Showing papers by "Carlos Bustamante published in 1995"

Scanning Force Microscopy in Biology

Abstract: Microscopes have played a fundamental role in the development of biology as an experimental science It was Robert Hooke who, when using a compound microscope in 1655, noticed that thin slices of cork were made up of identical and small self‐contained units, which he called “cells” The generalization of this observation and its acceptance, though, had to wait until the late 1830s, when German microscopists Matthias Schleiden and Thcodor Schwann—working independently—introduced the “cell theory” of complex organisms By the second half of the 19th century Magnus Retzius, Santiago Ramon y Cajal and Camillo Golgi were busy completing the microscopic anatomical description of the cell

Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy.

Abstract: Gene activation frequently requires an array of proteins bound to sites distal to the transcription start site. The assembly of these protein-bound sites into specialized nucleoprotein complexes is a prerequisite for transcriptional activation. Structural analysis of these higher order complexes will provide crucial information for understanding the mechanisms of gene activation. We have used both electron microscopy and scanning force microscopy to elucidate the structure of complexes formed between DNA and heat-shock transcription factor (HSF) 2, a human heat-shock transcriptional activator that binds DNA as a trimer. Electron microscopy reveals that HSF2 will bring together distant DNA sites to create a loop. We show that this association requires only the DNA binding and trimerization domains of HSF2. Metal shadowing techniques used for electron microscopy obscure details of these nucleoprotein structures. Greatly increased resolution was achieved by directly imaging the complexes in the scanning force microscope, which reveals that at least two trimers are required for the association of HSF2-bound DNA sites.

Scanning tunneling microscopy. II. Calculation of images of atomic and molecular adsorbates.

Abstract: A theory of the scanning-tunneling-microscopy (STM) current of adsorbates with focus on effects of coherence in electron motion in the STM junction has been developed in the preceding paper [Phys. Rev. B 51, 11 074 (1995)]. That theory is illustrated through an application to the computation of several realistic STM images. The tip, the substrate, and the adsorbate are treated as finite clusters of atoms. Extended H\"uckel theory is used to calculate the STM current. The substrate used is Au (111). Images that can be compared directly with experimental observations are constructed for the bare Au (111) surface as well as for atomic and molecular adsorbates. The atomic adsorbates treated are Au, Na, H, C, and O. The molecular adsorbates treated are ${\mathrm{O}}_{2}$, CO, and benzene. A comparison is made with observations reported in the literature and agreement with a number of features is found. Our calculated images suggest that observations of giant corrugation in STM of metals might be arising from nonperturbative electron transport between localized surface states. Nontopographical artifacts due to adsorbates and the dependence of the image on the tip-sample separation are discussed.

Physical parameters that control the imaging of purple membranes with the scanning tunneling microscope

Abstract: The dominant physical and geometrical parameters for imaging hydrated purple membranes with the scanning tunneling microscope are identified and characterized. These are sample voltage, current density, and tip radius. Purple membranes are imaged with vertical and lateral resolution of 0.7 nm and 15 nm, respectively. We also found that the apparent height of the membranes depends on the tip-substrate separation. These experiments pose the problem of electron transport through 5-10 nm thick insulating materials. We propose a model where the contrast mechanism is controlled by two factors, the electric field at the interface and the transmission through empty states in the membrane.

A very low current scanning tunneling microscope

Abstract: The applications of the scanning tunneling microscope (STM) in air are usually restricted to good conducting materials as clean metals, doped and passivated semiconductors, or to some molecular adsorbates deposited onto graphite. In order to study poor conducting materials as biological molecules, we have built a very low current STM. This instrument can routinely be operated at 0.1 pA while having a bandwidth of 7 kHz. The advantages of using very low currents are illustrated by imaging 5‐nm‐thick purple membranes. These membranes can only be imaged at currents smaller than 2 pA.

Role of the structural domains of linker histones and histone H3 in the chromatin fiber structure at low-ionic strength: scanning force microscopy (SFM) studies on partially trypsinized chromatin

Abstract: Chromatin fibers have been hydrolyzed by trypsin and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and scanning force microscopy (SFM) At early points of hydrolysis, when mostly only the tails of the linker histones have been cleaved, nucleosomes appear to pile upon each other within a fiber Later, once significant hydrolysis of the N-terminal tail of histone H3 has occurred, fibers exhibit an open, three-dimensional arrangement of nucleosomes Linker DNA between adjacent nucleosomes is observed for the first time Adjacent nucleosomes appear to have a 'zig-zag' arrangement Finally, when all the tails of the linker histones and the N-terminal tails of H3 have been cleaved, then the fibers exhibit (i) a flat two- dimensional arrangement of nucleosomes, (ii) linker DNA between nearly all nucleosomes, and (iii) a zig-zag arrangement among some nucleosomes We suggest that (i) the linker histone globular domains help fix the angle of the DNA entering and exiting the nucleosome, (ii) the angle, however, is not sufficient to maintain the three-dimensionality of the fiber, and (iii) the N-terminal tails of histone H3 ares necessary for the three-dimensional conformation of the fiber at low ionic strength© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering Downloading of the abstract is permitted for personal use only

Scanning force microscopy study of native and linker histone depleted chromatin fibers

Abstract: Scanning force microscopy has been used to study the structure of chromatin fibers at low salt concentrations. Chicken erythrocyte chromatin fibers in low ionic strength buffer solutions were deposited on mica and imaged in ambient conditions with a tapping mode scanning force microscope. Individual nucleosomes can be clearly discerned in the images of the fibers. Native chromatin fibers show an asymmetrical, 3D structure of sinuous fiber trajectory with irregularly positioned nucleosomes. Fibers depleted of linker histones H1 and H5 have a completely extended 'beads-on-a-string' structure, with linker DNA visible between single nucleosomes. Molecular modeling of the fiber architecture and computer simulation of the imaging process provided more evidence on the observed organization of chromatin at low salt conditions. These results have implications on mechanisms of transcription control and chromatin compaction.