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PubMed1990

A role for the aspartyl protease from the human immunodeficiency virus type 1 (HIV-1) in the orchestration of virus assembly.

Functional HIV-1 protease (PR) is required for the maturation of viral proteins, for the appearance of characteristic structural features in the virion (as determined by electron microscopy), and for the final assembly of mature virus. Most importantly, HIV-1 PR activity is required for the development of infectivity. Still largely undefined, however, is the timing and control of protease action in this assembly process. Based on the three-dimensional structure of HIV-1 PR2,3 and experimental data reported in the literature, we propose a comprehensive virus assembly model that highlights the role of HIV-1 PR, suggests further experiments to verify the validity of the model, and poses specific questions relevant to the ultimate exploitation of HIV-1 protease as a therapeutic target.

Animals↗
PubMed1988

Studies on DNA topoisomerase activity during in vitro chromatin assembly.

The in vitro assembly of chromatin, promoted by the Xenopus cell-free extract (S-150), can be inhibited by oxolinic acid and to a lesser extent by nalidixic acid. Both of these antibiotics have been shown to block the activity of the specialized type II Topoisomerase, bacterial DNA Gyrase. Oxolinic acid induces a DNA cleavage by Micrococcal Nuclease at specific sequences in the multiple cloning vector pGEM-4. Nalidixic acid does not inhibit DNA supercoiling, but does diminish the extent of chromatin formation achieved by the S-150 on circular DNA templates. The Topoisomerase I inhibitor, berenil, does not inhibit extensive chromatin assembly, although it does diminish the level of supercoiling. Taken together, these results suggest that both topoisomerases play a role in the assembly process. Topoisomerase I may catalyze both the introduction of unconstrained supercoils into relaxed DNA and the formation of monosomes, while Topoisomerase II may promote extended chromatin assembly.

Animals↗
PubMed1991

Evidence for the existence of two assembly domains within the sea urchin fertilization envelope.

The sea urchin fertilization envelope (FE) is a complex, macromolecular aggregate assembled by the addition of cortical granule secretions to the vitelline layer. The completed, trilaminar structure has a dense layer sandwiched between surface coats of paracrystalline material. Two cortical granule enzymes, ovoperoxidase and protease, and a cell surface transglutaminase are required for the assembly process. We have examined, by quick-freeze, deep-etch, rotary-shadow electron microscopy, the effects of inhibiting each of these enzymes upon FE assembly. These experiments reveal two domains within the FE, distinguishable by their enzymatic requirements for proper maturation. The first domain consists of the microvillar casts which require both protease and transglutaminase activities to obtain a normal paracrystalline coat. The second domain comprises the regions between casts and appears to mature by ovoperoxidase-mediated cross-linking of paracrystalline material to the envelope.

Animals↗
PubMed2025

Colora: a Snakemake workflow for complete chromosome-scale de novo genome assembly.

MOTIVATION: De novo assembly creates reference genomes that underpin many modern biodiversity and conservation studies. Large numbers of new genomes are being assembled by labs around the world. To avoid duplication of efforts and variable data quality, we desire a best-practice assembly process, implemented as an automated portable workflow. RESULTS: Here, we present Colora, a Snakemake workflow that produces chromosome-scale de novo primary or phased genome assemblies complete with organelles using Pacific Biosciences HiFi, Hi-C, and optionally Oxford Nanopore Technologies reads as input. Colora is a user-friendly, versatile, and reproducible pipeline that is ready to use by researchers looking for an automated way to obtain high-quality de novo genome assemblies. AVAILABILITY AND IMPLEMENTATION: The source code of Colora is available on GitHub (https://github.com/LiaOb21/colora) and has been deposited in Zenodo under DOI https://doi.org/10.5281/zenodo.13321576. Colora is also available at the Snakemake Workflow Catalog (https://snakemake.github.io/snakemake-workflow-catalog/? usage=LiaOb21%2Fcolora).

Software↗
PubMed1985

Templeting and self-assembly.

Templeting and self-assembly represent the two extremes of the spectrum of determinate pattern-assembly processes. A templeted pattern can be defined as one that requires a prepattern or templet explicitly specifying the final topology of the pattern. Conversely, a self-assembling pattern can be defined as one for which the inherent constraints of the precursor elements alone are sufficient to specify the final pattern. Both concepts can be directly expressed in matrix notation, and a simple matrix measure, the templeting index, characterizes the relative amount of templeting or of self-assembly in any particular system. With this language, a fundamental principle of pattern-assembly becomes evident: in the determinate realm, some patterns can only be assembled using the same-sized templets--templets that are at least as large as the final pattern.

Mathematics↗
PubMed1992

Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting.

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).

Chromosomes, Human, Pair 19↗
PubMed1991

Contribution to the thermodynamics of protein folding from the reduction in water-accessible nonpolar surface area.

Protein folding and the transfer of hydrocarbons from a dilute aqueous solution to the pure liquid phase are thermodynamically similar in that both processes remove nonpolar surface from water and both are accompanied by anomalously large negative heat capacity changes. On the basis of a limited set of published surface areas, we previously proposed that heat capacity changes (delta C degrees p) for the transfer of hydrocarbons from water to the pure liquid phase and for the folding of globular proteins exhibit the same proportionality to the reduction in water-accessible nonpolar surface area (delta Anp) [Spolar, R.S., Ha, J.H., & Record, M.T., Jr. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8382-8385]. The consequence of this proposal is that the experimental delta C degrees p for protein folding can be used to obtain estimates of delta Anp and of the contribution to the stability of the folded state from removal of a nonpolar surface from water. In this paper, a rigorous molecular surface area algorithm [Richmond, T.J. (1984) J. Mol. Biol. 178, 63-89] is applied to obtain self-consistent values of the water-accessible nonpolar surface areas of the native and completely denatured states of the entire set of globular proteins for which both crystal structures and delta C degrees p of folding have been determined and for the set of liquid and liquefiable hydrocarbons for which delta C degrees p of transfer are known. Both processes (hydrocarbon transfer and protein folding) exhibit the same direct proportionality between delta C degrees p and delta Anp. We conclude that the large negative heat capacity changes observed in protein folding and other self-assembly processes involving proteins provide a quantitative measure of the reduction in the water-accessible nonpolar surface area and of the contribution of the hydrophobic effect to the stability of the native state and to protein assembly.

Protein Conformation↗
PubMed1976

Studies on the ability of partially iodinated 16S RNA to participate in 30S ribosome assembly.

Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C. Under these conditions 33 moles of iodine are incorporated per mole of RNA. As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S. The iodinated RNA was examined for its ability to reconstitute with total 30S proteins. Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S. In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts. The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.

Centrifugation, Density Gradient↗
PubMed1983

Regulation of protein expression in differentiation by subunit assembly. Human membrane and secreted IgM.

Newly synthesized IgM heavy chains are either expressed as functional IgM or catabolized, depending on the stage of differentiation of the B cell. Heavy chains of the membrane type are rapidly degraded in pre-B cells, but expressed on the cell surface as monomeric IgM in resting and secreting B cells. Secreted-type heavy chains are catabolized in pre-B and resting B cells, but secreted as pentameric IgM by secreting B cells. The differences between the heavy chains that are expressed and those that are catabolized are post-translational. Stable membrane and secreted heavy chains have been covalently assembled with light chains, terminally glycosylated, and removed from intracellular proteases by insertion into the plasma membrane or by secretion. The carboxylic ionophores monensin and nigericin have been used here to determine the relative importance of these post-translational events in stabilizing newly synthesized heavy chains. Monensin and nigericin inhibited both the rates and extents of terminal glycosylation and of intracellular transport of these proteins, without affecting the covalent assembly processes. These ionophores did not affect the rate of catabolism of heavy chains in either a resting or a secreting B cell line. IgM heavy chains thus appear to be stabilized against intracellular proteolysis by full covalent assembly to monomeric membrane IgM and to secreted pentameric IgM. The fully assembled IgM can then be terminally glycosylated and transported to the cell surface.

B-Lymphocytes↗
PubMed1990

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro.

We analysed the soluble form in which the nuclear pore complex protein p68 is stored in Xenopus laevis eggs and its involvement in pore complex assembly processes. We have shown previously that p68, which is the major wheat germ agglutinin (WGA)-binding glycoprotein of nuclear pore complexes from Xenopus oocytes, is located in the pore channel and participates in mediated transport of karyophilic proteins. Using a monoclonal antibody directed against p68 (PI1) we removed this protein from Xenopus egg extract by immunoadsorption. On addition of lambda DNA the immuno-depleted extract supported reconstitution of nuclei which were surrounded by a continuous double-membrane envelope but lacked pore complexes and were unable to import karyophilic proteins such as nucleoplasmin or lamin LIII. Essentially identical results were obtained with extract depleted of WGA-binding proteins. Our finding that both the anti-p68 antibody and WGA efficiently removed components from the extract necessary for pore complex assembly but did not interfere with nuclear membrane formation demonstrates that these processes are independent of each other. Analysis of the immunoprecipitate on silver-stained SDS-polyacrylamide gels indicated that the antibody adsorbed other proteins besides p68, notably two high molecular weight components. By sucrose gradient centrifugation and gel filtration we showed that p68 together with associated protein(s) forms a stable, approximately globular complex with an Mr of 254,000, a Stokes radius of 5.2 nm and a sedimentation coefficient of 11.3 S. Our finding that p68 occurs in the form of larger macromolecular assemblies offers an explanation for the distinctly punctate immunofluorescence pattern observed in the cytoplasm of mitotic cells after staining with antibodies to p68.

Animals↗
PubMed1990

Dynamic Monte Carlo simulations of globular protein folding/unfolding pathways. II. Alpha-helical motifs.

Dynamic Monte Carlo simulations of the folding pathways of alpha-helical protein motifs have been undertaken in the context of a diamond lattice model of globular proteins. The first question addressed in the nature of the assembly process of an alpha-helical hairpin. While the hairpin could, in principle, be formed via the diffusion-collision-adhesion of isolated performed helices, this is not the dominant mechanism of assembly found in the simulations. Rather, the helices that form native hairpins are constructed on-site, with folding initiating at or near the turn in almost all cases. Next, the folding/unfolding pathways of four-helix bundles having tight bends and one and two long loops in the native state are explored. Once again, an on-site construction mechanism of folding obtains, with a hairpin forming first, followed by the formation of a three-helix bundle, and finally the fourth helix of the native bundle assembles. Unfolding is essentially the reverse of folding. A simplified analytic theory is developed that reproduces the equilibrium folding transitions obtained from the simulations remarkably well and, for the dominant folding pathway, correctly identifies the intermediates seen in the simulations. The analytic theory provides the free energy along the reaction co-ordinate and identifies the transition state for all three motifs as being quite close to the native state, with three of the four helices assembled, and approximately one turn of the fourth helix in place. The transition state is separated from the native conformation by a free-energy barrier of mainly energetic origin and from the denatured state by a barrier of mainly entropic origin. The general features of the folding pathway seen in all variants of the model four-helix bundles are similar to those observed in the folding of beta-barrel, Greek key proteins; this suggests that many of the qualitative aspects of folding are invariant to the particular native state topology and secondary structure.

Algorithms↗
PubMed1988

Expression of type I procollagen genes.

All of the type I collagen in connective tissue is the product of one structural gene for the pro alpha 1(I) chain and another for the pro alpha 2(I) chain of type I procollagen. An intriguing question therefore is how the expression of the two genes differs in mineralizing and non-mineralizing tissues. One approach that our laboratory has pursued to answer this and related questions is to develop a new system whereby one can examine the self-assembly of collagen fibrils de novo by controlled enzymic cleavage of procollagen to collagen under physiological conditions. The system has made it possible for the first time to define thermodynamic parameters for the self-assembly process. We are now using the system to define the normal kinetics for fibril formation. The results should make it possible to study the effects of other components of extracellular matrix on fibril assembly, including the effects of bone-specific components that initiate mineralization. A second approach has been to define mutations in type I procollagen genes that cause increased brittleness of bone. Over a dozen mutations in type I procollagen genes have been found in probands with osteogenesis imperfecta. One of the surprises has been that at least 25% of the probands with lethal variants of osteogenesis imperfecta have mutations in type I procollagen genes. Another surprise has been the observation that a number of the mutations are tissue specific in terms of their phenotypic manifestations even though the same abnormal pro alpha chains are being synthesized in a variety of tissues.

Amino Acid Sequence↗
PubMed1990

Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein.

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1972

Ribosome synthesis in thermally shocked cells of Staphylococcus aureus.

Thermally shocked cells of Staphylococcus aureus rapidly synthesized ribonucleic acid (RNA) during the early stages of recovery. During this period, protein synthesis was not observed and occurred only after RNA had reached a maximum level. Even in the absence of coordinated protein synthesis, a large portion of the RNA appeared in newly synthesized ribosomes. Although the 30S subunit was specifically destroyed by the heating process, both ribosomal particles were reassembled during recovery. The addition of chloramphenicol did not inhibit the formation of the ribosomal subunits, nor was the presence of immature chloramphenicol particles detected. Extended recovery with highly prelabeled cells showed that the original ribosomal proteins present before heating are conserved and recycled. Furthermore, the data indicate that the 50S subunit is turned over and used as a source of protein for new ribosome assembly. Kinetic studies of the assembly process by pulse labeling have not revealed the presence of the normally reported precursor particles. Rather, the data suggest that assembly may occur, in this system, in a manner similar to that reported for in vitro assembly of Escherichia coli subunits.

Arginine↗
PubMed1989

Reversible inhibition of mammalian tubulin assembly in vitro and effects in Saccharomyces cerevisiae D61.M by mitomycin C.

Gaulden reported a novel and unexpected mitomycin C (MMC) effect, namely a pronounced retardation of very late prophase and loss of chromosome orientation in neuroblasts of the grasshopper Chortophaga viridifasciate. Because this effect may be due to interactions of MMC with non-DNA targets, MMC was tested for its interaction with porcine brain tubulin assembly in vitro and for the induction of chromosomal malsegregation in the diploid yeast Saccharomyces cerevisiae strain D61.M. A reversible dose-dependent inhibition of tubulin assembly was observed. Since no biological activation system was present in the incubation mixture this inhibition seems to result from an interaction of unactivated MMC with the assembly process. The possible chemical activation of MMC by reduction with 1,4-dithioerythritol (DTE) was investigated by omission of this compound during isolation and polymerization of tubulin. The absence of DTE resulted in a strong reduction of the net tubulin assembly. Also under these conditions MMC led to a dose-dependent inhibition of the assembly, indicating that the effect of MMC on tubulin assembly is independent of a reductive chemical modification. In S. cerevisiae D61.M, MMC did not induce chromosome loss, but induced other genetic events (possibly mutations, deletions or mitotic recombination) as was detected by an increase of the total number and of the frequency of cycloheximide-resistant colonies. This effect could be observed with and without the addition of rat liver S9 as an exogenous activation system.

Animals↗
PubMed1988

Nonrandom assembly of chromatin during hydroxyurea inhibition of DNA synthesis.

Incubation of MSB-1 chicken lymphoblastoid cells with hydroxyurea leads to a rapid 25-fold decrease in the incorporation of [3H]thymidine into DNA and a 5-fold decrease [3H]lysine into the nucleosome core histones. I have investigated whether the distortion in the normal proportion of histone-DNA synthesis results in alterations in the nucleosome assembly process and find that neither the stoichiometry of new histone synthesis nor the deposition is appreciably changed during hydroxyurea incubation. Protein cross-linking and micrococcal nuclease digestion show that the histones synthesized during hydroxyurea treatment form octamer structures and are assembled into typical nucleosome particles. Minor nucleosome subpopulations are found which exhibit altered sensitivity to nuclease digestion and which are depleted in new histones H3 and H4. When MSB-1 cells incubated in hydroxyurea are pulsed briefly with density-labeled amino acids and [3H]lysine, the radiolabeled core histone octamers formed are as dense as individual monomer histones. These results suggest that the newly synthesized histone octamers are uniformly dense and do not contain mixtures of new and old histones. Thus, histones synthesized during hydroxyurea incubation are deposited nonrandomly and do not exchange with preexisting histones.

Animals↗
PubMed1979

Extracts of Drosophila embryos mediate chromatin assembly in vitro.

Extracts of Drosophila embryos can mediate the assembly of a chromatinlike structure from histones and DNA under physiological conditions. The histone-DNA complex formed in vitro contains micrococcal nuclease-sensitive sites spaced at 200-base pair intervals. More extensive digestion of the complex by micrococcal nuclease generates 11S particles which cosediment with nucleosome core particles isolated from native chromatin. These particles contain 140-base pair DNA fragments which upon further cleavage with micrococcal nuclease give rise to a pattern of discretely sized DNA fragments characteristic of nucleosome core particles. We have assayed the chromatin assembly process both qualitatively by measuring the induction of supertwists into a relaxed circular DNA (a process requiring a nicking-closing enzyme) and quantitatively by measuring the formation of micrococcal nuclease-resistant DNA fragments from radioactively labeled linear DNA. The amount of chromatin formed depends primarily on the amount of histones, whereas the rate of assembly depends on the amount of extract protein added. The factors in the extract that mediate chromatin assembly appear to interact first with the DNA because preincubation of the DNA with the extract markedly increases the extent of assembly.

Animals↗
PubMedDate not supplied

[alphaC domains of fibrinogen molecules as the structures accelerating fibrin assembly].

Preparation of monomeric fibrin lacking intact alpha C-domains (monomeric X1-fragment), but fully clottable, is described. The assembly process of both monomeric fibrin and monomeric X1-fragment has been studied by electron microscopy and light scattering methods. It was shown that both proteins form similar fibrils with characteristic cross-banding. Upon dilution a sharp elevation of the differences between the assembly rates of monomeric X1-fragment and monomeric fibrin was revealed. The results obtained show that alpha C-domains take part in fibrin clot formation not as structural components but as the factor accelerating the ordered assembly of complex fibrin structure. The possible mechanism of alpha C-domains participation in fibrin clot formation are regarded.

Animals↗