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Results for “Faith Assembly”

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PubMed1995

Degradation of ornithine decarboxylase by the mammalian and yeast 26S proteasome complexes requires all the components of the protease.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is an extremely short-lived protein. This attribute is important for the regulation of the activity of the enzyme and implies that the mechanisms involved in its degradation play an important role in the control of the cellular processes in which the enzyme is involved. Recently, it has been shown that ODC is degraded by the 26S proteasome complex in a process that requires antizyme, but not ubiquitin. With one reported exception, ODC, the 26S complex recognizes and degrades specifically ubiquitinated proteins. Their unconjugated counterparts are not targeted. The 26S complex is composed of a core catalytic unit, the 20S proteasome complex, and two additional, and apparently distinct, subcomplexes. The two additional subcomplexes are regulatory subunits that are required in order to confer specificity and control. In this study, we demonstrate that, like the degradation of ubiquitin-conjugated proteins, ubiquitin-independent degradation of ODC also requires prior assembly of the mammalian 26S proteasome from all the three subunits, the 20S proteasome and the two subcomplexes. The combination of any two subunits does not support generation of a proteolytically active complex. This is also true for the yeast 26S complex. Like the mammalian 20S proteasome, the yeast 20S complex can cleave short peptides in an ATP-independent mode, but cannot degrade ODC or ubiquitin-conjugated proteins. These proteins are degraded only following addition of the regulatory subunits and generation of the high-molecular-mass 26S complex. In a distinct, but related, set of experiments, we demonstrate that the degradation of ODC by the assembled 26S proteasome in vitro reproduces faithfully proteolysis of the enzyme in the intact cell. Namely, (a) a C-terminal-deleted mouse ODC and trypanosome ODC are stable both in vitro and in vivo, and (b) like proteolysis in the intact cell, degradation in the reconstituted cell-free system is also dependent upon the addition of antizyme.

Animals↗

PubMed1998

'No other logical choice': global malaria eradication and the politics of international health in the post-war era.

In 1955 the Eighth World Health Assembly voted to initiate a program for the global eradication of malaria. The global eradication of malaria represented a remarkable leap of faith. Many health authorities, both within and outside the Assembly, viewed eradication as at best fool hardy, and at worst, potentially disastrous. To understand why the World Health Assembly went ahead with a Global Eradication strategy, despite these concerns, it is necessary to examine the politics of international health and development in the post-war era. This political context shaped decisions about the adoption of DDT as a primary tool in the fight against malaria, as well as the adoption of the Malaria Eradication Program. It is equally important to understand how the advocates of an eradication strategy shaped arguments and developed support for their cause in the years leading up to the Eighth World Health Assembly meeting.

DDT↗

PubMed2005

The fission yeast Crb2/Chk1 pathway coordinates the DNA damage and spindle checkpoint in response to replication stress induced by topoisomerase I inhibitor.

Living organisms experience constant threats that challenge their genome stability. The DNA damage checkpoint pathway coordinates cell cycle progression with DNA repair when DNA is damaged, thus ensuring faithful transmission of the genome. The spindle assembly checkpoint inhibits chromosome segregation until all chromosomes are properly attached to the spindle, ensuring accurate partition of the genetic material. Both the DNA damage and spindle checkpoint pathways participate in genome integrity. However, no clear connection between these two pathways has been described. Here, we analyze mutants in the BRCT domains of fission yeast Crb2, which mediates Chk1 activation, and provide evidence for a novel function of the Chk1 pathway. When the Crb2 mutants experience damaged replication forks upon inhibition of the religation activity of topoisomerase I, the Chk1 DNA damage pathway induces sustained activation of the spindle checkpoint, which in turn delays metaphase-to-anaphase transition in a Mad2-dependent fashion. This new pathway enhances cell survival and genome stability when cells undergo replicative stress in the absence of a proficient G(2)/M DNA damage checkpoint.

Alleles↗

PubMed2005

Loss of Drosophila borealin causes polyploidy, delayed apoptosis and abnormal tissue development.

The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. Here, we report the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), a pronounced attenuation at prometaphase and multipolar spindles. Our evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium that involve Wingless signalling, which ultimately perturb the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy.

Animals↗

PubMed2024

One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system.

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

Infectious bronchitis virus↗

PubMed1984

A system for the translation of receptor messenger-RNA and the study of the assembly of functional receptors.

The Xenopus oocyte translation system has been developed for the recognition of receptor messenger-RNA molecules. Observations with the nicotinic acetylcholine, GABA, glycine, glutamate and serotonin receptors show that the mRNAs coding for their subunits can be faithfully translated in the oocyte and the products processed, glycosylated, assembled to a receptor structure, inserted correctly in the cell membrane and organised to form the functional ion channel. The specific regulatory interactions between different sites on one receptor, as exemplified in the GABAA receptor, are established in this process. This system is of value for the detection of mRNAs for receptor gene cloning and for the study of receptor assembly and of the expression of multiple receptor genes. The receptor ion channels formed can also be studied in their own right in a highly accessible and controlled situation.

Acetylcholine↗

PubMed2005

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.

Anaphase↗

PubMed1991

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.

Formaldehyde↗

PubMed2000

Stoichiometry of P1 plasmid partition complexes.

The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1. We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.

Bacterial Proteins↗

PubMed1999

Functional characterization of ABC10alpha, an essential polypeptide shared by all three forms of eukaryotic DNA-dependent RNA polymerases.

ABC10alpha is a small polypeptide shared by the three yeast RNA polymerases. Homologous polypeptides in higher eukaryotes have a zinc-binding CX(2)C. CX(2)C motif and a conserved basic C-terminal end. These features are also found in archaeal gene products that may encode an RNA polymerase subunit. The CX(2)C. CX(2)C motif is partly dispensable, since only its first cysteine is essential for growth, whereas the basic C-terminal end is critical in vivo. A mutant in the latter domain has an RNA polymerase III-specific defect and, in vitro, impairs RNA polymerase III assembly. Polymerase activity was, however, not affected in various faithful transcription assays. The mutant is suppressed by a high gene dosage of the second largest subunit of RNA polymerase III, whereas the homologous subunits of RNA polymerase I and II have aggravating effects. In a two-hybrid assay, ABC10alpha binds to the C-terminal half of the second largest subunit of RNA polymerase I, in a way that requires the integrity of the CX(2)C. CX(2)C motif. Thus, ABC10alpha appears to interact directly with the second largest subunit during polymerase assembly. This interaction is presumably a major rate-limiting step in assembly, since diploid cells containing only one functional gene copy for ABC10alpha have a partial growth defect.

Amino Acid Sequence↗

PubMed2004

Centromere DNA, proteins and kinetochore assembly in vertebrate cells.

The centromere is a specialized region of the chromosome that is essential for faithful chromosome segregation during mitosis and meiosis in eukaryotic cells. It is the site at which the kinetochore, the functional nucleoprotein complex responsible for microtubule binding and chromosome movement, is assembled through complex molecular mechanisms. Herein, I review recent advances in our understanding of centromeric DNAs as sites for kinetochore assembly and the mechanisms underlying kinetochore assembly in vertebrate cells.

Animals↗

PubMedDate not supplied

Device for preparing combinatorial libraries in powder metallurgy.

This paper describes a powder-metering, -mixing, and -dispensing mechanism that can be used as a method for producing large numbers of samples for metallurgical evaluation or electrical or mechanical testing from multicomponent metal and cermet powder systems. It is designed to make use of the same commercial powders that are used in powder metallurgy and, therefore, to produce samples that are faithful to the microstructure of finished products. The particle assemblies produced by the device could be consolidated by die pressing, isostatic pressing, laser sintering, or direct melting. The powder metering valve provides both on/off and flow rate control of dry powders in open capillaries using acoustic vibration. The valve is simple and involves no relative movement, avoiding seizure with fine powders. An orchestra of such valves can be arranged on a building platform to prepare multicomponent combinatorial libraries. As with many combinatorial devices, identification and evaluation of sources of mixing error as a function of sample size is mandatory. Such an analysis is presented.

Journal Article↗

PubMed2001

Multi-step control of spindle pole body duplication by cyclin-dependent kinase.

Organelles called centrosomes in metazoans or spindle pole bodies (SPBs) in yeast direct the assembly of a bipolar spindle that is essential for faithful segregation of chromosomes during mitosis. Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumour cells and cells with targeted mutations in tumour-suppressor genes. The defects that lead to centrosome amplification are not understood. We have recapitulated the multiple-centrosome phenotype in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes. Our observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication.

Cell Cycle↗

PubMed1986

Analysis of RNA polymerase III transcription complexes by gel filtration.

We studied the in vitro assembly and stability of RNA polymerase III transcription complexes on the 5 S RNA and tRNAMet genes of Xenopus and the VA genes of adenovirus. Complete and partial assemblies were formed on these genes using transcription factor IIIA from Xenopus ovaries and factors IIIB and IIIC from HeLa cells. The complexes were purified away from unbound factors by filtration through Sepharose 4B columns and then assayed for transcription in the presence of Xenopus polymerase III. The 5 S gene complexes were also investigated using a postlabeling DNase I footprinting technique that we devised. The binding of factor IIIA to the 5 S gene facilitated the binding of factor IIIC; this subassembly was required for factor IIIB to bind. On the VA I and tRNA genes, factor IIIC alone bound and allowed IIIB to bind. RNA polymerase bound last to form a preinitiation complex, but it was less stably affixed than any of the factors. The complete factor complexes on the 5 S and VA I genes were strikingly stable to brief exposure to high salt concentrations, and the stability of the factor IIIB interaction was limiting. Two modes of IIIC binding were distinguished that differed in stability and specificity. Assembly of the complexes did not require ATP, and faithful transcription occurred when adenyl-5'-yl imidodiphosphate was substituted for ATP.

Adenosine Triphosphate↗

PubMed2005

The mitotic checkpoint in cancer and aging: what have mice taught us?

The spindle assembly checkpoint is a cellular surveillance mechanism that functions to ensure faithful chromosome segregation during mitosis. Failure of this checkpoint can result in aneuploidy, a state of having abnormal numbers of chromosomes. Most human cancers consist of aneuploid cells, but it is unclear if the aneuploidy is a cause or a consequence of tumorigenesis. Over recent years, mouse models for spindle assembly checkpoint failure have been generated to investigate the biological relevance of the different spindle assembly checkpoint genes and the pathologies associated with chromosome number instability. Most of these models exhibit susceptibility to carcinogenesis. Moreover, one model has led to the identification of the spindle checkpoint protein BubR1 as a regulator of the normal aging process.

Aging↗

PubMed2001

Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast.

Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.

Amyloid↗

PubMed2004

Assembly of kinetochores in vertebrate cells.

The centromere is a specialized region of each chromosome that is essential for faithful chromosome segregation during mitosis and meiosis in eukaryotic cells. Centromeres are the site at which kinetochores are formed. The kinetochore is responsible for microtubule binding and chromosome movement. In this review, I will focus on recent advances in our understanding of centromere DNAs as sites for kinetochore assembly and the mechanism underlying kinetochore assembly in vertebrate cells.

Animals↗

PubMed2000

Cardiomyocyte cytoskeleton and myofibrillogenesis in healthy and diseased heart.

The unique cytoarchitecture of cardiomyocytes arises by complex interactions of different filamentous structures of the cytoskeleton. Intermediate filaments of the non-sarcomeric cytoskeleton are not essential for development but important for maintenance of myofibrils. Myofibrils consist of contractile proteins involved in force generation and the muscle cytoskeleton framework. The latter is essential for proper assembly and maintenance as well as for interaction with other cardiomyocytes or the extracellular matrix, thus being involved in force transmission. The information for sarcomere assembly is encoded in the proteins and some domains essential for faithful incorporation have been identified by epitope tagging experiments. Many KO mutations result in embryonic lethal phenotypes and new techniques e.g. using cardiomyocytes derived from ES cell-lines will have to be developed that allow to study such mutations in cardiomyocytes rather than whole organisms. Alterations in the expression levels of several proteins of the muscle cytoskeleton or impairment of their function by point mutations can result in increased mechanical stress in the cardiomyocytes which finally leads to cellular responses such as the development of dilated cardiomyopathy (DCM). MLP (muscle-LIM-protein) deficient mice develop DCM and changes in the mechanical coupling of cardiomyocytes result in alterations at the intercalated disks and enhanced accumulation of adherens junction proteins. Therefore, controlled interactions between proteins of the muscle cytoskeleton and contractile proteins are essential to ensure proper cardiac function and a more detailed insight in these processes might provide new tools to improve the contractile efficiency of the cardiomyocytes and thus working output in cardiomyopathies.

Animals↗