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PubMed1991

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.

Amino Acid Sequence↗
PubMed1993

Complementation of a poliovirus defective genome by a recombinant vaccinia virus which provides poliovirus P1 capsid precursor in trans.

Defective interfering (DI) RNA genomes of poliovirus which contain in-frame deletions in the P1 capsid protein-encoding region have been described. DI genomes are capable of replication and can be encapsidated by capsid proteins provided in trans from wild-type poliovirus. In this report, we demonstrate that a previously described poliovirus DI genome (K. Hagino-Yamagishi and A. Nomoto, J. Virol. 63:5386-5392, 1989) can be complemented by a recombinant vaccinia virus, VVP1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991), which expresses the poliovirus capsid precursor polyprotein, P1. Stocks of defective polioviruses were generated by transfecting in vitro-transcribed defective genome RNA derived from plasmid pSM1(T7)1 into HeLa cells infected with VVP1 and were maintained by serial passage in the presence of VVP1. Encapsidation of the defective poliovirus genome was demonstrated by characterizing poliovirus-specific protein expression in cells infected with preparations of defective poliovirus and by Northern (RNA) blot analysis of poliovirus-specific RNA incorporated into defective poliovirus particles. Cells infected with preparations of defective poliovirus expressed poliovirus protein 3CD but did not express capsid proteins derived from a full-length P1 precursor. Poliovirus-specific RNA encapsidated in viral particles generated in cells coinfected with VVP1 and defective poliovirus migrated slightly faster on formaldehyde-agarose gels than wild-type poliovirus RNA, demonstrating maintenance of the genomic deletion. By metabolic radiolabeling with [35S]methionine-cysteine, the defective poliovirus particles were shown to contain appropriate mature-virion proteins. This is the first report of the generation of a pure population of defective polioviruses free of contaminating wild-type poliovirus. We demonstrate the use of this recombinant vaccinia virus-defective poliovirus genome complementation system for studying the effects of a defined mutation in the P1 capsid precursor on virus assembly. Following removal of residual VVP1 from defective poliovirus preparations, processing and assembly of poliovirus capsid proteins derived from a nonmyristylated P1 precursor expressed by a recombinant vaccinia virus, VVP1 myr- (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 66:4556-4563, 1992), in cells coinfected with defective poliovirus were analyzed. Capsid proteins generated from nonmyristylated P1 did not assemble detectable levels of mature virions but did assemble, at low levels, into empty capsids.(ABSTRACT TRUNCATED AT 400 WORDS)

Capsid↗
PubMed2001

Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis.

In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503-1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.

Dithiothreitol↗
PubMed1978

Structure of the leech nerve cord: distribution of neurons and organization of fiber pathways.

The abdominal nerve cord of the leech Macrobdella decora was studied under the light and electron microscopes. The ganglionic cortex consists of six hemicone-shaped packets of neuronal perikarya and apical processes regularly assembled in bilaterally symmetric rows. The orderly projection of the apical processes into the hilum of the packets is also followed by an orderly distribution of their branches across the neuropile. This part of the ganglion is made of two symmetrical halves or hemineuropiles enclosing two types of nerve tissue: coarse and fine neuropiles. The coarse neuropile has seven longitudinal and four commissural tracts of fibers and a distinctively segregated synaptic zone. Nerve processes in this neuropile mostly proceed from the neurons in the ganglia and some are the branches of giant afferent axons. The fine neuropile includes several longitudinal tracts of fibers and a non-segregated synaptic zone. Most nerve processes in this neuropile are small afferent axons and some come from neurons in the ganglia. Bundles of axons in the connectives result by the orderly projection of the neuropile longitudinal tracts and together form fiber pathways connecting the synaptic zones of successive ganglia. Pathways of through-ganglia giant axons, linking the coarse neuropile synaptic zones, and of small axons, linking the fine neuropile synaptic zones, are described.

Animals↗
PubMed1997

Dissecting the assembly pathway of the 20S proteasome.

Proteasomes reach their mature active state via a complex cascade of folding, assembly and processing events. The Rhodococcus proteasome offers a means to dissect the assembly pathway and to characterize intermediates; its four subunits (alpha1, alpha2, beta1, beta2) assemble efficiently in vitro with any combination of alpha and beta. Assembly studies with wild-type and N-terminally truncated beta-subunits in conjunction with refolding studies allowed to define the role of the propeptide which is two-fold: It supports the initial folding of the beta-subunits and it promotes the maturation of the holoproteasomes.

Cloning, Molecular↗
PubMed1996

Occupational disease surveillance using disability insurance at an automotive stamping and assembly complex.

Sickness and accident-insurance (sick leave) claims at an automotive stamping and assembly complex were analyzed using Poisson regression over a 4-year period to identify occupational health problems. The incidence of lower-respiratory disability (excluding asthma) was higher in painting operations (rate ratio [RR] = 2.9, 95% confidence interval [CI] = 1.2 to 6.8), and final assembly and processing areas (RR = 2.7, 95% CI = 1.0 to 7.4) at the assembly plant, and in metal assembly (welding) areas (RR = 2.8, 95% CI = 1.4 to 5.7) at the stamping plant. Disability rates for upper-extremity musculoskeletal disorders were statistically significantly higher (RR = 3.1 to 3.8) in major assembly plant production areas, as were back disability rates (RR = 1.5). During the first 6 months of new work assignments in painting or final assembly, respiratory problem rates were four times higher than in other areas. Upper-extremity musculoskeletal rate ratios ranged from 4.4 to 5.7 for new assignments in body, hard trim, and chassis areas. Higher rates in new assignments appeared to result from assignment changes precipitated by developing health problems, or from routine assignments to new tasks, some of which conferred high risk and were tolerated for less than 6 months. Musculoskeletal disability was consistent with known ergonomic hazards and paralleled that reported on the Occupational Safety and Health Administration log. Work-related musculoskeletal and other problems can be readily identified from disability insurance claims without dependence on plant medical visits or workers' compensation records. Disability insurance appears to absorb considerable work-related medical and absence costs.

Accidents, Occupational↗
PubMed1999

Separate assembly and transport domains within the Gag precursor of Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus (M-PMV), the prototypical type D retrovirus, assembles immature capsids within the cytoplasm of the cell prior to plasma membrane interaction. Several mutants of M-PMV Gag have been described which display altered transport, assembly, or both. In this report, we describe the use of an in vitro synthesis and assembly system to distinguish between defects in intracellular transport and the process of assembly itself for two previously described gag gene mutants. Matrix domain mutant R55W converts the type D morphogenesis of M-PMV particles into type C and has been hypothesized to alter the transport of Gag, redirecting it to the plasma membrane where assembly subsequently occurs. We show here that R55W can assemble in both the in vitro translation-assembly system and within inclusion bodies in bacteria and thus has retained the capacity to assemble in the cytoplasm. This supports the concept that R55 is located within a domain responsible for the transport of Gag to an intracellular site for assembly. In contrast, deletions within the p12 domain of M-PMV Gag had previously been shown to affect the efficiency of particle formation such that under low-level expression conditions, Gag would fail to assemble. We demonstrate here that the efficiency of assembly in the in vitro system mirrors that seen in cells under expression conditions similar to that of an infection. These results argue that the p12 domain of this D-type retrovirus plays a critical role in the membrane-independent assembly of immature capsids.

Amino Acid Sequence↗
PubMed1993

Similar attentional, frequency, and associative effects for pseudohomophones and words.

Between the presentation and recall of 1 or 5 digits, Ss performed a secondary task of naming a visually presented letter string--a pseudohomophone (e.g., FOLE, HOAP) or its real-word counterpart (FOAL, HOPE). Memory load interacted with frequency (HOPE vs. FOAL, HOAP vs. FOLE) but not with lexicality (HOPE vs. HOAP, FOAL vs. FOLE). This outcome counters models in which nonwords are named by a slow (resource-expensive) process that assembles phonology and words are named by a fast (resource-inexpensive) process that accesses lexical phonology. When the associative priming-of-naming task was secondary to the memory task, pseudohomophone associative priming (HOAP-DESPAIR, FOLE-HORSE) equaled associative priming (HOPE-DESPAIR, FOAL-HORSE) and was affected in the same way by memory load. Assembled phonology seems to underlie the naming of both words and nonwords.

Adult↗
PubMed2005

Regulation of histone synthesis and nucleosome assembly.

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.

Cell Cycle↗
PubMed1993

Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.

We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted. In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid. These strains thus grow in galactose but not glucose media. By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies [MNBs]) that stained with these antibodies were seen in the nucleus. Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures. These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus. Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs. Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts. Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants.

Cell Nucleolus↗
PubMed2007

pH-controlled reversible assembly of peptide-functionalized gold nanoparticles.

The assembly/disassembly process of carboxylated peptide-functionalized gold nanoparticles (peptide-GNPs) was studied within the pH interval of 2.5 to 10. The assembly process was not well controlled at pH 2.5, leading to the formation of 3D structures of GNPs, whereas at pH 4 we observed controlled assembly with the formation of only a network of 1D chains. In the pH range of 2.5 to 4, the assembly proceeded with the formation of a combination of two extremes (i.e., having both 1D and 2D nanostructures). The assembly process was reversed on changing the pH of the medium to 10. The assembly/disassembly process was monitored using UV-vis spectroscopy and finally confirmed by TEM analysis. This assembly resulted from the intermolecular H-bonding between two carboxylic acid groups of peptides bound to the two adjacent GNPs and were confirmed by FTIR spectroscopy.

Gold↗
PubMed1993

Assembly of the matrix protein of simian immunodeficiency virus into virus-like particles.

To obtain a better understanding of the processes of assembly and morphogenesis of simian immunodeficiency virus (SIV), recombinant vaccinia viruses containing regions of the gag-pol open reading frame were constructed and their intracellular expression as well as the ability of the Gag polypeptides to be released into the culture medium as constituents of virus-like particles were studied. Biochemical and electron microscopy analyses of cells infected with a recombinant expressing only the SIV matrix (MA) domain of the Gag polyprotein (v-p17 gag) showed that this protein self-assembles into 100-nm virus-like particles which are released into the culture medium. Interestingly, coexpression of SIV MA and Env proteins resulted in incorporation of gp120 and gp41 proteins into the recombinant p17-made particles. In addition when a positively charged domain of SIV MA (residues 26-33), which is highly conserved among all HIV and SIV MA proteins, was mutated into an acidic region, particle release was abolished without affecting protein expression, processing, or stability. Further characterization of the phenotype of this mutant by electron microscopy indicated that this mutant was blocked at the stage of assembly. These results suggest that SIV MA protein, along with its function in myristic acid-mediated membrane targeting, has intrinsic information for self-assembly as well as incorporation of viral Env glycoproteins into particles.

Amino Acid Sequence↗
PubMed1994

20 S proteasomes are assembled via distinct precursor complexes. Processing of LMP2 and LMP7 proproteins takes place in 13-16 S preproteasome complexes.

The non-essential mouse proteasome beta-type subunits LMP2 and LMP7 are thought to connect proteasomes to the MHC class I antigen processing pathway. Both subunits are synthesized as proproteins. We have studied the processing of both subunits, correlated with the maturation of 20 S proteasomes in mouse T cells. Our data show that proteasome assembly occurs via 13-16 S precursor complexes which possess a protein pattern distinct from that of 20 S proteasomes. Both LMP2 and LMP7 proproteins are processed within these preproteasome complexes and only their processed forms become part of active 20 S proteasomes. Our data show that the maturation and assembly of 20 S proteasomes via precursor particles is a translation-dependent gradual process, that processing of subunit proproteins takes place in these 13-16 S complexes and that subunit processing and proteasome formation occur together.

Animals↗
PubMed2003

Products transcribed from rearranged rrn genes of Escherichia coli can assemble to form functional ribosomes.

To examine the flexibility of rRNA operons with respect to fundamental organization, transcription, processing, and assembly of ribosomes, operon variations were introduced by a plasmid into an Escherichia coli strain that has deletions of all chromosomal copies of rRNA genes. In the reconstructed operons, a Salmonella intervening sequence (IVS) from 23S helix 45 was introduced into the E. coli 23S gene at the same position. Three different constructs of the E. coli 16S gene were then placed wholly within the IVS sequence, and the 16S gene was deleted from its normal position. The resulting plasmids thus had the normal operon promoters and the leader region followed by the 5' one-third of the 23S gene, the entire 16S gene within the IVS, the last two-thirds of the 23S gene, and the normal end of the operon. The three constructs differed in the amount of 16S leader and spacer regions they contained. Only two of the three constructs, those with redundant leader and spacer antiterminator signals, resulted in viable cultures of the rrn deletion strain. Electron micrographs of the variant operon suggest that the 23S rRNA is made in two separate parts which then must form subassemblies before assembling into a functional 50S subunit. Cells containing only the reshuffled genes were debilitated in their growth properties and ribosome contents. The fact that such out of the ordinary manipulation of rRNA sequences in E. coli is possible paves the way for detailed analysis of ribosome assembly and evolution.

DNA, Ribosomal Spacer↗
PubMed2000

Mechanisms of action of transcription activation and repression domains.

Transcriptional regulators contain domains that either activate or repress transcription. Indeed, many cellular transcriptional regulators contain both activation and repression domains. Transcriptional regulators act at several stages in the transcription process, including assembly of the transcription complex, initiation and elongation. In order to influence these processes, the regulatory domains must interact with components of the transcription apparatus. This review will focus on our current understanding of the nature of transcriptional regulatory domains and their targets in the transcription machinery.

Animals↗
PubMed2002

Designing supramolecular protein assemblies.

Many natural proteins self-assemble, either to fulfill their biological function or as part of a pathogenic process. Biological assembly phenomena such as amyloidogenesis, domain swapping and symmetric oligomerization are inspiring new strategies for designing proteins that self-assemble to form supramolecular complexes. Recent advances include the design of novel proteins that assemble into filaments, symmetric cages and regular arrays.

Amyloid↗
PubMed2001

Hierarchic patterning: architectures beyond 'giant molecular wheels'.

Based on symmetry breaking steps under one-pot conditions, simple molybdenum oxide-based building blocks initially assemble to 'giant molecular wheels' in a fast process followed by further slower assembly processes leading stepwise to more complex mesoscopic architectures including spherical ones and finally to those with a size larger than 500 nm.

Journal Article↗
PubMed1984

Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

Animals↗