Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Four-Stranded Intercalated Cytosine-Rich Molecules: Novel Insights into DNA Structure and Stability

Abstract

DNA fragments with stretches of cytosine residues can form four-stranded intercalated i-DNA molecules stabilized by hemiprotonated cytosine·cytosine⁺ (C·C⁺) base pairs. Intriguing features of this motif are the accomodation of base stacking that is unfavorable due to electrostatic repulsion and the close approach of phosphates in narrow grooves of the molecule. Unusual sources of stability in this structure involve sugar-base stacking and CH-O interribose short contacts between the backbones of adjacent strands.     

Conformational dynamics of the human propeller telomeric DNA quadruplex on a microsecond time scale

The human telomeric DNA sequence with four repeats can fold into a parallel-stranded propeller-type topology. NMR structures solved under molecular crowding experiments correlate with the crystal structures found with crystal-packing interactions that are effectively equivalent to molecular crowding. This topology has been used for rationalization of ligand design and occurs experimentally in a number of complexes with a diversity of ligands, at least in the crystalline state. Although G-quartet stems have been well characterized, the interactions of the TTA loop with the G-quartets are much less defined. To better understand the conformational variability and structural dynamics of the propeller-type topology, we performed molecular dynamics simulations in explicit solvent up to 1.5 μs. The analysis provides a detailed atomistic account of the dynamic nature of the TTA loops highlighting their interactions with the G-quartets including formation of an A:A base pair, triad, pentad and hexad. The results present a threshold in quadruplex simulations, with regards to understanding the flexible nature of the sugar-phosphate backbone in formation of unusual architecture within the topology. Furthermore, this study stresses the importance of simulation time in sampling conformational space for this topology.     

Refinement of the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers

We present here the parmbsc0 force field, a refinement of the AMBER parm99 force field, where emphasis has been made on the correct representation of the alpha/gamma concerted rotation in nucleic acids (NAs). The modified force field corrects overpopulations of the alpha/gamma = (g+,t) backbone that were seen in long (more than 10 ns) simulations with previous AMBER parameter sets (parm94-99). The force field has been derived by fitting to high-level quantum mechanical data and verified by comparison with very high-level quantum mechanical calculations and by a very extensive comparison between simulations and experimental data. The set of validation simulations includes two of the longest trajectories published to date for the DNA duplex (200 ns each) and the largest variety of NA structures studied to date (15 different NA families and 97 individual structures). The total simulation time used to validate the force field includes near 1 mus of state-of-the-art molecular dynamics simulations in aqueous solution.     

Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

The self-cleaving hepatitis delta virus (HDV) ribozyme is essential for the replication of HDV, a liver disease causing pathogen in humans. The catalytically critical nucleotide C75 of the ribozyme is buttressed by a trefoil turn pivoting around an extruded G76. In all available crystal structures, the conformation of G76 is restricted by stacking with G76 of a neighboring molecule. To test whether this crystal contact introduces a structural perturbation into the catalytic core, we have analyzed approximately 200 ns of molecular dynamics (MD) simulations. In the absence of crystal packing, the simulated G76 fluctuates between several conformations, including one wherein G76 establishes a perpendicular base quadruplet in the major groove of the adjacent P1 stem. Second-site mutagenesis experiments suggest that the identity of the nucleotide in position 76 (N76) indeed contributes to the catalytic activity of a trans-acting HDV ribozyme through its capacity for hydrogen bonding with P1. By contrast, in the cis-cleaving genomic ribozyme the functional relevance of N76 is less pronounced and not correlated with the P1 sequence. Terbium(III) footprinting and additional MD show that the activity differences between N76 mutants of this ribozyme are related instead to changes in average conformation and modified cross-correlations in the trefoil turn.     

Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine

The ability of the four-stranded guanine (G)-DNA motif to incorporate nonstandard guanine analogue bases 6-oxopurine (inosine, I), 6-thioguanine (tG), and 6-thiopurine (tI) has been investigated using large-scale molecular dynamics simulations. The simulations suggest that a G-DNA stem can incorporate inosines without any marked effect on its structure and dynamics. The all-inosine quadruplex stem d(IIII)(4) shows identical dynamical properties as d(GGGG)(4) on the nanosecond time scale, with both molecular assemblies being stabilized by monovalent cations residing in the channel of the stem. However, simulations carried out in the absence of these cations show dramatic differences in the behavior of d(GGGG)(4) and d(IIII)(4). Whereas vacant d(GGGG)(4) shows large fluctuations but does not disintegrate, vacant d(IIII)(4) is completely disrupted within the first nanosecond. This is a consequence of the lack of the H-bonds involving the N2 amino group that is not present in inosine. This indicates that formation of the inosine quadruplex could involve entirely different intermediate structures than formation of the guanosine quadruplex, and early association of cations in this process appears to be inevitable. In the simulations, the incorporation of 6-thioguanine and 6-thiopurine sharply destabilizes four-stranded G-DNA structures, in close agreement with experimental data. The main reason is the size of the thiogroup leading to considerable steric conflicts and expelling the cations out of the channel of the quadruplex stem. The G-DNA stem can accommodate a single thioguanine base with minor perturbations. Incorporation of a thioguanine quartet layer is associated with a large destabilization of the G-DNA stem whereas the all-thioguanine quadruplex immediately collapses.     

Phylogeographic analysis of the brooding brittle star Amphipholis squamata (Echinodermata) along the coast of New Zealand reveals high cryptic genetic variation and cryptic dispersal potential

Abstract.— Direct development in benthic marine invertebrates is usually associated with narrow geographical range, low rates of colonization, and low levels of gene flow. Paradoxically, the small brittle star Amphipholis squamata broods its larvae to a crawl-away juvenile stage, yet has a cosmopolitan distribution. Using sequence and restriction-fragment-length-polymorphisms (RFLP) analyses of nuclear and mitochondrial DNA from 16 coastal populations throughout New Zealand, we tested whether the species is indeed a poor disperser, as may be expected from its brooding habit. We predicted that local and regional populations would be genetically structured according to isolation by distance. We also suspected that this ubiquitous "species" is composed of a variety of cryptic taxa in different geographic areas, as has been discovered in an increasing number of marine invertebrates. We found evidence of four genetically divergent and reproductively isolated lineages that can exist in syntopy. Lineages vary in abundance, haplotype diversity, and geographic distribution. The partitioning of genetic variation within the most common lineage, as well as the geographic distribution of the four lineages, suggest a north/south split. This pattern is consistent with known New Zealand marine biogeographic zones and appears to be linked to the regime of oceanic circulation, which is characterized by subtropical, southward-moving water masses in the north, and sub-Antarctic, northward-moving water in the south. We conclude that the dispersal ability of A. squamata is regionally restricted but with sporadic long-distance dispersal, which serves to increase local genetic variation. Our results support the idea that dispersal occurs through passive transport by drifting or rafting on macroalgae, which A. squamata commonly inhabits, and emphasize that poor dispersal ability is not necessarily a corollary of direct development.     

The influence of the thymine C5 methyl group on spontaneous base pair breathing in DNA

Sequences of four or more AT base pairs without a 5'-TA-3' step, so-called A-tracts, influence the global properties of DNA by causing curvature of the helix axis if phased with the helical repeat and also influence nucleosome packaging. Hence it is interesting to understand this phenomenon on the molecular level, and numerous studies have been devoted to investigations of dynamical and structural features of A-tract DNA. It was early observed that anomalously slow base pair-opening kinetics were a striking physical property unique to DNA A-tracts (Leroy, J. L., Charretier, E., Kochoyan, M., and Gueron, M. (1988) Biochemistry 27, 8894-8898). Furthermore, a strong correlation between DNA curvature and anomalously slow base pair-opening dynamics was found. In the present work it is shown, using imino proton exchange measurements by NMR spectroscopy that the main contribution to the dampening of the base pair-opening fluctuations in A-tracts comes from the C5 methylation of the thymine base. Because the methyl group has been shown to have a very limited effect on the DNA curvature as well as the structure of the DNA helix, the thymine C5 methyl group stabilizes the helix directly. Empirical potential energy calculations show that methylation of the tract improves the stacking energy of a base pair with its neighbors in the tract by 3-4 kcal/mol.     

Critical effect of the N2 amino group on structure, dynamics, and elasticity of DNA polypurine tracts

Unrestrained 5-20-ns explicit-solvent molecular dynamics simulations using the Cornell et al. force field have been carried out for d[GCG(N)11GCG]2 (N, purine base) considering guanine*cytosine (G*C), adenine*thymine (A*T), inosine*5-methyl-cytosine (I*mC), and 2-amino-adenine*thymine (D*T) basepairs. The simulations unambiguously show that the structure and elasticity of N-tracts is primarily determined by the presence of the amino group in the minor groove. Simulated A-, I-, and AI-tracts show almost identical structures, with high propeller twist and minor groove narrowing. G- and D-tracts have small propeller twisting and are partly shifted toward the A-form. The elastic properties also differ between the two groups. The sequence-dependent electrostatic component of base stacking seems to play a minor role. Our conclusions are entirely consistent with available experimental data. Nevertheless, the propeller twist and helical twist in the simulated A-tract appear to be underestimated compared to crystallographic studies. To obtain further insight into the possible force field deficiencies, additional multiple simulations have been made for d(A)10, systematically comparing four major force fields currently used in DNA simulations and utilizing B and A-DNA forms as the starting structure. This comparison shows that the conclusions of the present work are not influenced by the force field choice.