Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.     

HLA-dependent GM effects in insulin-dependent diabetes: evidence from pairs of affected siblings.

In a recent study of GM allotype frequencies in HLA-defined subsets of patients with insulin-dependent diabetes mellitus (IDDM) and similarly defined healthy sibling controls, we found evidence for an HLA-dependent GM effect on IDDM susceptibility. To circumvent problems inherent in such patient-control studies of complex diseases, we have now examined sharing of genes in the HLA and GM regions in 26 informative pairs of siblings who were both affected with IDDM. We found that: (1) in the total sample of affected sib-pairs, sharing of two HLA haplotypes was increased compared to Mendelian expectations, in agreement with many previous studies; (2) in the total sample, sharing of GM region genes (as measured by GM phenotype concordance) was not different from Mendelian expectations, given the distribution of parental mating types; and (3) affected sib-pairs who shared two HLA haplotypes showed significantly increased sharing of GM region genes compared to affected sib-pairs who shared one or zero HLA haplotypes (P = .018). These results provide new evidence for HLA-dependent effects of a locus at or near GM on susceptibility to IDDM.     

Sample-size considerations and strategies for linkage analysis in autosomal recessive disorders.

The opportunity raised by recombinant DNA technology to develop a linkage marker panel that spans the human genome requires cost-efficient strategies for its optimal utilization. Questions arise as to whether it is more cost-effective to convert a dimorphic restriction enzyme marker system into a highly polymorphic system or, instead, to increase the number of families studied, simply using the available marker alleles. The choice is highly dependent on the population available for study, and, therefore, an examination of the informational content of the various family structures is important to obtain the most informative data. To guide such decisions, we have developed tables of the average sample number of families required to detect linkage for autosomal recessive disorders under single backcross and under "fully informative" matings. The latter cross consists of a marker locus with highly polymorphic codominant alleles such that the parental marker genotypes can be uniquely distinguished. The sampling scheme considers families with unaffected parents of known mating types ascertained via affected offspring, for sibship sizes ranging from two to four and various numbers of affected individuals. The sample-size tables, calculated for various values of the recombination fractions and lod scores, may serve as a guide to a more efficient application of the restriction fragment length polymorphism technology to sequential linkage analysis.     

Variation in antigenic determinants of p53 transformation-related protein obtained from various species

p53 is a cellular-encoded transformation-related protein. It is synthesized at elevated levels in tumor cells but has also been detected at low concentrations in several types of nontransformed cells. The p53 of tumor cells is immunogenic and elicits specific antibody production. The antigenic determinants of the p53 protein were studied by specific binding to anti-p53 monoclonal antibodies obtained from the RA3-2C2, PAb122, and PAb421 established hybridoma cell lines, and their conservation was followed in various animal species. We found that whereas mouse p53 efficiently immunoprecipitated with all three anti-p53 monoclonal antibodies, human and rat p53 bound PAb122 and PAb421 but lacked a determinant binding RA3-2C2. The hamster p53 molecule represented a third category, which immunoprecipitated with polyclonal anti-p53 antibodies but failed to bind all three monoclonal antibodies analyzed here. Using these monoclonal antibodies, we detected no variations between p53 found in transformed and p53 found in nontransformed cells, within a given species. The results also showed that RA3-2C2, which recognizes a mouse-specific determinant, binds a site located at a proteolytic digestion fragment of the p53 molecule that differs from that containing PAb122 and PAb421 recognition site(s). p53 is a single protein that can be immunoprecipitated through different antigenic determinants that vary between species.     

Involvement of ethylene in the abscission of flowers and petals of Leptospermum scoparium

The growth of cotyledons and primary leaves of I-day-old Sinapis alba L. plants were studied under various light conditions and action spectra produced. For both responses blue and red light are most effective and a strong fluence rate dependency can be observed. The red light effect appears to be mediated through phytochrome, that of blue light being due to a separate blue light receptor, although this receptor requires the presence of far-red absorbing phytochrome (P_fr) in order to be effective.     

P53 transformation-related protein accumulates in the nucleus of transformed fibroblasts in association with the chromatin and is found in the cytoplasm of non-transformed fibroblasts.

The subcellular localization of the p53 molecule was studied in transformed and non-transformed fibroblasts. A newly established transformed cell line obtained by treating primary embryonic mouse cells in vitro with the chemical carcinogen methylcholanthrene was compared with the embryonic parent fibroblasts. The transformed cells lost the spindle shape characteristic of the parent fibroblasts, acquired an accelerated growth rate, developed into tumors when injected into syngeneic mice and expressed high levels of p53 synthesis estimated by immunoprecipitation of [35S]methionine-labeled cell extracts. The cellular localization of the p53 molecule was studied by immunofluorescent staining of fixed cells with monoclonal antibodies and by immunoprecipitation of [35S]-methionine-labeled p53 from various subcellular fractions. p53 was mainly found in the nucleus of the transformed fibroblast, while in the parent non-transformed primary embryonic cells, p53 was detected in the cytoplasm in a Triton X-100 soluble fraction, and associated with the cytoskeleton. The modulated distribution of p53 was also confirmed by analyzing a wide range of independently established transformed and non-transformed fibroblastic cell lines growing in vitro. The switch from the cytoplasmic localization of p53 in the non-transformed fibroblasts to a chromatin-associated accumulation in the transformed cells suggests a possible mechanism by which this protein may function in the transformed fibroblasts.     

Evidence against superoxide involvement in tyrosine hydroxylation by mushroom tyrosinase

The possible involvement of superoxide anions in the hydroxylation of tyrosine by mushroom tyrosinase was studied. Superoxide dismutase and scavengers of superoxide ions of smaller MW than superoxide dismutase, such as nitroblue tetrazolium and copper salicylate, had no direct effect on the monohydroxyphenolase activity of mushroom tyrosinase. The kinetics of tyrosine hydroxylation, but not of DOPA oxidation, by mushroom tyrosinase was atrected by the addition of a xanthine-xanthine oxidase system. In the presence of the xanthine-xanthine oxidase system, the lag period of tyrosine hydroxylation was shortened compared to the lag period in the absence of the xanthine-xanthine oxidase system. The xanthine- xanthine oxidase system alone (without mushroom tyrosinase) had no effect on tyrosine conversion to dopachrome. Superoxide dismutase, catalase and hydroxyl radical scavengers counteracted to some extent the shortening of the lag period of tyrosine hydroxylation by mushroom tyrosinase caused by the xanthin e-xanthine oxidase system. It is suggested that the shortening of the lag period is due mainly to hydroxyl radicals generated by the xanthine-xanthine oxidase system via interaction of O₂^?. and hydrogen paroxide (a Haber-Weiss type reaction). The data do not support the direct participation of superoxide anions in tyrosine hydroxylation by mushroom tyrosinase.     

Histochemical localization of ornithine decarboxylase with a labelled suicidal enzyme inhibitor

We describe a new technique for cytochemical localization of ornithine decarboxylase by the use of a synthesized conjugate of rhodamine bound to α-difluoromethylornithine a suicidal inhibitor of the enzyme. The labelled inhibitor retained its specificity and irreversibility towards ornithine decarboxylase inhibition. Using this technique we have localized the enzyme in specific regions of the developing rat cerebellum. This novel technique may be generally applicable to other enzymes.