible GBACKGROUND: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.Item Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon(Oxford University Press on behalf of The Society for Molecular Biology and Evolution (SMBE), 2021-07) Karkare K; Lai H-Y; Azevedo RBR; Cooper TF; Wittkopp PPopulations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.Item Dynamics of bacterial adaptation(Portland Press Limited on behalf of the Biochemical Society, 2021-04-12) Lai H-Y; Cooper TFDetermining pattern in the dynamics of population evolution is a long-standing focus of evolutionary biology. Complementing the study of natural populations, microbial laboratory evolution experiments have become an important tool for addressing these dynamics because they allow detailed and replicated analysis of evolution in response to controlled environmental and genetic conditions. Key findings include a tendency for smoothly declining rates of adaptation during selection in constant environments, at least in part a reflection of antagonism between accumulating beneficial mutations, and a large number of beneficial mutations available to replicate populations leading to significant, but relatively low genetic parallelism, even as phenotypic characteristics show high similarity. Together, there is a picture of adaptation as a process with a varied and largely unpredictable genetic basis leading to much more similar phenotypic outcomes. Increasing sophistication of sequencing and genetic tools will allow insight into mechanisms behind these and other patterns.Item Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor(MDPI (Basel, Switzerland), 2021-02-12) Barzak FM; Ryan TM; Kvach MV; Kurup HM; Aihara H; Harris RS; Filichev VV; Harjes E; Jameson GB; Chelico LIn normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.Item Identifying colorectal cancer caused by biallelic MUTYH pathogenic variants using tumor mutational signatures(Springer Nature Limited, 6/06/2022) Georgeson P; Harrison TA; Pope BJ; Zaidi SH; Qu C; Steinfelder RS; Lin Y; Joo JE; Mahmood K; Clendenning M; Walker R; Amitay EL; Berndt SI; Brenner H; Campbell PT; Cao Y; Chan AT; Chang-Claude J; Doheny KF; Drew DA; Figueiredo JC; French AJ; Gallinger S; Giannakis M; Giles GG; Gsur A; Gunter MJ; Hoffmeister M; Hsu L; Huang W-Y; Limburg P; Manson JE; Moreno V; Nassir R; Nowak JA; Obón-Santacana M; Ogino S; Phipps AI; Potter JD; Schoen RE; Sun W; Toland AE; Trinh QM; Ugai T; Macrae FA; Rosty C; Hudson TJ; Jenkins MA; Thibodeau SN; Winship IM; Peters U; Buchanan DDCarriers of germline biallelic pathogenic variants in the MUTYH gene have a high risk of colorectal cancer. We test 5649 colorectal cancers to evaluate the discriminatory potential of a tumor mutational signature specific to MUTYH for identifying biallelic carriers and classifying variants of uncertain clinical significance (VUS). Using a tumor and matched germline targeted multi-gene panel approach, our classifier identifies all biallelic MUTYH carriers and all known non-carriers in an independent test set of 3019 colorectal cancers (accuracy = 100% (95% confidence interval 99.87-100%)). All monoallelic MUTYH carriers are classified with the non-MUTYH carriers. The classifier provides evidence for a pathogenic classification for two VUS and a benign classification for five VUS. Somatic hotspot mutations KRAS p.G12C and PIK3CA p.Q546K are associated with colorectal cancers from biallelic MUTYH carriers compared with non-carriers (p = 2 × 10-23 and p = 6 × 10-11, respectively). Here, we demonstrate the potential application of mutational signatures to tumor sequencing workflows to improve the identification of biallelic MUTYH carriers.