Control of the gua operon of Escherichia coli K-12

114P dehydrogenase-less (guaB) mutants of Escherichia coli were isolated following chemical mutagenesis. Two ua mutants (I:S1021 and PL1138) were found to be temperate-sensitive. A cell-free extract of NS1021 gave only weak precipitin lines on Ouchterlony double diffusion gels whereas a cell-free extract of PL1138 gave precipitin lines as intense as those from a g„RT strain. Complementation in vitro between paB mutants revealed two complementing groups. One group has only two memmers NS1021 and PL1138, the other group is comprised of all other CR14 mutants. Complementation is only observed between the two groups and not within either group. Complementation using NS1021 always resulted in lower enzyme activities compared with PL1138. Alkylation of mutant extracts using Cl-31D prior to complementation revealed that both NS1021 and PL1138 provide the catalytic subunits in the hybrid enzyme. The regulation of the gua operon of E.coli was investigated using lac fusion strains isolated by the general method of Casadaban 1976, J.Nol.Biol. 104, 51+1). The lao genes were shown to be regulated as part of the gua,opercn by cnse to the addition of purines and purine analogues to the growth medium. Expression of the lac genes was derepressed during growth in guanine-limited medium. Both guaA-lac and guaB-lac fusions exhibited the same degree of derepxession of j3-galactesidase synthesis upon guanine starvation (approximately seven-fold) compared with fifty-fold for IMP dehydrogenase (in a guaA mutant) and twenty-fold for GMP synthetase (in a guaB mutant). The reason for the similar low derepression ratios may be due to the presence of a rho-dependent transcription termination sequence between Lua and lac. Regulatory mutants were sought from the ua--lac fusions. Mutants with increased levels of lac expression were isolated using a variety of procedures, however none of these mutants had altered expression of R. The apparent regulatory mutants were probably due to mutations in the -L--D DNA beeween gua and lac. The introduction of a placid carrying the R promoter and promoter proTimal portion of guaB into a strain with a aaa-lac fusion caused an alteration in expression of the fused lac genes which was dependent upon the growth medium. In minimal glucose medium the introduction of of the gua plasmid caused an increase in chromosomal gua expression whereas in L-broth the presence of the plasmid had the opposite effect. This suggests that gua operon expression may be under metabolic control. Analysis of transcription in vitro revealed only one promoter on the 2.1 kb DNA fragment used to construct the gua plasmid mentioned above. The transcript produced from this isolated DNA fragment was approximately 630 bases. Experiments to demonstrate the specific nucleoside or nucleotide involved in control of the qua, operon proved inconclusive.</p

Control of the gua operon of Escherichia coli K-12

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The receptor binding site of feline leukemia virus surface glycoprotein is distinct from the site involved in virus neutralization

The external surface glycoprotein (SU) of feline leukemia virus (FeLV) contains sites which define the viral subgroup and induce virus-neutralizing antibodies. The subgroup phenotypic determinants have been located to a small variable region, VR1, towards the amino terminus of SU. The sites which function as neutralizing epitopes in vivo are unknown. Recombinant SU proteins were produced by using baculoviruses that contained sequences encoding the SUs of FeLV subgroup A (FeLV-A), FeLV-C, and two chimeric FeLVs (FeLV-215 and FeLV-VC) in which the VR1 domain of FeLV-A had been replaced by the corresponding regions of FeLV-C isolates. The recombinant glycoproteins, designated Bgp70-A, -C, -215, and -VC, respectively, were similar to their wild-type counterparts in several immunoblots and inhibited infection of susceptible cell lines in a subgroup-specific manner. Thus, Bgp70-A interfered with infection by FeLV-A, whereas Bgp70-C, -VC, and -215 did not. Conversely, Bgp70-C, -VC, and -215 blocked infection with FeLV-C, while Bgp70-A had no effect. These results indicate that the site on SU which binds to the FeLV cell surface receptor was preserved in the recombinant glycoproteins. It was also found that the recombinant proteins were able to bind naturally occurring neutralizing antibodies. Bgp70-A, -VC, and -215 interfered with the action of anti-FeLV-A neutralizing antibodies, whereas Bgp70-C did not. Furthermore, Bgp70-C interfered with the action of anti-FeLV-C neutralizing antibodies, while the other proteins did not. These results indicate that the neutralizing epitope(s) of FeLV SU lies outside the subgroup-determining VR1 domain

Development of a Novel Canine Parvovirus Vaccine Capable of Stimulating Protective Immunity in Four-Week-Old Puppies in the Face of High Levels of Maternal Antibodies

Many highly effective vaccines have been developed to protect dogs against disease caused by canine parvovirus, but despite this vaccine interference by maternally derived antibodies continues to cause immunisation failure. To help overcome this limitation we have developed a novel, recombinant canine parvovirus type 2c vaccine strain, based on the structural and non-structural elements of an established type 2 vaccine. This novel CPV-2c vaccine strain has unique efficacy in the field, it is able to induce sterilising immunity in naïve animals 3 days after vaccination and is able to overcome very high levels of maternally derived antibodies from 4 weeks of age—thus closing the immunity gap to canine parvovirus infection in young puppies. The vaccine strain, named 630a, has been combined with an established canine distemper virus Onderstepoort vaccine strain to produce a new bivalent vaccine (Nobivac DP PLUS), intended to immunise very young puppies in the face of high levels of maternally derived antibody. Here, we describe the onset of immunity and maternal antibody interference studies that support the unique efficacy of the strain, and present overdose studies in both dogs and cats that demonstrate the vaccine to be safe

Cutaneous lesions in pet rabbits following subcutaneous administration of a novel bivalent vaccine against myxomatosis and rabbit haemorrhagic disease

BACKGROUND: A novel bivalent vaccine to protect against myxomatosis and rabbit haemorrhagic disease is commercially available for pet rabbits. HYPOTHESIS/OBJECTIVES: To describe the appearance of cutaneous lesions arising in pet rabbits positive for myxoma virus (MV) by RT-PCR evaluation shortly after vaccination. ANIMALS: Four pet rabbits presenting with papular, crusting skin lesions ~10 days after vaccination. METHODS: Histological evaluation of formalin-fixed skin biopsies obtained from lesional skin (case 1). Real-time polymerase chain reaction (RT-PCR) evaluation of paraffin-embedded tissue from skin biopsies (case 1) and crusts obtained from the lesion surface (cases 2-4) for myxoma virus are reported as cycle threshold (Ct ) values. RESULTS: Lesions affecting the ear pinna, dorsal aspect of the nose, vulva and/or conjunctiva are reported. Histopathological findings included severe ulcerative, necrotizing dermatitis and intralesional cytoplasmic inclusion bodies in myxoma cells. DNA was amplified from all the paraffin-embedded skin biopsies (Ct  = 34-35) and crusts (Ct  = 20-24). CONCLUSIONS AND CLINICAL IMPORTANCE: Although a wild virus challenge cannot be definitively excluded, veterinarians and pet-owners should be aware that cutaneous lesions have been observed after vaccination with this novel vaccine in low numbers of rabbits

Canine Parvovirus Type 2 Vaccine Protects Against Virulent Challenge with Type 2c Virus

Abstract The ability of dogs vaccinated with a live attenuated CPV type 2 (Nobivac Intervet) vaccine to resist challenge with a current CPV2c isolate was investigated. Six SPF beagle dogs were given the minimum recommended course of vaccination, comprising a single inoculation of vaccine (Nobivac Lepto + Nobivac Pi) at 8-10 weeks of age followed 3 weeks later with a parvovirus vaccine in combination with distemper, adenovirus and parainfluenza virus (Nobivac DHPPi) and a repeat leptospirosis vaccine. Six control dogs were kept unvaccinated. All animals were challenged orally with a type 2c isolate of CPV and monitored for clinical signs, virus shedding, white blood cell fluctuations and serological responses. All vaccinated dogs were fully protected; showing no clinical signs nor shedding challenge virus in the faeces, in contrast to control animals, which displayed all the typical signs of infection with pathogenic CPV and shed challenge virus in the faeces.