The tumor-suppressor gene p53 is altered by missense mutation in numerous human malignancies. However, the biochemical properties of p53 and the effect of mutation on these properties are unclear. A human DNA sequence was identified that binds specifically to wild-type human p53 protein in vitro. As few as 33 base pairs were sufficient to confer specific binding. Certain guanines within this 33-base pair region were critical, as methylation of these guanines or their substitution with thymine-abrogated binding. Human p53 proteins containing either of two missense mutations commonly found in human tumors were unable to bind significantly to this sequence. These data suggest that a function of p53 may be mediated by its ability to bind to specific DNA sequences in the human genome, and that this activity is altered by mutations that occur in human tumors.

The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatfises (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) function as growth suppressors, we have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B

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Spec 1 mRNAs increase 100-fold in abundance per embryo during early sea urchin development. Previous studies indicated an enrichment of this mRNA in ectoderm fractions of gastrulae and plutei. We have determined the precise localization of this mRNA by in situ hybridization techniques. In pluteus larvae, the mRNA is highly restricted to a set of morphologically uniform ectoderm cells in the dorsal part of the embryo.The mRNA is not detectable in other regions of ectoderm or in endoderm and mesoderm. The pattern of localization is already established at the gastrula stage, before these cells are distinguishable by morphological criteria. This pattern of distribution of Spec 1 mRNA is distinct from that of bulk poly(A)+ mRNA. Measurements of the amount of Spec 1 mRNA per embryo and the number of cells containing this RNA indicate that there are about 500 Spec 1 mRNA molecules per cell at the pluteus stage and probably twice as many at the gastrula stage. These results indicate that the sensitivity of the in situ hybridization method allows detection of sequences that comprise '-0.05% of the embryo mRNA nucleotides.The sea urchin embryo is perhaps the best-characterized system with respect to developmental modulation in gene expression (1-4). Recent analysis of a large number of different mRNAs indicates that few undergo greater than 10-fold modulation in abundance per embryo during early development (5). It is likely that such measurements of whole embryo concentration underestimate larger cell lineage-specific differences in gene activity that either reflect or effect determination and differentiation. To date, no set of gene products has been shown to be cell type specific during sea urchin embryogenesis. More accurate analyses of determination and the onset of differentiation require studies of expression of individual genes in different cell lineages of developing embryos.Recently, cell fractionation techniques have been used to identify a set of mRNAs and their associated proteins that are enriched in ectoderm of plutei (1,6). These studies showed that mRNAs complementary to the cDNA clone pSpecl increase markedly in concentration during embryogenesis beginning at early blastula stage. In vitro translation of mRNAs hybridizing to pSpecl revealed a family of similar small acidic proteins whose quantitative regulation during development parallels that of their mRNAs.In the experiments reported here, we used an improved in situ hybridization technique to map the distribution of Spec 1 mRNA precisely and to quantitate its concentrations in individual cells of developing embryos.MATERIALS AND METHODS Embryo Culture and Tissue Preparation. Strongylocentrotus purpuratus were obtained from Pacific Biomarine (Venice, CA) or from Patrick Leahy (Kerckhoff Marine Laboratory, California Institute of Technology). Embryos were cultured by standard techniques, fixed in 1% glutaraldehyde, embedded in paraffin, and sectioned and the sections were treated with proteinase K and acetic anhydride as described (7,8).Prepar...