Shell Assembly Research Articles

On the mechanism of horse spleen apoferritin assembly: a sedimentation velocity and circular dichroism study.

The apoferritin shell is known to assemble spontaneously from its subunits obtained at acid pH upon neutralization. The reassembly of apoferritin from horse spleen has been followed by means of sedimentation velocity and circular dichroism experiments as a function of the pH and the nature of the assembly buffer in order to obtain information on the assembly pathway. In all the buffer systems tested the subunits sediment as a single peak of varying sedimentation and diffusion coefficients, and shell assembly starts at pH values around 3.5. In dilute glycine-acetate buffers the subunits are essentially dimeric up to this pH value. Therefore, the dimeric building blocks of the apoferritin shell that are apparent in the X-ray structure represent the first assembly intermediates. When the pH is increased to 4.0-4.3, the weight-average sedimentation velocity of the subunits increases to 3.6-4.7 S, respectively, and the subunit population becomes heterogeneous. Concomitantly, significant changes in the circular dichroism properties of the aromatic residues take place. On the basis of the X-ray structure, where aromatic residues appear to be located at or near the fourfold symmetry axes, these data suggest that assembly proceeds from dimers through tetramers and octamers. In the pH range 4.5-6.5 the reassembly process cannot be followed due to reversible precipitation of the subunits near their isoelectric point; at neutral pH values essentially quantitative reassembly is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones.

We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.

Autoregulation of the bacteriophage P22 scaffolding protein gene.

During the formation of each bacteriophage P22 head, about 250 molecules of the product of gene 8, scaffolding protein, coassemble with and dictate correct assembly of the coat protein into a proper shell structure. At approximately the time that DNA is inserted inside the coat protein shell, all of the scaffolding protein molecules leave the structure. They remain active and participate in several subsequent rounds of shell assembly. Previous work has shown that scaffolding protein gene expression is affected by the head assembly process and has generated the hypothesis that unassembled scaffolding protein negatively modulates the expression of its own gene but that it lacks this activity when complexed with coat protein in proheads. To test this model, a P22 restriction fragment containing the scaffolding and coat protein genes was cloned under control of the lac promoter. These cloned genes were then expressed in an in vitro DNA-dependent transcription-translation reaction. The addition of purified scaffolding protein to this reaction resulted in reduced scaffolding protein synthesis relative to coat and tail protein synthesis to an extent and at a protein concentration that was consistent with the observed reduction in vivo. We conclude that scaffolding protein synthesis is autoregulated and that scaffolding protein is the only phage-coded protein required for this process. In addition, these experiments provide additional evidence that this autoregulation is posttranscriptional.

Laminas cilíndricas circulares. Una solución aproximada

This work presents a simplification of the analysis of cylindrical shells with circular directrix and proposes an approximate solution obtained by factorizing the total work into two simpler ones, longitudinal and transversal. This factorizing process is made possible by introducing a virtual load for longitudinal work which is subtracted from that which is applied for transversal work. The virtual load (new, unknown function of the problem) enables one of the balance equations to be factorized into a further two. In this way, a group of equations is obtaines which only depend on the angular variant with which the problem of transversal work can arise. The theory is mathematically developed by accepting the substitution of some functions for others with an acceptable degree of approximation. The application for symmetrical multifoil sets is examined. Assembly of the concrete shells is proposed in three directions, which avoids isostatic tracing, justified by the formulae of the method itself

Formation of the prohead core of bacteriophage T4 in vivo

Formation of the prohead core of bacteriophage T4 was not dependent on shell assembly. In mutant infections, where the production or assembly of active shell protein was not possible, naked core structures were formed. The particles were generally attached to the bacterial inner membrane and possessed defined prolate dimensions. The intracellular yield varied between 15 and 71% of a corresponding prohead yield and was dependent on the temperature of incubation. The products of genes 21 and 22 were found to be essential for in vivo core formation, whereas those of genes 20, 23, 24, 31, and 40, as well as the internal proteins I to III, were dispensable.

Elastic behaviour of isolated column-supported ringbeams

Abstract Containment structures such as bins and tanks commonly consist of an elevated cylindrical vessel with a conical closure at the base. The complete structure is typically supported by a ringbeam placed at the intersection of the cylinder and cone, and this in turn is supported on columns. In this paper, a comparative study is made of three ringbeam geometries under radial and vertical loading. The axial, flexural, torsional and distortional responses of the ringbeams are modelled by treating the ringbeam as an assembly of axisymmetric shells. The behaviour is shown to be complex and the stresses are found to be poorly described by existing ringbeam design methods.

Work Structuring in Automated Manufacturing Systems Exemplified by Use of Industrial Robots for Body Assembly

With the setting up of a new highly automated body shell assembly unit at AUDI NSU, Ingolstadt, technological and working structure aspects were combined at a very early stage in the planning.The result is the introduction of so-called manufacturing teams of direct production personnel for technologically based system sections. In addition to manual insertion operations and control and supervisory tasks these manufacturing teams uill also have certain maintenance and repair operations as uell as checking operations for uhich they are responsible. The general objective of this is, by concentrating the tasks, to ensure preventive trouble recognition and quick trouble elimination on the part of personnel employed directly on the plant and thus to increase the availability of the plant. At the same time the idea is to enrich the residual operations on this kind of automatic system, the pure insertion functions, with additional qualified functions. The team philosophy promotes the cooperation, starts off further qualification processes and makes possible a change of strain by job rotation.

Strength evaluation for a composite rod structure made of brittle material with a glued joint

1. The data obtained should be considered as a first step in the direction of proven selection of structural and production parmaters for joints in shells assembled from essentially different-modulus and homogeneous materials of the glass, glass ceramic, and cermic type. 2. Modeled structural solutions for butt joints in composite rod systems made of glass and glass ceramic were considered, with some exceptions, to be efficient and suitable for proving during assembly of shell elements made of materials designed for loading with uniform compressive forces. 3. The strength of joints depends essentially on geometric parameters of the elements in contact and deformation conditions for the basic and intermediate elements in areas where article surfaces are in contact. Joint structure determines to a considerable extent the stress-strain state and the average value and scatter of individual values of supporting capacity. 4. A study of the supporting capacity of rod elements made of glass MKR-1 and glass ceramic STL-10 and force proving of standard glass articles made it possible to recommend these materials for use in highly stressed shell assemblies operating under the action of high external pressure. 5. It has been noted that construction of strong composite systems made of brittle materials by means of the recommended procedure holds promise, including the fundamental introduction of numerical methods which provide analysis of the stress state of articles in the joint assemblies.

Passive Neutron Multiplication Measurements

We have developed an instrument to measure neutron multiplication by statistical analysis of the timing of neutrons emitted from fissionable material. This instrument is capable of repeated analysis of the same recorded data with selected algorithms, graphical displays showing statistical properties of the data, and preservation of raw data on disk for future comparisons. In our measurements we have made a comparison of the covariance to mean and Feynman variance to mean analysis algorithms to show that the covariance avoids a bias term and measures directly the effect due to the presence of neutron chains. A spherical assembly of enriched uranium shells and acrylic resin reflector/moderator components used for the measurements is described. Preliminary experimental results of the Feynman variance to mean measurements show the expected correlation with assembly multiplication.

Automatic welding in consolidated blast-furnace shell assembly

Automatic welding in consolidated blast-furnace shell assembly

Heterogeneity of infection-associated particles in foot-and-mouth disease virus.

Abstract The overall pattern of picornavirus shell assembly is fairly well understood, but the role of 75 S empty capsids and the stage in which the genomic RNA is buried within the coat protein have not yet been elucidated. To further investigate these questions, different samples taken from sucrose density gradients during purification of foot-and-mouth disease virus, were analyzed by electron microscopy and polyacrylamide gel electrophoresis. The results showed the consistent presence of different virus-related structures with sedimentation coefficients of 50 to 190 S. The protein composition of each structure was determined. Two polypeptides of about 76,000 and 66,000 daltons were found associated with 90 and 85 S structures. Some of these newly identified particles may represent intermediates in the assembly process of foot-and-mouth disease virus.

Scaffolding proteins and the genetic control of virus shell assembly.

Historically a gap has existed between the study of the one-dimensional organization of hereditary information in genes, and of the three-dimensional organization of macromolecules in biological structures. In this article we describe progress in closing this gap through the genetic and biochemical analysis of the assembly of the icosahedral shells of spherical viruses, a class of subcellular structures whose subunit organization is relatively well understood. The genes specifying the proteins required for capsid assembly have been identified for many bacterial viruses. By using mutants defective in these genes, it has been possible to identify intermediates in shell morphogenesis and DNA condensation, and to unravel the different levels of the genetic control of macromolecular assembly processes. In general, a precursor shell or procapsid is first constructed, and the DNA is subsequently coiled within it. The construction of a closed shell poses as difficult a problem for a virus as for an architect. In the well-studied bacteriophage P22 of Salmonella typhimurium, the construction of the procapsid requires the interaction of about 200 molecules of the gene-8 scaffolding protein with 420 molecules of the gene-5 coat protein, forming a double-shelled structure with the scaffolding protein on the inside. Once completed, procapsids undergo substantial alteration in the course of encapsulating the viral DNA. In P22, the initiation of DNA packaging triggers the exit of all of the scaffolding molecules from within the capsid, probably through the coat-protein lattice. These released molecules are re-utilized, interacting with newly synthesized coat subunits to form further procapsids. Thus, the scaffolding protein functions catalytically in capsid assembly. All of the well-studied DNA phages require a scaffolding protein species for procapsid assembly, though their properties vary. Purified coat and scaffolding subunits by themselves show little tendency to polymerize, and are stable as monomers in solution. Upon mixing together under the appropriate conditions, however, the proteins copolymerize into double shells. Their interaction with each other appears to be critical for efficient assembly; this interaction probably occurs on the edges of growing shells, and not among subunits in solution. We have termed this kind of process, which we previously described in T4 tail morphogenesis, self-regulated assembly. The subunits are synthesized in a nonreactive form and are activated, not in solution, but upon incorporation into the growing substrate structure. A number of further transformations of the capsid subunits occur only within the organized structure and not as free subunits. Thus, aspects of the genetic information controlling the assembly process are not fully expressed at the level of the properties of protein subunits, but become manifest only through interactions with other proteins, or at a higher level, after completion of the correct organized structure.

Regulation of coat protein polymerization by the scaffolding protein of bacteriophage P22

In the morphogenesis of double stranded DNA phages, a precursor protein shell empty of DNA is first assembled and then filled with DNA. The assembly of the correctly dimensioned precursor shell (procapsid) of Salmonella bacteriophage P22 requires the interaction of some 420 coat protein subunits with approximately 200 scaffolding protein subunits to form a double shelled particle with the scaffolding protein on the inside. In the course of DNA packaging, all of the scaffolding protein subunits exit from the procapsid and participate in further rounds of procapsid assembly (King and Casjens. 1974. Nature (Lond.). 251:112-119). To study the mechanism of shell assembly we have purified the coat and scaffolding protein subunits by selective dissociation of isolated procapsids. Both proteins can be obtained as soluble subunits in Tris buffer at near neutral pH. The coat protein sedimented in sucrose gradients as a roughly spherical monomer, while the scaffolding protein sedimented as if it were an elongated monomer. When the two proteins were mixed together in 1.5 M guanidine hydrochloride and dialyzed back to buffer at room temperature, procapsids formed which were very similar in morphology, sedimentation behavior, and protein composition to procapsids formed in vivo. Incubation of either protein alone under the same conditions did not yield any large structures. We interpret these results to mean that the assembly of the shell involves a switching of both proteins from their nonaggregating to their aggregating forms through their mutual interaction. The results are discussed in terms of the general problem of self-regulated assembly and the control of protein polymerization in morphogenesis.

Control of the synthesis of phage p22 scaffolding protein is coupled to capsid assembly

The assembly of the precursor shells of bacteriophage P22 entails the co-polymerization of gene 5 coat protein with gene 8 scaffolding protein into double shell structures. During DNA encapsidation, the inner shell of scaffolding molecules dissociates and exits from the prohead. These molecules then recycle, catalyzing the assembly of newly synthesized coat protein to form new proheads (King and Casjens, 1974). Although gene 5 and gene 8 are adjacent on the phage chromosome, we find that the synthesis of the two proteins is differentially regulated. In productively infected cells, scaffolding protein is synthesized at a low rate relative to the coat protein. In contrast, cells that are infected with mutants blocked in DNA packaging and accumulate precursor shells synthesize scaffolding protein at a much higher rate. If a mutation is introduced into the coat protein gene, however, preventing shell assembly, the rate of scaffolding protein synthesis decreases to less than the wild-type rate. The experiments are consistent with models in which either continued synthesis of scaffolding protein depends upon co-polymerization with coat subunits, or soluble scaffolding subunits (but not assembled subunits) depress their own further synthesis. The finding that amber fragments of the scaffolding protein are synthesized at a very low rate is inconsistent with the second model. There is evidence, however, that fragments of the protein may have regulatory activity. The regulatory circuit couples scaffolding protein synthesis to morphogenesis. Gene dosage experiments show that regulation results in the maintenance of coat and scaffolding subunits in the proper ratio for shell assembly.

Universal stand for the assembly of shells by circumferential seam welding

Universal stand for the assembly of shells by circumferential seam welding

Elastic Interactions at the Junction of an Assembly of Axi-Symmetric Shells

Based on certain limiting assumptions a general solution in matrix form is given for the self-equilibrated loads caused by discontinuity at the junction of axi-symmetric shells. Formulae and computation forms are given for the practical derivation of quantities defining local bending of cylindrical, spherical and conical shells and the components of their membrane displacements under several standard types of loading. Derivation of some formulae is indicated and a numerical example illustrating the use of the method is included. In the discussion this particular ‘computation procedure’ approach to calculation of shells is compared with ‘explicit formula’ approach and the influence of normally neglected membrane rotations is assessed.