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Biomedical subjects

G Harauz

Publications and source records attributed to G Harauz.

At least 19 recordsLinked to original sources
PubMed1992

Electron microscopic visualisation of the 5S rRNA-YL3 complex from Saccharomyces cerevisiae.

The complex comprising 5S ribosomal RNA and the ribosomal protein YL3 (5S rRNP) was isolated from yeast (Saccharomyces cerevisiae), and positively contrasted preparations were imaged by transmission electron microscopy. The overall dimensions of the 5S rRNP complex in the micrographs were 10 nm by 6 nm. Three predominant projections were selected from several hundred putative particles for digitisation and computer averaging to yield two-dimensional constructions with reproducible spatial resolutions exceeding 2 nm. The enhanced projection images were compatible with structural models of this complex based on biochemical studies.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Electron microscopical projections of the large ribosomal subunit from Thermomyces lanuginosus.

Multivariate statistical analysis and hierarchical ascendant classification have been used to construct averages of two fundamental electron microscopical views of large ribosomal subunits from the thermophilic fungus Thermomyces lanuginosus. The first was roughly pentagonal in shape and corresponded to the canonical crown view seen in images of large subunits from prokaryotic species. The second and more prevalent projection was elliptical in shape, and by matching protuberances could be interpreted as the complex rotated from the crown orientation. Because of its ubiquity and consistency, this elongated view could potentially serve as the standard for structural comparisons of the large ribosomal subunit from eukaryotic organisms, and as the basis for a three-dimensional reconstruction.

Microscopy, Electron↗
PubMed1992

Characteristic electron microscopical projections of the small ribosomal subunit from Thermomyces lanuginosus.

Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of small ribosomal subunits from the thermophilic fungus Thermomyces lanuginosus into their characteristic views. Three predominant modes of adsorption to the support were elucidated: right-lateral, left-lateral and asymmetric, showing reproducible detail approaching 1.8 nm resolution. The projections of the fungal complexes appeared almost identical to those of HeLa cells, rat liver and rabbit reticulocytes studied previously in this manner. This result contrasts with the greater variation in fine structural features observed between ribosomal subunits from different prokaryotic species.

Animals↗
PubMed1992

Electron image analysis of ribosomal subunits from Thermus aquaticus.

Electron micrographs of ribosomal subunits from the thermophilic bacterium Thermus aquaticus were analysed using multivariate statistical analysis and characteristic views constructed to reproducible spatial resolutions ranging from 1.9 to 3.6 nm. These views were comparable to morphological classes of Escherichia coli ribosomal subunits, albeit with differences in fine features also found in archaebacterial ribosomes.

Bacterial Proteins↗
PubMed1992

Participation of a cyanobacterial S layer in fine-grain mineral formation.

Cyanobacteria belonging to the Synechococcus group are ubiquitous inhabitants of diverse marine and freshwater environments. Through interactions with the soluble constituents of their aqueous habitats, they inevitably affect the chemistry of the waters they inhabit. Synechococcus strain GL24 was isolated from Fayetteville Green Lake, New York, where it has a demonstrated role in the formation of calcitic minerals. In order to understand the detailed interactions which lead to mineral formation by this organism, we have undertaken detailed ultrastructural studies of its cell surface and the initial events in mineral growth using a variety of electron microscopic and computer image enhancement techniques. Synechococcus strain GL24 has a hexagonally symmetrical S layer as its outermost cell surface component. The constituent protein(s) of this structure appears as a double band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r)s of 104,000 and 109,000. We demonstrate that the S layer acts as a template for fine-grain gypsum and calcite formation by providing discrete, regularly arranged nucleation sites for the critical initial events in the mineralization process. To our knowledge, this is the first time that a bacterial S layer has been shown to have a role in mineral formation in a natural environment, and this report provides conclusive evidence for the specific involvement of bacterial surfaces in natural mineral formation processes.

Cyanobacteria↗
PubMed1992

Visualization of a mammalian transcription initiation complex.

Various proteins required for the initiation of eukaryotic gene transcription by RNA polymerase II have been identified and characterized, but little is known about their organization into a functional unit. Here, we describe the appearance of the murine ribosomal protein (rp) L32 gene transcription initiation complex as determined by transmission electron microscopy. Using a fractionated nuclear extract enriched for transcription factors necessary for rpL32 gene transcription in vitro and a DNA fragment containing the rpL32 gene promoter, the transcription initiation complex was imaged by standard transmission electron microscopy. Quantitative image analysis demonstrated that the complex is a multilobed structure whose two-dimensional projections are approximately 24 x 34 nm in size. Looping of the DNA seen in these images suggests that the proteins residing at the promoter region associate with proteins several hundred base pairs distant to the RNA start site, with bending of the DNA allowing these interactions to occur.

Animals↗
PubMed1992

Multivariate statistical analysis of electron micrographs of a mammalian transcription initiation complex.

We describe the appearance of the murine ribosomal protein (rp) L32 gene transcription initiation complex as determined by electron image analysis. Using a fractionated nuclear extract enriched for transcription factors necessary for rpL32 gene transcription in vitro and a DNA fragment containing the rpL32 gene promoter, the transcription initiation complex was prepared and viewed by standard transmission electron microscopy. Image analysis demonstrated that the complex was a multilobed structure.

Cells, Cultured↗
PubMed1991

Covering events in eigenimages of biomolecules.

Multivariate statistical analysis of a large set of micrographs of biological macromolecules involves the computation of eigenimages representing principal features, on the basis of which similar views of the complexes can be grouped. It is not generally clear what these eigenimages represent physically and which ones should be used in the classification process. In this paper, hierarchical maximum entropy discretisation and event covering are used to (1) detect statistically significant relationships in the eigenimages, (2) select the most relevant eigenimages for classifying biomolecular projections, and (3) build a prototype of the biomolecular complex under study.

Algorithms↗
PubMed1990

Structure of ribosomes from Thermomyces lanuginosus by electron microscopy and image processing.

Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of ribosomes from the thermophilic fungus Thermomyces lanuginosus into their characteristic views. Three predominant views were elucidated, called here overlap, non-overlap and top, showing reproducible detail approaching 1.8 nm resolution. The overlap and non-overlap forms of the fungal ribosomes appeared to be similar to those from the eubacterium Escherichia coli, despite differences in rRNA composition. The non-overlap projection predominated for the fungal complexes, suggesting different adsorption properties for ribosomes from the two species. Additionally, the top view has not been previously described for eubacteria. No major morphological differences could be detected between the fungal and eubacterial ribosomes at the resolution achieved in this study, suggesting a strong conservation of tertiary structure of this macromolecular complex despite the evolutionary gap between these two organisms.

Escherichia coli↗
PubMed1989

Automatic selection of macromolecules from electron micrographs by component labelling and symbolic processing.

A new solution to the problem of extracting images of individual biological macromolecules from electron micrographs is described. There are three distinct steps in the process. The initial stage of low-level image processing consists of noise suppression and edge detection. An intermediate stage of component labelling and feature computation bridges the gap between the iconic (low-level) processing and the final phase of symbolic (high-level) processing. Simple symbolic objects (bounding boxes) are derived from the edges, and are easily represented and manipulated in the decision-making process. The efficacy of the algorithm is demonstrated using electron micrographs of ribosomes and ribosomal subunits. The hierarchical nature of the analysis embodies a reduction in the amount of data and a change in its nature. Initially, thousands of pixels of continuous gray levels must be dealt with. After component labelling, there are fewer than a hundred bounding boxes whose manipulation can easily be defined and articulated by an expert. The software package that has been written can thus serve as a basis for applying artificial intelligence methodologies to analysis of electron micrographs.

Algorithms↗
PubMed1988

Packing of the 30 nm chromatin fiber in the human metaphase chromosome.

The human genetic material is packed hierarchically within the metaphase chromosome: the DNA molecule together with histone proteins form 11 nm diameter nucleosomes, which are then ordered into the 30 nm thick chromatin fiber. Little is known about the packing of this fiber within the chromosome. We have developed a tracking algorithm with which we followed its path within a three-dimensional reconstruction of a human chromosome computed from a series of electron micrographic projections. Fiber segments were seen to form loops of 100-350 nm diameter. Our observations indicate that these loops--which themselves show no preferred orientation--are organised into regions of roughly 200 nm axial extent.

Chromatin↗
PubMed1988

Biological macromolecules explored by pattern recognition.

Electron microscopy represents a very direct method for determining the structure of biological macromolecules, and complements both the well-established technique of X-ray crystallography (c.f. Blundell and Johnson, 1976) and the still in-its-infancy field of structure prediction (Kolata, 1986, Blundell et al., 1987). Our research has involved establishing a precise methodology based on computerised image analysis and pattern recognition to enhance the visibility of statistically significant structural features in electron images of isolated macromolecules. Our newly developed technique of angular reconstitution enables us to orient in three dimensions the commonly occurring projection forms of the macromolecules and thus perform a 3D reconstruction. In this paper, we describe the steps involved in determining the structure of a biological macromolecule by electron microscopy and image analysis, including pattern recognition and three dimensional reconstruction.

Image Processing, Computer-Assisted↗
PubMed1987

Three-dimensional reconstruction of a human metaphase chromosome from electron micrographs.

A complete human metaphase chromosome has been reconstructed from a series of electron microscopical projections obtained by tilting the specimen stage at 3 degree intervals from -60 to +60 degrees. The reconstructed structure is about 3.0 microns long, 1.6 micron wide, and 0.8 micron thick. The mass distribution was fairly homogeneous within the chromatids and neither a hollow nor a dense core was observed. The distribution and course of fibers observed are most consistent with a looping model of chromosome structure.

Algorithms↗
PubMed1987

Characteristic views of prokaryotic 50S ribosomal subunits.

Multivariate statistical analysis and classification techniques are powerful tools in sorting noisy electron micrographs of single particles according to their principal features, enabling one to form average images with an enhanced signal-to-noise ratio and a better reproducible resolution. We apply this methodology here to determining the characteristic views of the large (50S) ribosomal subunits from the eubacterium Escherichia coli and the archaebacteria Methanococcus vannielii, Sulfolobus solfataricus, and Halobacterium marismortui. Average images were obtained of the subunit in the common crown and kidney projections, but views of the particle in orientations intermediate between these two extremes were also elucidated for all species. These averages show reproducible detail of up to 2.0 nm resolution, thus enabling the visualization and interspecies comparison of many structural features as a first step toward comparing the actual three-dimensional structures. Our results disprove evolutionary lineages recently postulated on the basis of electron microscopical images of ribosomal subunits.

Archaea↗
PubMed1984

Nucleosome reconstruction via phosphorus mapping.

Electron spectroscopic imaging was combined with reconstruction algorithms to derive the three-dimensional structure of the nucleosome core particle to a resolution of 1.5 nanometers. Images of phosphorus distributions within individual nucleosomes were interpreted as projections of a supercoil of DNA. These were used to orient the corresponding individual nucleosome images, making it possible to reconstruct the entire nucleosome in three dimensions. The structure is consistent with known biochemical and biophysical data and explains site-specific nuclease sensitivity, although differing in part with other nucleosome models.

DNA↗