Mouse p53 blocks SV40 DNA replication in vitro and downregulates T antigen DNA helicase activity.

Immunopurified mouse p53 proteins were used to gain experimental access to the mechanisms underlying nonprimate p53 directed suppression of SV40 origin directed DNA replication in vivo. In replication competent HeLa cell extracts containing exogenous T antigen, mouse p53 blocks T antigen dependent DNA synthesis as in vivo. However, in transcription competent HeLa extracts, mouse p53 has no effect either on overall transcription or on the ability of immunopurified T antigen to downregulate SV40 early transcription. We show that although mouse p53 has no significant effect on T antigen encoded activities such as ATPase and DNA binding, helicase activity is somewhat reduced suggesting that the in vivo suppression by mouse p53 of SV40 replication may be due, at least in part, to direct modulation of T antigen function.

p53 interacts with p34cdc2 in mammalian cells: implications for cell cycle control and oncogenesis.

The p53 gene product has been implicated in both human and animal tumorigenesis. p53 complexes with the transforming proteins encoded by several different DNA tumour viruses. We demonstrate that human p53 is phosphorylated by the mammalian p34cdc2 kinase in vitro and coprecipitates with p34cdc2 in vivo. Our observations suggest that phosphorylation of p53 by p34cdc2 kinase may regulate the known activities of p53 in the initiation steps of DNA replication in mammalian cells.

A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization.

The p53 gene product has been implicated in both human and animal tumorigenesis. p53 forms heterologous complexes with the transforming proteins encoded by several different DNA tumor viruses. p53 also assembles into stable homo-oligomers. We demonstrate that the major structural determinant for the tetramerization of p53 is an alpha-helical plus basic region motif near the C-terminus of the protein. A monomeric p53 mutant adopts a conformation distinct from both 'wild-type' and 'mutant' form as defined by PAb1620 and PAb240 monoclonal antibody recognition. Nevertheless, monomeric and dimeric mutant p53 proteins retain the ability to suppress SV40 origin-directed DNA replication in vivo. Thus, p53-p53 interaction and expression of the PAb1620 epitope is not a prerequisite for such activity. We present data suggesting that suppression of replication by p53 may occur by a mechanism that is independent of detectable p53-T antigen association.

Natural paratuberculosis infection in rabbits in Scotland.

Natural paratuberculosis infection of rabbits (Oryctolagus cuniculus) was recently diagnosed in Scotland, and an investigation into the pathology of the disease in wild rabbits is reported in this paper. Evidence of Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) infection was detected in 22% of 110 rabbits; the organism was cultured from 17 of 110 rabbits, and histopathological lesions consistent with M.a. paratuberculosis infection were noted in 18 of 98 rabbits examined. No macroscopical lesions suggestive of M.a. paratuberculosis infection were observed. The histopathological lesions were either severe or mild. Severe lesions consisted of extensive macrophage granulomata and numerous giant cells, with many intracellular acid-fast bacteria in the small intestine. For the examination of formalin-fixed, paraffin wax-embedded tissues, neither immunohistochemistry nor the polymerase chain reaction was as sensitive a method of diagnosis as histopathology.

Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53.

Malignant transformation of primary cells requires at least two distinct and characteristic alterations in cellular behaviour. The first, cellular immortality, can be induced by chemical carcinogens or by cloned oncogenes such as polyoma large T (ref. 4), adenovirus early region 1A (E1A) or the oncogene from avian (MC29) myelocytomatosis virus, v-myc. Cells whose in vitro life-span has been extended by these procedures can be fully transformed by transfection with oncogenes belonging to a different complementation group, including genes of the ras family, adenovirus E1b and polyoma virus middle T (refs 4, 5). The unstable cellular phosphoprotein p53 is frequently present at elevated levels in transformed cells and is stabilized by the formation of complexes with simian virus 40 (SV40) large T or adenovirus E1b 57K protein. Although several reports have associated p53 with cell proliferation, its role remains obscure. We have cloned complementary DNA sequences encoding murine p53 and report here that transfection of p53 expression constructs into cells of finite lifespan in vitro results in cellular immortality and susceptibility to transformation by a ras oncogene.

The cellular oncogene p53 can be activated by mutagenesis.

P53 is a cellular phosphoprotein of short half-life (t1/2) which is present at elevated levels in cells transformed by various stimuli including viruses, chemicals and radiation. p53 forms specific stable complexes with simian virus 40 (SV40) large-T antigen and an adenovirus E1b protein of relative molecular mass (Mr) 57,000. A number of reports have associated p53 with cell proliferation, and p53 complementary DNA expression constructs immortalize primary cells in vitro and render them sensitive to transformation by an activated ras oncogene. We have examined the biological properties of a set of p53 expression constructs, and report here that cellular immortalization by a wild-type p53 cDNA gene is conditional upon the promoter/enhancer construction used, but that p53 can extend cellular lifespan by a second distinct mechanism involving rearrangements of the coding sequence which give rise to stable protein products. Cells immortalized by one of these mutants are refractory to subsequent transformation by a ras oncogene, indicating that cellular immortalization and ras cooperation are separate activities.

Cloning and expression analysis of full length mouse cDNA sequences encoding the transformation associated protein p53.

We have cloned and sequenced overlapping cDNA fragments which together encode the entire mouse protein p53. Using these cDNA's we have reconstructed the full length coding region for the protein, and have analysed its coding potential by expression in vitro, both as a full length sequence and as a subfragment contained in a fusion protein. The predicted amino acid sequence contains no obvious homologies to any known oncogenes but includes a possible tyrosine kinase acceptor site.

Mouse p53 inhibits SV40 origin-dependent DNA replication.

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.

Paratuberculosis infection of nonruminant wildlife in Scotland.

Recent reports of natural paratuberculosis (or Johne's disease) in rabbits, foxes, and stoats has focused debate on the presence and importance of wildlife reservoirs in the epidemiology of this disease. This paper describes an extensive study investigating 18 nonruminant wildlife species for evidence of paratuberculosis. Using both culture and histopathological analysis, fox, stoat, weasel, crow, rook, jackdaw, rat, wood mouse, hare, and badger were found to harbor Mycobacterium avium subsp. paratuberculosis, the causative organism of paratuberculosis, suggesting that the epidemiology of this disease is more complex than previously realized.

Experimental paratuberculosis in calves following inoculation with a rabbit isolate of Mycobacterium avium subsp. paratuberculosis.

The role of wildlife species in the epidemiology of paratuberculosis has been the subject of increased research efforts following the discovery of natural paratuberculosis in free-living rabbits from farms in east Scotland. This paper describes the experimental inoculation of young calves with an isolate of Mycobacterium avium subsp. paratuberculosis recovered from a free-living rabbit. After a 6-month incubation period, all eight calves inoculated with the rabbit isolate had developed histopathological and/or microbiological evidence of M. avium subsp. paratuberculosis infection. Similar results were obtained from a group of calves infected with a bovine isolate of M. avium subsp. paratuberculosis. The virulence of the rabbit isolate for calves demonstrated in this study suggests that rabbits are capable of passing paratuberculosis to domestic ruminants and that wildlife reservoirs of M. avium subsp. paratuberculosis should therefore be considered when formulating control plans for the disease.