Mike Boursnell

Non-essential Genes in the Vaccinia Virus HindIII K Fragment: a Gene Related to Serine Protease Inhibitors and a Gene Related to the 37K Vaccinia Virus Major Envelope Antigen

The complete nucleotide sequence of a cloned copy of the HindIII K fragment of the WR strain of vaccinia virus has been determined. Eight open reading frames (ORFs) have been identified, on the basis of size and codon usage. The predicted amino acid sequences of the putative genes have been compared to the Protein Identification Resource and to published vaccinia virus sequences. One gene, predicted to encode a 42.2K protein, is highly related to the family of serine protease inhibitors. It shows approximately 25% identity to human antithrombin III and 19% identity to the cowpox virus 38K protein gene which is also related to serine protease inhibitors. The product of another gene shows a similar high level of identity to the 37K vaccinia virus major envelope antigen. The existence of viable deletion mutants and recombinants containing foreign DNA inserted into both these genes indicates that they are non-essential.

Sequence analysis of an 11.2 kilobase, near-terminal, BamHI fragment of fowlpox virus.

The nucleotide sequence of an 11.2 kilobase fragment of the fowlpox virus genome is presented. The fragment comes from near one end of the genome and contains part of the terminal inverted repeat. Twenty open reading frames (ORFs) are predicted from the sequence and are classified into 13 major and seven minor ORFs. The 100 base pairs immediately upstream of each ORF are up to 83% AT-rich, with some motifs similar to those seen in vaccinia virus early gene promoters. The TTTTTNT element which has been identified as a termination signal for vaccinia virus early genes is also found downstream of several ORFs. Three ORFs are predicted to specify polypeptides with significant homology to proteins coded by genes near termini of orthopoxvirus genomes: the vaccinia virus 42K early gene and 32.5K host range gene, and the cowpox virus 38K red pock gene. In addition, there are two families of ORFs within the fragment which potentially encode related polypeptides. One of these, family B, contains three ORFs which are related to those of chicken and rat hepatic lectins.

A frameshift mutation in golden retriever dogs with progressive retinal atrophy endorses SLC4A3 as a candidate gene for human retinal degenerations.

Progressive retinal atrophy (PRA) in dogs, the canine equivalent of retinitis pigmentosa (RP) in humans, is characterised by vision loss due to degeneration of the photoreceptor cells in the retina, eventually leading to complete blindness. It affects more than 100 dog breeds, and is caused by numerous mutations. RP affects 1 in 4000 people in the Western world and 70% of causal mutations remain unknown. Canine diseases are natural models for the study of human diseases and are becoming increasingly useful for the development of therapies in humans. One variant, prcd-PRA, only accounts for a small proportion of PRA cases in the Golden Retriever (GR) breed. Using genome-wide association with 27 cases and 19 controls we identified a novel PRA locus on CFA37 (praw = 1.94×10−10, pgenome = 1.0×10−5), where a 644 kb region was homozygous within cases. A frameshift mutation was identified in a solute carrier anion exchanger gene (SLC4A3) located within this region. This variant was present in 56% of PRA cases and 87% of obligate carriers, and displayed a recessive mode of inheritance with full penetrance within those lineages in which it segregated. Allele frequencies are approximately 4% in the UK, 6% in Sweden and 2% in France, but the variant has not been found in GRs from the US. A large proportion of cases (approximately 44%) remain unexplained, indicating that PRA in this breed is genetically heterogeneous and caused by at least three mutations. SLC4A3 is important for retinal function and has not previously been associated with spontaneously occurring retinal degenerations in any other species, including humans.

Expression of the Infectious Bronchitis Virus Spike Protein by Recombinant Vaccinia Virus and Induction of Neutralizing Antibodies in Vaccinated Mice

A cDNA clone of the infectious bronchitis virus (IBV) spike protein gene has been recombined into vaccinia virus. Cells infected with the recombinant virus synthesized IBV spike antigen which was recognized by antibody raised against purified spike protein. Immunofluorescence showed that the IBV spike antigen was transported to the infected cell surface membrane and immunoprecipitation showed the presence of the glycosylated 180K mol. wt. polypeptide precursor of the two spike subunits S1 and S2 that comigrated with this antigen from IBV-infected cells. Vaccinated mice produced antibody that recognized the IBV spike antigen by ELISA and which neutralized IBV infectivity as shown by ciliostasis tests on tracheal organ cultures.

Copper toxicosis in non-COMMD1 Bedlington terriers is associated with metal transport gene ABCA12

Wilsons disease, caused by a mutation in the ATP-ase 7B gene, is the only genetically characterised human disease with inhibition of biliary copper excretion and toxic copper accumulation in liver and occasionally brain. A similar copper toxicosis occurs in Bedlington terriers (CT) with liver damage only. Although CT has been associated with a defect in the COMMD1 gene (COMMD1 (del/del)), Bedlington terriers with CT and lacking this mutation are also recognised (non-COMMD1 (del/del)). A study was designed to identify any other gene polymorphisms associated with copper toxicity in Bedlington terriers employing genome wide association studies (GWAS) followed by deep sequencing of the candidate region. Blood for DNA analysis and liver for confirmation of the diagnosis was obtained from 30 non-COMMD1 (del/del) Bedlington terriers comprising equal numbers of CT-affected dogs and controls. DNA was initially subjected to GWAS screening and then further sequencing to target the putative mutant gene. The study has identified a significant disease association with a region on chromosome 37 containing identified SNPs which are highly significantly associated with non-COMMD1 (del/del) Bedlington terrier CT. This region contains the ABCA12 gene which bears a close functional relationship to ATP-ase 7B responsible for Wilsons disease in man.

Genome sequencing reveals a splice donor site mutation in the SNX14 gene associated with a novel cerebellar cortical degeneration in the Hungarian Vizsla dog breed

BackgroundCerebellar cortical degeneration (CCD) is an increasingly recognised neurodegenerative disease process affecting many dog breeds. Typical presentation consists of a progressive cerebellar ataxia, with a variable age at onset and rate of progression between different breeds. Cerebellar histopathological findings typically consist of primary Purkinje neuronal degeneration and loss, with variable secondary depletion of the granular and molecular cell layers. Causative genes have been identified associated with CCD in several breeds, allowing screening for selective breeding to reduce the prevalence of these conditions. There have been no previous reports of CCD in Hungarian Vizslas.ResultsTwo full-sibling Hungarian Vizsla puppies from a litter of nine presented with a history of progressive ataxia, starting around three months of age. Clinical signs included marked hypermetric and dysmetric ataxia, truncal sway, intention tremors and absent menace responses, with positional horizontal nystagmus in one dog. Routine diagnostic investigations were unremarkable, and magnetic resonance imaging performed in one dog revealed mild craniodorsal cerebellar sulci widening, supportive of cerebellar atrophy. Owners of both dogs elected for euthanasia shortly after the onset of signs. Histopathological examination revealed primary Purkinje neuron loss consistent with CCD. Whole genome sequencing was used to successfully identify a disease-associated splice donor site variant in the sorting nexin 14 gene (SNX14) as a strong causative candidate. An altered SNX14 splicing pattern for a CCD case was demonstrated by RNA analysis, and no SNX14 protein could be detected in CCD case cerebellum by western blotting. SNX14 is involved in maintaining normal neuronal excitability and synaptic transmission, and a mutation has recently been found to cause autosomal recessive cerebellar ataxia and intellectual disability syndrome in humans. Genetic screening of 133 unaffected Hungarian Vizslas revealed the presence of three heterozygotes, supporting the presence of carriers in the wider population.ConclusionsThis is the first report of CCD in Hungarian Vizsla dogs and identifies a highly associated splice donor site mutation in SNX14, with an autosomal recessive mode of inheritance suspected.

The Complete Nucleotide Sequence of Avian Infectious Bronchitis Virus: Analysis of the Polymerase-Coding Region

Avian infectious bronchitis virus (IBV) is the type species of the family Coronaviridae (Siddell et al., 1983). It has a large positive-stranded RNA genome which has been estimated at 20–24 kilobases (Lomniczi & Kennedy, 1977). As with other coronaviruses, a number of subgenomic messenger RNA species are produced in infected cells which form a 3′-coterminal nested set (Stern & Kennedy, 1980a; 1980b). In the case of IBV there are six mRNA species in total, which are designated mRNAs A-F, mRNA A being the smallest, and mRNA F being of genome size. mRNAs A, C and E encode the three main structural components of the virion, the nucleocapsid polypeptide, the membrane polypeptide and the precursor polypeptide to the spike (Stern & Sefton, 1984). mRNA D encodes at least one product, a 12.4 kilodalton polypeptide of unknown function (Smith et al., this volume), but no product has yet been detected for mRNA B. The coding regions of mRNAs A-E are situated in the 3′-most 7.3 kilobases of the IBV genome, the nucleotide sequence of which has been determined previously from cDNA clones (Boursnell et al., 1984, 1985a, 1985b; Boursnell & Brown, 1984; Binns et al., 1985b).

Defining the ABC of gene essentiality in streptococci

BackgroundUtilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci.ResultsSix barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species.ConclusionsThe use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

Expression of IBV Spike Protein by a Vaccinia Virus Recombinant

The spike protein of IBV is a target for virus neutralising antibodies in the chicken (Cavanagh et al, 1984; Mockett, 1985). Vaccinia virus, a poxvirus, has been shown to be a useful and efficient eukaryotic expression vector. Here we describe the expression of the IBV spike from a cDNA clone inserted into vaccinia virus and demonstrate that mice vaccinated with the recombinant produce antibodies to the spike which can neutralise virus infectivity.