P.R. Smith

Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly

Abstract The groE protein, which is involved in the morphogenesis of several bacteriophages, was isolated using a hybrid bacteriophage λ strain which overproduces it. The protein was characterized using biophysical methods, electron microscopy and digital image processing. We postulate that the gp groE complex contains 14 subunits in a cylindrical aggregate with 7-fold rotational symmetry. Possible mechanisms are discussed for the action of this complex in phage morphogenesis.

Image reconstruction from finite numbers of projections

Several results are obtained appertaining to the reconstruction of a two- dimensional image from a finite number of projections. Several schemes are considered for interpolating between the given data. When a trigonometrical Fourier series is used for angular interpolation then one finds, firstly, a consistency condition whereby a posteriori estimates can be made of the errors in the given data, and secondly, a basic image which contains only that information common to all physically permissible interpolation schemes. This basic image is necessarily free of misleading artefacts but it is computationally slow. Several computationally rapid interpolation schemes (based on the fast Fourier transform algorithm) are found to give good quality images, provided the given number of projections is sufficient to resolve the major details of the true image. A computational example is presented showing that a contrived image can be accurately reconstructed from a single projection.

AN INTEGRATED SET OF COMPUTER PROGRAMS FOR PROCESSING ELECTRON MICROGRAPHS OF BIOLOGICAL STRUCTURES

A set of computer programs is described which has been developed for processing electron micrographs of biological structures. The programs provide facilities for efficient image storage, enhancement and display. Extensive programs are available for analysis of images of structures with translational, rotational and helical symmetry, and for performing 3-dimensional reconstruction. The considerations which went into the design of the system are listed. A description is provided of the means of control of the main program, which performs the majority of the image processing operations, and an example is given to illustrate its use. These programs are compared with other program systems also developed for micrograph image processing.

Surface reliefs computed from micrographs of heavy metal-shadowed specimens

A method has been developed which allows a topographical map of a specimen surface to be computed from a digitized micrograph of a heavy metal-shadowed specimen. Preliminary results from the application of the method to micrographs of tungsten-shadowed freeze-dried T-layers (Kistler, J., Aebi, U., and Kellenberger, E. (1997) J. Ultrastruct. Res. 59, 76) show that qualitatively consistent results for the T-layer surface structure can be obtained from specimens shadowed from different directions. The method and its drawbacks are discussed with regard to the known difficulties in interpretation of the heavy metal deposit on a specimen surface.

Structure of the actin molecule determined from electron micrographs of crystalline actin sheets with a tentative alignment of the molecule in the actin filament.

Electron microscopy and image processing of negatively stained crystalline sheets induced from Acanthamoeba actin have been used to yield a three-dimensional reconstruction of the actin molecule, including data to a maximum resolution of 15 A. This model shows actin to be an asymmetric, wedge-shaped molecule. A three-dimensional reconstruction of an averaged, polar actin filament from negatively stained polylysine-induced actin filament paracrystals has also been computed. We show two possible ways in which the wedge-shaped actin molecule from the sheets can be placed into such a filament reconstruction. In both, the major intermolecular contacts are formed on complementary surfaces of the actin subunit and follow the left-handed genetic helix of the filament, a feature also found in the filament reconstruction.

The structure of the adenovirus capsid: III. Hexon packing determined from electron micrographs of capsid fragments

The orientation and relative positions of all 240 hexons in the icosahedral outer capsid of adenovirus have been determined. Two types of capsid fragments, obtained after selective disruption of the virion, were analyzed using electron microscopy and image-processing techniques. Planar inverted groups-of-nine, arising from the central region of the capsid facet, were minimally stained to reveal the morphology of restricted regions of their component hexons. Images shown to be related by correspondence analysis were averaged and features of the individual hexon molecule, known from an X-ray crystallographic investigation, were used in their interpretation. The study confirms earlier observations that the hexons in the group-of-nine are distributed on a p3 net, shows that the hexons form a close-packed array using the pseudo-hexagonal shape of the hexon base, and provides their relative positions. Twenty interlocking groups-of-nine account for 180 of the 240 hexons present in the viral capsid. The orientation of the remaining 60 peripentonal hexons was obtained from a rotationally averaged image of a quarter-capsid, a novel viral fragment comprising five complete facets. Each peripentonal hexon forms planar asymmetric interactions with two neighbors in an adjacent group-of-nine so that it lies on an extension of the p3 net. The complete facet thus consists of 12 hexons arranged on a planar p3 net, with a shape that permits interlocking of hexons at the capsid edge. The relative positions of the hexons have been determined to within 5 A using the molecular model, and indicate that the pseudo-hexagonal basal regions are close-packed in a manner that maximizes the hexon-hexon contacts. The results confirm the model proposed earlier for the arrangement of hexons within the adenovirus capsid (Burnett, 1985), and show the power of the inter-disciplinary approach.

Effects of Nickel Treatment on H3K4 Trimethylation and Gene Expression

Occupational exposure to nickel compounds has been associated with lung and nasal cancers. We have previously shown that exposure of the human lung adenocarcinoma A549 cells to NiCl(2) for 24 hr significantly increased global levels of trimethylated H3K4 (H3K4me3), a transcriptional activating mark that maps to the promoters of transcribed genes. To further understand the potential epigenetic mechanism(s) underlying nickel carcinogenesis, we performed genome-wide mapping of H3K4me3 by chromatin immunoprecipitation and direct genome sequencing (ChIP-seq) and correlated with transcriptome genome-wide mapping of RNA transcripts by massive parallel sequencing of cDNA (RNA-seq). The effect of NiCl(2) treatment on H3K4me3 peaks within 5,000 bp of transcription start sites (TSSs) on a set of genes highly induced by nickel in both A549 cells and human peripheral blood mononuclear cells were analyzed. Nickel exposure increased the level of H3K4 trimethylation in both the promoters and coding regions of several genes including CA9 and NDRG1 that were increased in expression in A549 cells. We have also compared the extent of the H3K4 trimethylation in the absence and presence of formaldehyde crosslinking and observed that crosslinking of chromatin was required to observe H3K4 trimethylation in the coding regions immediately downstream of TSSs of some nickel-induced genes including ADM and IGFBP3. This is the first genome-wide mapping of trimethylated H3K4 in the promoter and coding regions of genes induced after exposure to NiCl(2). This study may provide insights into the epigenetic mechanism(s) underlying the carcinogenicity of nickel compounds.

Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2

Treatment of purified preparations of Na,K-ATPase by phospholipase A2 has led to the formation of two-dimensional crystals of the protein. Control tests with another phospholipase and two detergents have shown that crystallization occurs as the result of hydrolysis and/or solubilization of the phospholipids in the enzyme vesicles. Experimentation with various buffer systems has indicated that reduction in the amount of phospholipids alone is sufficient for inducing the formation of crystalline sheets. Inclusion of crystal inducing ions in the buffer facilitates the crystallization process, resulting in more extensive arrays. The new crystalline sheets are exclusively dimeric with average unit cell dimensions: a = 15.8 +/- 0.4 nm, b = 4.9 +/- 0.2 nm, and gamma = 64 +/- 3 degrees. Examination of the micrographs shows that the initial intermolecular interaction leading to the formation of sheets is between the alpha subunits. Results from this study suggest that removal and/or modification of phospholipids by phospholipases could prove successful in crystallizing those membrane proteins in which excess lipid is the main barrier to the formation of two-dimensional arrays.

Surface reliefs computed from micrographs of isolated heavy metal shadowed particles.

A method of reconstructing the surface topography of isolated heavy metal shadowed particles has been devised which employs both the distribution of metal deposit on the specimen, and the lengths of shadows cast within the specimen or onto the support film beyond it. It is shown that in ideal conditions the Fourier transform of an image of a shadowed object will show a zero line perpendicular to the direction of metal deposit, independent of whether shadows are cast or not. The method has been applied to an image of a critical point-dried tungsten-tantalum-shadowed eukaryotic small ribosomal subunit. The surface topography of this particle was found to be in qualitative agreement with the model employed by Emanuilov, Sabatini, Lake, and Freinstein, 1978 , Proc. Nat. Acad. Sci. USA, 75, 1389–1393. The reconstruction revealed, however, that the particle was flattened to 60% of its original dimension parallel to the electron beam. The prospects of using this method as a means to obtain the structure of the surfaces of other isolated particles is discussed.

Combining multiple ChIP-seq peak detection systems using combinatorial fusion

BackgroundDue to the recent rapid development in ChIP-seq technologies, which uses high-throughput next-generation DNA sequencing to identify the targets of Chromatin Immunoprecipitation, there is an increasing amount of sequencing data being generated that provides us with greater opportunity to analyze genome-wide protein-DNA interactions. In particular, we are interested in evaluating and enhancing computational and statistical techniques for locating protein binding sites. Many peak detection systems have been developed; in this study, we utilize the following six: CisGenome, MACS, PeakSeq, QuEST, SISSRs, and TRLocator.ResultsWe define two methods to merge and rescore the regions of two peak detection systems and analyze the performance based on average precision and coverage of transcription start sites. The results indicate that ChIP-seq peak detection can be improved by fusion using score or rank combination.ConclusionOur method of combination and fusion analysis would provide a means for generic assessment of available technologies and systems and assist researchers in choosing an appropriate system (or fusion method) for analyzing ChIP-seq data. This analysis offers an alternate approach for increasing true positive rates, while decreasing false positive rates and hence improving the ChIP-seq peak identification process.