Paul V. C. Hough

Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy

For inorganic crystals such as calcite (CaCO3), Atomic Force Microscopy (AFM) has provided surface structure at atomic resolution (Ohnesorge and Binnig, 1993). As part of a broad effort to obtain high resolution for an individual protein or protein assembly (Binnig et al., 1986; Rugar and Hansma, 1990; Radmacher et al., 1992), we applied AFM to study the ATP-dependent double hexamer of SV40 large T antigen, which assembles around the viral origin of DNA replication. Multimeric mass has been determined in two-dimensional projected images by Scanning Transmission Electron Microscopy (STEM) (Mastrangelo et al., 1989). By AFM, if the DNA-protein preparation has been stained positively by uranyl acetate, the contour at the junction between hexamers is visible as a cleft, 2-4 nm deep. The cleft, whether determined as a fraction of height by AFM or as a fraction of mass thickness by STEM, is of comparable magnitude. On either side of the cleft, hexamers attain a maximum height of 13-16 nm. Monomers found in the absence of ATP show heights of 5-7 nm. Taken together, the z coordinates provide a surface profile of complete and partial replication assemblies consistent with the spatial distribution of recognition pentanucleotides on the DNA, and they contribute direct geometrical evidence for a ring-like hexamer structure.

DNA-protein complexes spread on N2-discharged carbon film and characterized by molecular weight and its projected distribution.

Abstract Omission of stain and shadow and the use of uncoated thin carbon film provide unparalleled contrast and resolution for protein-nucleic acid complexes studied by scanning transmission electron microscopy. A new technique, electrical discharge of the film in an atmosphere of pure N 2 , provides the required adhesion and spreading. Molecular weight determination by electron scattering achieves nearly its minimum possible error under these circumstances, as shown by test measurement of Escherichia coli RNA polymerase bound to bacteriophage T7 DNA. DNA contour length is within a few per cent of form B length and shows a relative standard deviation of 2 to 3%, several-fold smaller than Kleinschmidt spreading for short fragments. These techniques have contributed to a demonstration of nucleosome formation on a fragment of adenovirus serotype 2 DNA during brief incubation with a soluble extract of HeLa cells known to give selective and accurate transcription by RNA polymerase II. An example indicates the effectiveness of this approach for study of polymerase subunits and protein factors involved in transcription initiation.

General purpose visual input for a computer.

A number of ingenious and useful instruments and techniques for biological measurements are described in this monograph. I have not myself worked specifically on medical or biological problems. However, in the course of recent rather extensive work on machine analysis of the Track Chamber photographs used in particle physics, certain principles have emerged which I should like to describe and propose for wider application.

Analysis of Eukaryotic Control Proteins at Their Recognition Sequences by Scanning Transmission Electron Microscopy

The capability of Scanning Transmission Electron Microscopy (STEM) for measurement of macromolecular mass and mass distribution (1–3) and the recent discovery of in vitro systems for eukaryotic transcription (4–7) have led us to explore the characterization of control proteins and initiation complexes by quantitative electron microscopy in the neighborhood of promoters and other important DNA sequences. Cloned individual genes and control sequences are used. This approach is feasible because of the adhesion and spreading of DNA fragments on thin carbon foil which results from activation of the foil by electrical discharge in pure N2 (8). A significant additional advantage of such preparations has proved to be the ability to determine position on unstained, unshadowed DNA to within a few base pairs (bp) (see below).

Excitation under the scanning electron microscope of DNA‐associated fluorescence from chicken erythrocyte nuclei, polytene chromosomes and adenovirus 2 virions

Biological structures not seen by conventional light microscopy, such as longitudinal striations in polytene chromosomes, and, at the limit of sensitivity, virions of adenovirus 2, have been detected via DNA‐associated fluorescence excited under the scanning electron microscope. The maximum sensitivity realized, about 1 detected photon per 700 base pairs, falls short by about an order of magnitude of that required to achieve, in unreplicated specimens, the 2 nm intrinsic resolution of the method. A combination of D2O‐H2O substitution with freeze‐drying provides the best unquenching procedure found for in situ DNA.