CELLULAR SYNTHESIS OF COLLAGEN: RIBOSOMAL AGGREGATES IN HYPERTHYROIDISM, HYPOTHYROIDISM, AND ASCORBIC ACID DEPLETION.
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Analysis of the multiple transcripts of the Dd ras gene during Dictyostelium discoideum development.
Transcripts from the Dd ras gene can only be detected once starved cells have begun to aggregate (Reymond et al., Cell 39: 141-148, 1984). We show in this report that the three transcripts which originate from Dd ras during normal development differ in their 5' ends. In suspension of starved single cells, one major Dd ras RNA accumulates upon addition of cAMP. It seems that the cAMP regulation of Dd ras expression happens both at the transcriptional and post-transcriptional level. An RNA secondary structure present in the 5' untranslated region of the gene is proposed to be important in this post-transcriptional regulation.
Base Sequence↗
Z* DNA, the left-handed helical form of poly[d(G-C)] in MgCl2-ethanol, is biologically active.
The interconversion between the right (R) and left (L) helical forms of poly[d(G-C)] occurs at low concentrations of MgCl2 and EtOH, acting together in a highly synergistic manner. Thus, the cooperative R---L transition is induced by only 0.4 mM and 4 MM MgCl2 in combination with 20% and 10% EtOH, respectively. The L form of poly[d(G-C)] formed under these conditions has the spectroscopic properties (absorption, circular dichroism) previously demonstrated under high salt conditions (Pohl and Jovin, 1972) and thought to correspond to the left-handed Z DNA structures recently established by X-ray crystallography (Wang et al., 1979; Drew et al., 1980). However, L DNA formed in Mg2+-EtOH (which we designate as Z* DNA) has unique properties: a) it can be sedimented readily out of solution at low speed, indicative of condensation and intermolecular aggregation; b) it supports the binding of several intercalating (ethidium bromide, actinomycin D) and non-intercalating (mithramycin) drugs, although these interact preferentially with the R (i.e., B) form of DNA; and c) it functions as a template for Escherichia coli RNA polymerase. B and Z* DNAs can be generated under identical ionic conditions and compared in a number of biochemical systems. Our results suggest that left-handed DNA may form under physiological conditions and serve a biological function.
Crystallography↗
A two step approach for semi-automated particle selection from low contrast cryo-electron micrographs.
Over recent years advances in cryo-electron microscopy for the study of macromolecular structure have resulted in resolutions in the range 10-15 A becoming routine. With this drive for increased resolution comes the need to collect larger datasets, commonly >10,000 particle images. Manual selection of particles from micrographs is often difficult and with such large numbers of particles now involved it is also laborious and a common bottleneck. Automated methods do exist but are normally restricted to specific samples or data, i.e., spherical particles, no aggregation, high contrast, and low noise. A two step approach has been developed that remains general and can be applied to low contrast, high noise micrographs of small molecules. Specifically, application of the approach is presented using micrographs of Escherichia coli RNA polymerase, which due to low contrast and the relatively small size of the molecule prove difficult to pick manually. To test the automated approach, independent reconstructions of RNA polymerase were carried out using manual and automatically picked data. The two reconstructions are shown to be comparable and the reconstruction from the automatically picked dataset is at a higher resolution, due to an increase in the number of particles picked.
Algorithms↗
Ultrasonic absorption evidence for enhanced volume fluctuations in the tobacco mosaic virus protein helical aggregate.
The increased ultrasonic absorption brought about by self-assembly of biomolecules is analyzed for the assembly process from the 20S aggregate to the helical rod of tobacco mosaic virus protein in solution, designated here as the 20S --> P-helix transition. The analysis is based on theoretical developments in ultrasonic relaxation spectrometry presented previously and illustrates the possibility that this technique can be used for characterizing fluctuations. The analysis makes use of NMR data for the system in solution and of x-ray diffraction data for the closely related transition from the two-ring disk to the virion. These x-ray data comprise the high-resolution structures and the Debye-Waller temperature factors of the main chain atoms of both the two-ring disk in crystals and the virion in oriented gel form. First, reduced ultrasonic spectra are obtained for the 4S, 20S, and helical rod aggregates. The fluctuation-enhancement factor for the helical rod is determined independently of any deconvolution into normal modes of relaxation and is shown not to depend on the particular procedure of reduction employed. The increase of ultrasonic absorption in the 20S --> P-helix transition primarily reveals enhancement of the relaxing system's normal-mode volume fluctuations. The observed relaxations probably involve one conformational process per subunit. The normal-mode volume fluctuations are then estimated from a bimodal least-squares best fit to the data, and a lower bound for the reaction volume associated with the fast steps is obtained. Two mechanisms are considered as follows: (i) a destabilization process in which the free-energy difference between two states is reduced and (ii) an increase in reaction volumes of local conformation changes in the helical aggregate, resulting from the formation of a "carboxyl cage-like" structure and from the change in environment produced inside the cage. Increased reaction volumes would not be detected with x-ray diffraction. The possible occurrence of fluctuations at the RNA binding site raises the question of whether a quaternary structure that exhibits significant conformational fluctuations must be present for the binding of the nucleic acid.
Journal Article↗
Basic polypeptides as histone models. Effect of conformation, base composition and methylation of nucleic acids on the interaction with H1 and histone models and on the circular dichroism of complexes.
Interaction of histone H 1 and models simulating histone chains was followed by monitoring the melting curves of supernatants after the sedimentation of aggregated complexes. In a mixture of two DNAs the histones reacted selectively with (A+T)-rich and non-methylated DNA, respectively. H 1 and (Ala-Lys-Pro)n also interacted preferentially with DNA in a mixture with double stranded RNA whereas (Lys30,Ala70)n did not show any selectivity. (G+C)-rich DNA in complexes showed CD spectra the intensity of which decreased with increasing DNA methylation to values comparable with these of complexes of (A+T)-rich DNA. In complexed with double stranded RNA only the polymer (Lys30,Ala70) displayed CD pattern similar to spectra of complexes with DNA. It was concluded that formation and structure of complexes depend selectively on the DNA conformation and base composition.
Binding Sites↗
Homozygous and compound heterozygous mutations in ZMPSTE24 cause the laminopathy restrictive dermopathy.
Restrictive dermopathy (RD) is a lethal human genetic disorder characterized by very tight, thin, easily eroded skin, rocker bottom feet, and joint contractures. This disease was recently reported to be associated with a single heterozygous mutation in ZMPSTE24 and hypothesized to be a digenic disorder (Navarro et al, Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy. Hum Mol Genet 13:2493-2503, 2004). ZMPSTE24 encodes an enzyme necessary for the correct processing and maturation of lamin A, an intermediate filament component of the nuclear envelope. Here we present four unrelated patients with homozygous mutations in ZMPSTE24 and a fifth patient with compound heterozygous mutations in ZMPSTE24. Two of the three different mutations we found are novel, and all are single base insertions that result in messenger RNA frameshifts. As a consequence of the presumed lack of ZMPSTE24 activity, prelamin A, the unprocessed toxic form of lamin A, was detected in the nuclei of both cultured cells and tissue from RD patients, but not in control nuclei. Abnormally aggregated lamin A/C was also observed. These results indicate that RD is an autosomal recessive laminopathy caused by inactivating ZMPSTE24 mutations that result in defective processing and nuclear accumulation of prelamin A.
Cell Nucleus↗
Raf, a trans-acting locus, regulates the alpha-fetoprotein gene in a cell-autonomous manner.
Genetic analysis provides an approach for identifying regulatory loci that govern the expression of specific genes within the context of the entire organism. Such analyses have defined two unlinked regulatory loci, termed raf and Rif, that modulate the levels of alpha-fetoprotein in liver. Of primary importance for the isolation and characterization of the raf product is to determine whether it is produced by the hepatocyte or whether it is produced by a different cell type. By means of analysis of alpha-fetoprotein expression in livers of embryo aggregation chimeras derived from mice of different raf genotypes it was possible to conclude that the product of the raf locus is expressed as a hepatocyte autonomous function that acts in trans to regulate the level of alpha-fetoprotein messenger RNA.
Animals↗
Association of CBFA2 mutation with decreased platelet PKC-theta and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin phosphorylation: proteins regulated by CBFA2 play a role in GPIIb-IIIa activation.
The mechanisms by which agonists activate glycoprotein (GP) IIb-IIIa function remain unclear. We have reported data on a patient with thrombocytopenia and impaired receptor-mediated aggregation, phosphorylation of pleckstrin (a protein kinase C [PKC] substrate), and activation of the GPIIb-IIIa complex. Abnormalities in hematopoietic transcription factors have been associated with thrombocytopenia and platelet dysfunction. To define the molecular mechanisms, we amplified from patient platelet RNA exons 3 to 6 of core-binding factor A2 (CBFA2) cDNA, which encompasses the DNA-binding Runt domain; a 13-nucleotide (nt) deletion was found (796-808 nt). The gDNA revealed a heterozygous mutation (G>T) in intron 3 at the splice acceptor site for exon 4, leading to a frameshift with premature termination in the Runt domain. On immunoblotting, platelet CBFA2, PKC-, albumin, and IgG were decreased, but pleckstrin, PKC-alpha, -betaI, -betaII, -eta, -epsilon, -delta, and -zeta, and fibrinogen were normal. Our conclusions are that (1) CBFA2 mutation is associated with not only thrombocytopenia, but also impaired platelet protein phosphorylation and GPIIb-IIIa activation; (2) proteins regulated by CBFA2 are required for inside-out signal transduction-dependent activation of GPIIb-IIIa; and (3) we have documented the first deficiency of a human PKC isozyme (PKC-), suggesting a major role of this isozyme in platelet production and function.
Amino Acid Sequence↗
Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1.
Specialised subapical junctions play a critical role in maintaining epithelial cell polarity and tissue integrity, and provide a platform for intracellular signalling. Here we analyse the roles of C. elegans genes let-413 and dlg-1, a homologue of Drosophila lethal discs large, in the assembly of the C. elegans apical junction (CeAJ), and provide the first characterisation of this structure. We have identified dlg-1 as an essential gene in an RNA interference screen against C. elegans homologues of genes encoding proteins involved in tight or septate junction formation. We show that DLG-1 colocalises with the junctional protein JAM-1 at CeAJs in a unit distinct from HMP-1/alpha-catenin, and apical to the laterally localised LET-413. Loss of dlg-1 activity leads to JAM-1 mislocalisation and the disappearance of the electron-dense component of the CeAJs, but only mild adhesion and polarity defects. In contrast, loss of let-413 activity leads to the formation of basally extended discontinuous CeAJs and strong adhesion and polarity defects. Interestingly, in LET-413-deficient embryos, CeAJ markers are localised along the lateral membrane in a manner resembling that observed in wild-type embryos at the onset of epithelial differentiation. We conclude that the primary function of LET-413 is to correctly position CeAJ components at a discrete subapical position. Furthermore, we propose that DLG-1 is required to aggregate JAM-1 and other proteins forming the electron-dense CeAJ structure. Our data suggest that epithelial adhesion is maintained by several redundant systems in C. elegans.
Animals↗
Subpopulations of human lymphoblastoid cell lines. Correlation with the expression of surface receptors and content of Epstein-Barr virus genome.
Forty lymphoblast cell lines derived from normal subjects, patients with infectious mononucleosis, leukemia, and Burkitt's lymphoma have been studied for surface receptors including surface Ig, complement receptors by the EAC rosette and fluorescent (Raji cell) techniques, and Fc (aggregate) receptor by direct and indirect immunofluorescence. Because of the B-cell tropism of the Epstein-Barr virus (EBV), an effort was made to correlate the expresion of various surface properties of lymphoblastoid cell lines with the content of EBV viral DNA as determined by complementary RNA-DNA (cNRA-DNA) hybridization on membrane filters or by DNA-DNA renaturation kinetic analysis. The only correlation established was with the Fc receptor determined by direct immunofluorescence. No correlation of EBV genome equivalents per cell with complement receptor or surface Ig was noted, suggesting that the expression of these receptors is not influenced by EBV viral DNA content. Subgroups of lymphoblastoid cell lines were on the basis of variable expression of surface receptors, designated B1, B2, B3, B4, and T. The distribution of lymphoblastoid cell lines into these subgroups were in the ratio of 14:4:1:4:1. The B1, B2, and B4 cell lines (except Molt 4F) were found to contain EBV. The B3 subgroup, for wich cell line 698 was the sole example, expressed surface immunoglobulins but no other B-cell characteristics, and H.S.B., a T-cell line, lacked detectable EBV.
Burkitt Lymphoma↗
Pressure induces folding intermediates that are crucial for protein-DNA recognition and virus assembly.
Protein-nucleic acid interactions are crucial for a variety of fundamental biological processes such as replication, transcription, restriction, translation and virus assembly. The molecular basis of protein-DNA and protein-RNA recognition is deeply related to the thermodynamics of the systems. We review here how protein-nucleic acid interactions can be approached in the same way as protein-protein interactions involved in protein folding and protein assembly, using hydrostatic pressure as the primary tool and employing several spectroscopic techniques, especially fluorescence, circular dichroism and high-resolution nuclear magnetic resonance. High pressure has the unique property of stabilizing partially folded states or molten-globule states of a protein. The competition between correct folding and misfolding, which in many proteins leads to formation of insoluble aggregates is an important problem in the biotechnology industry and in human diseases such as amyloidosis, Alzheimer's, prion and tumor diseases. The pressure studies reveal that a gradient of partially folded (molten globule) conformations is present between the unfolded and fully folded structure of several bacteria, plant and mammalian viruses. Using pressure, we have detected the presence of a ribonucleoprotein intermediate, where the coat protein is partially unfolded but bound to RNA. These intermediates are potential targets for antiviral compounds. Pressure studies on viruses have direct biotechnological applications. The ability of pressure to inactivate viruses has been evaluated with a view toward the applications of vaccine development and virus sterilization. Recent studies demonstrate that pressure causes virus inactivation while preserving the immunogenic properties. There is substantial evidence that a high-pressure cycle traps a virus in the 'fusion intermediate state', not infectious but highly immunogenic.
Amyloid↗
Regulation of IgD-receptor expression on murine T cells. I. Characterization and metabolic requirements of the process leading to their expression.
Receptors for IgD (IgD-R) are found on murine CD4+ T cells and T cell clones. Previous work has shown that incubation with aggregated (but not monomeric) IgD causes the rapid upregulation of IgD-R and enables the T cells to respond with augmented helper function in antibody production. In the present study, IgD-R upregulation is shown to be (a) rapid, reaching plateau levels by 60 min, (b) independent of de novo protein or RNA synthesis, and (c) only slightly reduced at 4 degrees C. The IgD-R+ T cells present both before and after upregulation of IgD-R expression are predominantly resting T cells, whose ability to rosette with IgD-SRBC is inhibited by soluble IgD. The upregulation of IgD-R, even after overnight exposure to IgD, does not cause any detectable change in the expression of other T cell surface markers. Also characteristic of resting T cells is that they exhibit IgD-R in response to IL-2 and IL-4 only after overnight incubation with these cytokines, and fail to respond at all to IL-1. In contrast, cloned Th2 cells, expressing IL-1 and IL-2 receptors, show IgD-R upregulation after a 2-hr exposure to IL-1 or IL-2. GM-CSF, TNF-alpha, IL-6, and IL-10 do not modulate IgD-R expression. T hybridoma cells constitutively express much higher IgD-R levels than resting splenic T cells and can be stained with aggregated IgD followed by FITC-anti-IgD. Their levels of IgD-R expression decrease, as assayed both by rosetting and by staining, on 4-14 hr of incubation with tunicamycin or deoxynojirimycin, suggesting that N-linked glycosylation and oligosaccharide processing, respectively, are needed for continued expression of IgD-R. Tunicamycin-treated cells without detectable IgD-R on their surface still show IgD-binding protein in the cell extracts, suggesting that surface expression is more dependent on glycosylation of the IgD-R molecules than on the ability to bind IgD. Ca2+ ions are needed for optimal binding of IgD to IgD-R, in line with previous findings showing IgD-R to be lectin-like in binding carbohydrate rather than peptide regions of the IgD molecule.
Animals↗
VP40 octamers are essential for Ebola virus replication.
Matrix protein VP40 of Ebola virus is essential for virus assembly and budding. Monomeric VP40 can oligomerize in vitro into RNA binding octamers, and the crystal structure of octameric VP40 has revealed that residues Phe125 and Arg134 are the most important residues for the coordination of a short single-stranded RNA. Here we show that full-length wild-type VP40 octamers bind RNA upon HEK 293 cell expression. While the Phe125-to-Ala mutation resulted in reduced RNA binding, the Arg134-to-Ala mutation completely abolished RNA binding and thus octamer formation. The absence of octamer formation, however, does not affect virus-like particle (VLP) formation, as the VLPs generated from the expression of wild-type VP40 and mutated VP40 in HEK 293 cells showed similar morphology and abundance and no significant difference in size. These results strongly indicate that octameric VP40 is dispensable for VLP formation. The cellular localization of mutant VP40 was different from that of wild-type VP40. While wild-type VP40 was present in small patches predominantly at the plasma membrane, the octamer-negative mutants were found in larger aggregates at the periphery of the cell and in the perinuclear region. We next introduced the Arg134-to-Ala and/or the Phe125-to-Ala mutation into the Ebola virus genome. Recombinant wild-type virus and virus expressing the VP40 Phe125-to-Ala mutation were both rescued. In contrast, no recombinant virus expressing the VP40 Arg134-to-Ala mutation could be recovered. These results suggest that RNA binding of VP40 and therefore octamer formation are essential for the Ebola virus life cycle.
Animals↗
Expression of metastasis-associated mts1 gene is co-induced with membrane type-1 matrix metalloproteinase (MT1-MMP) during oncogenic transformation and tubular formation of Madin Darby canine kidney (MDCK) epithelial cells.
Madin-Darby canine kidney (MDCK) epithelial cells form branching tubules in three-dimensional collagen gel in the presence of hepatocyte growth factor (HGF). Membrane type-1 matrix metalloproteinase (MT1-MMP), expression of which was induced by collagen-gel culture, was demonstrated to play an essential role in tubular formation (Y. Kadono et al. Biochem Biophys Res Commun 1988; 251: 681-7 [13]). Oncogenic transformation of MDCK cells by erbB2 and v-src induced expression of MT1-MMP, loss of cell-cell adhesion and scattered invasion into collagen gel, mRNA differential display and Northern hybridization identified metastasis-associated mts1 as one of the genes co-induced with MT1-MMP by oncogenic transformation or collagen-gel culture of MDCK cells. Expression of antisense RNA to mts1 in MDCK cells interfered with the extension of tubules into the collagen gel, however, it did not affect the morphological changes induced by HGF in culture on plastic dishes. ErbB2-transformant transfected with mts1 antisense construct, which showed unaltered morphology in culture on plastic dishes, did not scatter into collagen gel but formed aggregates. These results suggested that Mts1 contributes not only to tumor invasion but also to kidney tubulogenesis in cooperation with MT1-MMP. The coordinated action of MT1-MMP and Mts1, which is responsible for the highly invasive properties of mesenchymal cells, may be involved in epithelial tubulogenesis and invasion of malignant carcinoma cells.
Amino Acid Sequence↗
Internal dynamics of tRNA(Phe) studied by depolarized dynamic light scattering.
The collective internal dynamics of transfer RNA(Phe) from brewer's yeast in solution was studied by depolarized dynamic light scattering (DDLS). Within the melting region of tRNA the depolarized spectra consist of two Lorentzian, where the narrow (slow) component describes the overall rotation of the macromolecule. The broad component is attributed to the collective reorientation of the bases within the biopolymer. At high temperature only this relaxation process is observed in the spectrum. The viscosity dependence of the collective internal relaxation process is described by the Stokes-Einstein-Debye equation for rotational diffusion. Estimates of the internal orientational pair correlation factor from the integral depolarized intensities of tRNA(Phe) solutions indicates that the observed dynamics correspond to the collective reorientation of approximately 5 bases. A comparison of the results presented with DDLS studies on the aggregation of the mononucleotide guanosine-5'-monophosphate confirms this result. For a further characterization of the relaxation process we studied the effect of hydrostatic pressure (1-1000 bar) on the depolarized spectra of tRNA. While other spectroscopic methods like nmr, fluorescence polarization anisotropy decay, or ESR give information about the very local motion of a single base within the DNA or RNA, this study shows that by DDLS one can characterize collective internal motions of macromolecules.
Light↗
Effect of template conversion from the B to the Z conformation on RNA polymerase activity.
Transition from the right-handed B to the left-handed Z conformation of DNA was studied by circular dichroism in parallel with the ability of the DNA to support RNA synthesis with Escherichia coli RNA polymerase. Since the B to Z transition is generally induced by a chemical agent, a definitive demonstration that a change in activity is due to the conformational change, and not to the agent itself or to other factors, requires the clear-cut correlation of template activity and conformation under a variety of conditions that result in conformational change. Such correlation was achieved by following the [Co(NH3)6]3+-induced transition of poly(dG-dC) X poly(dG-dC) and poly(dG-dm5C) X poly(dG-dm5C) and the Mg2+-induced transition of poly(dG-dm5C) X poly(dG-dm5C). In addition, conditions were chosen to minimize possible aggregation. In each of these three systems, the B to Z conformational transition was accompanied by a substantial decrease in transcription activity. While the conversion from B to Z of poly(dG-dm5C) X poly(dG-dm5C) is induced by a 25-fold lower concentration of [Co(NH3)6]3+ than that required for the conversion of unmethylated polymer, in both cases the RNA polymerase activity is decreased at the same cation concentration as that producing the conformational transition. Neither [Co(NH3)6]3+ nor Mg2+ inhibits RNA synthesis with control templates that are not converted to Z under the same conditions, such as poly(dA-dT) X poly(dA-dT) or calf thymus DNA with [Co(NH3)6]3+ or poly(dG-dC) X poly(dG-dC) with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Circular Dichroism↗
Characterization of two recombination-complementation groups of Uukuniemi virus temperature-sensitive mutants.
With the aim of isolating temperature-sensitive (ts) mutants defective in virus maturation or glycoprotein transport, Uukuniemi virus, a bunyavirus, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. Out of 13 initial clones unable to grow at 39 degrees C (non-permissive temperature), five mutants which grew to titres above 10(7) p.f.u./ml at 32 degrees C (permissive temperature) were selected for further studies. The mutants fell into two coinciding recombination-complementation groups. Three group I mutants ( ts7 , 8 and 12) and two group II mutants ( ts6 and 11) synthesized all three RNA segments and were able to form the corresponding nucleoproteins at 39 degrees C. Thus, members of these two recombination groups had a RNA-positive phenotype. All five mutants showed immunofluorescence when cells were stained at 39 degrees C using a double-staining technique employing monoclonal antibodies against the glycoproteins G1 or G2, and polyclonal antibodies against the nucleoprotein, N. We have previously shown that in cells infected with wild-type virus both the G1/G2 and the N proteins accumulate in the Golgi complex, the site of virus maturation. In cells infected with ts12 , accumulation of G1 and G2, but not N protein, was observed in the Golgi complex at 39 degrees C. The N protein was found evenly scattered in the cytoplasm, suggesting lack of interaction between the G1/G2 and N proteins. With ts6 and 11, G1 and G2 appeared to accumulate and aggregate in the endoplasmic reticulum (ER) at 39 degrees C. The location of the N protein coincided with that of the aggregated glycoproteins, suggesting that the N protein interacted with G1/G2 already in the ER. Thus, these mutants may prove valuable tools in studying the mechanism of Uukuniemi virus maturation.
Bunyaviridae↗