Autonomous replication sequences in an extrachromosomal element of a pathogenic Entamoeba histolytica.

Entamoeba histolytica possesses a 24.5 kilobase plasmid-like molecule which encodes for the organism's ribosomal RNAs. Sequence analysis of this extrachromosomal element revealed the presence of AT rich sequences which show homology to the origin of replication of other lower eucaryotes. An 802 bp fragment containing these sequences was cloned into a yeast shuttle vector lacking the origin of replication and the construct tested for its ability to replicate autonomously in yeast. Mitotic stability tests as well as evidence for plasmid maintenance indicate that the transformed cells contained self-replicating episomes and not stably integrated molecules. The nucleotide sequence of this ARS-containing fragment is presented.     

The taxonomic status of "Halobacterium marismortui" from the Dead Sea: a comparison with Halobacterium vallismortis

A Halobacterium strain, isolated by Ginzburg et al. from the Dead Sea in the late 1960's, often referred to as "Halobacterium marismortui" or "Halobacterium of the Dead Sea" (deposited in the American Type Culture Collection as ATCC 43049) was compared with Halobacterium (Haloarcula) vallismortis ATCC 29715. The strains appeared to be very closely related, as shown by the near identity of their 5S and 16S ribosomal RNA's, and a large number of other common properties. Distinct differences exist, however, in cell morphology, and in their potency to utilize different sugars and other compounds.     

Enhanced binding of a 95 kDa protein to p53 in cells undergoing p53-mediated growth arrest.

To explore the biochemical functions of p53, we have initiated a search for cellular p53-binding proteins. Coprecipitation of three polypeptides was observed when cell lines overexpressing a temperature-sensitive (ts) p53 mutant were maintained at 32.5 degrees C (wild-type p53 activity, leading to growth arrest) but not at 37.5 degrees C (mutant p53 activity). One of these three proteins, designated p95 on the basis of its apparent molecular mass, was highly abundant in p53 immune complexes. We demonstrate herein that p95 is a p53-binding protein, which exhibits poor p53-binding in cells overproducing several distinct mutant p53 proteins. Yet, p95 associates equally well with both the wild-type (wt) and the mutant conformations of the ts p53 in transformed cells growth-arrested at 32.5 degrees C. On the basis of our findings we suggest that wt p53 activity increases p53-p95 complex formation and that such interaction may play a central role in p53 mediated tumour suppression.     

Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding.

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.     

Simian virus 40 can overcome the antiproliferative effect of wild-type p53 in the absence of stable large T antigen-p53 binding.

In simian virus 40 (SV40)-transformed cells, a tight complex is formed between the viral large T antigen (large T) and p53. It has been proposed that this complex interferes with the antiproliferative activity of p53. This notion was tested in primary rat fibroblasts by assessing the ability of SV40-mediated transformation to be spared from the inhibitory effect of wild-type (wt) p53. The data indicate that relative to transformation induced by myc plus ras, SV40-plus-ras-mediated focus formation was indeed much less suppressed by p53 plasmids. A majority of the resultant cell lines made a p53 protein with properties characteristic of a wt conformation. Furthermore, cell lines expressing stably both SV40 large T and a temperature-sensitive p53 mutant continued to proliferate at a temperature at which this p53 assumes wt-like properties and normally causes a growth arrest. Surprisingly, at least partial resistance to the growth-inhibitory effect of wt p53 was also evident when transformation was mediated by an SV40 deletion mutant, encoding a large T which does not bind p53 detectably. In addition to supporting the idea that SV40 can overcome the growth-restrictive activity of wt p53, these findings strongly suggest that at least part of this effect does not require a stable association between p53 and large T.     

Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein.

A temperature-sensitive mutant of p53, p53Val-135, was found to be able to arrest cell proliferation when overexpressed at 32.5 degrees C. While much of the protein was cytoplasmic in cells proliferating at 37.5 degrees C, it became predominantly nuclear at 32.5 degrees C. Concomitantly, p53Val-135 became destabilized, although not to the extent seen in primary fibroblasts.     

Nonlinear cable properties of the giant axon of the cockroach Periplaneta americana

The steady state nonlinear properties of the giant axon membrane of the cockroach Periplaneta americana were studied by means of intracellular electrodes. The resistivity of this membrane markedly decreases in response to small subthreshold depolarizations. The specific slope resistance is reduced by twofold at 5 mV depolarization and by a factor of 14 at 20 mV depolarization. As a result, the spatial decay, V(X), of depolarizing potentials is enhanced when compared with the passive (exponential) decay. This enhancement is maximal at a distance of 1-1.5 mm from a point of subthreshold (0-20 mV) depolarizing perturbation. At that distance, the difference between the actual potential and the potential expected in the passive axon is approximately 30%. The effects of membrane rectification on V(X) were analyzed quantitatively with a novel derivation based on Cole's theorem, which enables one to calculate V(X) directly from the input current-voltage (I0-V) relation of a long axon. It is shown that when the experimental I0-V curve is replotted as (I0Rin)-1 against V (where Rin is the input resistance at the resting potential), the integral between any two potentials (V1 greater than V2) on this curve is the distance, in units of the resting space constant, over which V1 attenuates to V2. Excellent agreement was found between the experimental V(X) and the predicted value based solely on the input I0-V relation. The results demonstrate that the rectifying properties of the giant axon membrane must be taken into account when the electrotonic spread of even small subthreshold potentials is studied, and that, in the steady state, this behavior can be extracted from measurements at a single point. The effect of rectification on synaptic efficacy is also discussed.     

p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors.

The relative levels of mRNA specific for the mouse p53 cellular tumor antigen were determined in various normal adult tissues, embryos, and tumors. All tumors studied contained concentrations of p53 mRNA well above those present in most normal tissues. Normal spleen, however, had p53 mRNA levels comparable to those found in some tumors, despite the fact that they contained barely detectable p53 protein. This apparent discrepancy was found to be due to the extremely rapid turnover rate of p53 in the spleen (half-life, approximately equal to 6 min). In developing fetuses, a marked reduction of p53 mRNA levels was manifest from day 11 onwards, whereas the levels during organogenesis (days 9 to 11) were comparable to those found in undifferentiated embryonic stem cells and in some tumors.     

Overproduction of protein p53 contributes to simian virus 40-mediated transformation.

The possible involvement of p53 overproduction in simian virus 40 (SV40)mediated transformation was studied by using the rat embryo fibroblast focus formation assay. Transformation by wild-type SV40 was enhanced two- to threefold by cotransfection of a plasmid overexpressing mouse p53. More significantly, such a plasmid could partially complement a transformation-defective deletion mutant of SV40. Hence, the ability of SV40 T antigen to induce high p53 levels may indeed be directly relevant to the viral transforming potential.