Scanning force microscopy of the complexes of p53 core domain with supercoiled DNA.

We used scanning force microscopy to analyse the interaction of the core domain of the tumor suppressor protein p53 (p53CD, amino acid residues 94 to 312), with supercoiled DNA (scDNA) molecules. The complexes were attached to a mica substrate by the divalent cation spreading technique. p53CD bound to supercoiled plasmid pPGM1 bearing the consensus sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON) was imaged as a globular complex. Only one such complex was observed with each scDNA molecule. In contrast, binding to supercoiled pBluescript II SK(-) DNA (lacking the consensus sequence) resulted in the appearance of multiple, variable size complexes of various sizes on single DNA molecules. Addition of p53CD to scDNA containing a cruciform-forming (AT)(34) insert resulted in the binding of the protein exclusively at the cruciform. The data presented here suggest that p53CD can form stable specific and non-specific complexes with supercoiled DNA molecules, albeit of variable multimeric organization.

DNA bending due to specific p53 and p53 core domain-DNA interactions visualized by electron microscopy.

We have used transmission electron microscopy to analyze the specificity and the extent of DNA bending upon binding of full-length wild-type human tumor suppressor protein p53 (p53) and the p53 core domain (p53CD) encoding amino acid residues 94-312, to linear double-stranded DNA bearing the consensus sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON). Both proteins interacted with high specificity and efficiency with the recognition sequence in the presence of 50 mM KCl at low temperature ( approximately 4 degrees C) while the p53CD also exhibits a strong and specific interaction at physiological temperature. Specific complex formation did not result in an apparent reduction of the DNA contour length. The interaction of p53 and the p53CD with p53CON induced a noticeable salt-dependent bending of the DNA axis. According to quantitative analysis with folded Gaussian distributions, the bending induced by p53 varied from approximately 40 degrees to 48 degrees upon decreasing of the KCl concentration from 50 mM to approximately 1 mM in the mounting buffer used for adsorption of the complexes to the carbon film surface. The p53CD bent DNA by 35-37 degrees for all salt concentrations used in the mounting buffer. The bending angle of the p53/DNA complex under low salt conditions showed a somewhat broader distribution (sigma approximately 39 degrees ) than at high salt concentration (sigma approximately 31 degrees ) or for p53CD (sigma approximately 24-27 degrees ). Together, these results demonstrate that the p53CD has a dominant role in complex formation and that the complexes formed both by p53 and p53CD under moderate salt conditions are similar. However, the dependence of the bending parameters on ambient conditions suggest that the segments flanking the p53CD contribute to complex formation as well. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed.

Feast or famine? Supplemental food programs and their impacts on two American Indian communities in California.

This article examines the use of supplemental food programs by two Native American populations and assesses some of the health and cultural impacts of these programs. A cross-sectional survey of 80 American Indian families, 40 families residing on the Round Valley Indian Reservation and 40 in Sacramento, California was conducted to determine access, use and appropriateness of supplemental feeding programs. Respondents at both the rural and urban geographic location showed considerable familiarity with available supplemental feeding programs. USDA Food Commodities were utilized most at Round Valley, raising the concern that provided staples which were highly processed and contained significant amounts of sodium, sucrose, and fat, could contribute to the problems of obesity and diabetes. Native Americans in Sacramento used food banks and food closets as their primary source of supplemental foods, and some expressed concern that the foods provided were highly sweetened and high in fat. While some nutrition advising was available at both geographical localities, access was inadequate. The study found that the foods provided by the supplemental food programs varied considerably in their nutritional quality and healthier foods such as fresh fruits, vegetables, and meats were either completely lacking or in short supply. In addition, culturally sensitive nutritional counseling and the development of education modules to instruct program recipients in the preparation of healthy meals and how to manage obesity and diabetes were needed and requested within the California Native American communities surveyed.

Snapshot blotting: transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to grids for electron microscopy.

We present a technique, "snapshot blotting," for the electrophoretic transfer of nucleic acids and nucleoprotein complexes in gel electrophoresis bands onto highly stable carbon film-coated grids for imaging by electron microscopy. The method permits structural analysis of macromolecular species that have been resolved by a gel mobility-shift assay. To demonstrate the efficiency and integrity of the transfer process for a multiprotein-DNA assembly, we have imaged various species of a prokaryotic transcription complex, using the cleavage-defective EcoRI(Q111) protein as an orientation marker and as a blockade of transcription elongation. Snapshot blotting should be of great utility in the structural characterization of nucleic acids and protein-nucleic acid interactions.