Folding of the Tetrahymena ribozyme by polyamines: importance of counterion valence and size

Polyamines are abundant metabolites that directly influence gene expression. Although the role of polyamines in DNA condensation is well known, their role in RNA folding is less understood. Non-denaturing gel electrophoresis was used to monitor the equilibrium folding transitions of the Tetrahymena ribozyme in the presence of polyamines. All of the polyamines tested induce near-native structures that readily convert to the native conformation in Mg(2+). The stability of the folded structure increases with the charge of the polyamine and decreases with the size of the polyamine. When the counterion excluded volume becomes large, the transition to the native state does not go to completion even under favorable folding conditions. Brownian dynamics simulations of a model polyelectrolyte suggest that the kinetics of counterion-mediated collapse and the dimensions of the collapsed RNA chains depend on the structure of the counterion. The results are consistent with delocalized condensation of polyamines around the RNA. However, the effective charge of the counterions is lowered by their excluded volume. The stability of the folded RNA is enhanced when the spacing between amino groups matches the distance between adjacent phosphate groups. These results show how changes in intracellular polyamine concentrations could alter RNA folding pathways.     

D-Titin: a giant protein with dual roles in chromosomes and muscles

Previously, we reported that chromosomes contain a giant filamentous protein, which we identified as titin, a component of muscle sarcomeres. Here, we report the sequence of the entire titin gene in Drosophila melanogaster, D-Titin, and show that it encodes a two-megadalton protein with significant colinear homology to the NH(2)-terminal half of vertebrate titin. Mutations in D-Titin cause chromosome undercondensation, chromosome breakage, loss of diploidy, and premature sister chromatid separation. Additionally, D-Titin mutants have defects in myoblast fusion and muscle organization. The phenotypes of the D-Titin mutants suggest parallel roles for titin in both muscle and chromosome structure and elasticity, and provide new insight into chromosome structure.     

Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa

Reversible phosphorylation of serine/threonine residues of cell cycle-regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28-related kinases) have been extensively studied, while the role of counter-acting protein phosphatases is less well understood. We used endothall (ET) as a cell-permeable inhibitor of serine/threonine-specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 microM) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 microM) of ET-treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 microM ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET-treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.     

Folding transition into a loosely collapsed state in plasmid DNA as revealed by single-molecule observation

The conformational transition of a plasmid DNA, pGEG.GL3 (12.5 kbp, circular), induced by spermine(4+) was studied through the observation of individual DNA by fluorescence microscopy. We deduced the change in the hydrodynamic radius R(H) from an analysis of the Brownian motion of single DNA molecules. R(H) decreases in a continuous manner with an increase in spermine(4+), in contrast to the large discrete on/off change for long linear DNA. Just after the transition to the collapsed state, a small number of DNA molecules tend to form an assembly, which disperses in the bulk solution without precipitation.     

Requirement of phosphatidylinositol-4,5-bisphosphate for HERC1-mediated guanine nucleotide release from ARF proteins

HERC1 is a giant multidomain protein involved in membrane trafficking through its interaction with vesicle coat proteins such as clathrin and ARF. Previously, it has been shown that the RCC1-like domain 1 (RLD1) of HERC1 stimulates guanine nucleotide dissociation on ARF1 and Rab proteins. In this study, we have analyzed whether HERC1 may also regulate ARF6 activity. We show that HERC1, through its RLD1, stimulates GDP release from ARF6 but, unexpectedly, it inhibits GDP/GTP exchange on ARF6 under conditions where ARNO stimulates it. Furthermore, we demonstrate that the activity of HERC1 as a guanine nucleotide release factor requires the presence of PI(4,5)P(2) bound to HERC1's RLD1. In agreement with this, we find that purified HERC1 contains PI(4,5)P(2) bound to the RLD1.     

Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina

The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.     

Components and Controls of Water Flux in an Old-growth Douglas-fir–Western Hemlock Ecosystem

We report measurements of rates of sap flow in dominant trees, changes in soil moisture, and evaporation from coarse woody debris in an old-growth Douglas-fir–western hemlock ecosystem at Wind River, Washington, USA, during dry periods in summer. The measurements are compared with eddy-covariance measurements of water-vapor fluxes above the forest (E_e) and at the forest floor (E_u) to examine the components of ecosystem water loss and the factors controlling them. Daily values of E_u were about 10% of E_e. Evaporation from coarse woody debris was only about 2% of E_e. Transpiration (E_t), estimated by scaling sap-flow measurements accounted for about 70% of (E_e– E_u); transpiration from subdominant trees may account for the remainder. The daily total change in soil moisture (E_s) in the top 30 cm was larger than the net change, probably because of hydraulic redistribution of soil water by roots. Observed differences between E_s and E_e were probably because roots also extract water from greater depth, and/or because the measuring systems sample at different spatial scales. The ratio of E_t to E_s decreased with decreasing soil water content, suggesting that partitioning in water use between understory and overstory changed during the season. The rate of soil drying exceeded E_e early in the day, probably because water vapor was being stored in canopy air space and condensed or adsorbed on tree stems, lichens, and mosses. The daily variation of E_e with vapor-pressure deficit showed strong hysteresis, most likely associated with transpiration of water stored in tree stems and branches.     

Localization of the gene-richest and the gene-poorest isochores in the interphase nuclei of mammals and birds

At a resolution of 850 bands, human chromosomes comprise two subsets of bands, the GC-richest H3⁺ and the GC-poorest L1⁺ bands, accounting for about 17 and 26%, respectively, of all bands. The former are a subset of the R bands and the latter are a subset of the G bands. These bands showed the highest and the lowest gene densities, respectively, as well as a number of other distinct features. Here we report that human and chicken interphase nuclei are characterized by the following features. (1) The gene-richest/GC-richest chromosomal regions are predominantly distributed in internal locations, whereas the gene-poorest/GC-poorest DNA regions are close to the nuclear envelope. (2) The interphase chromosomes seem to be characterized by a polar arrangement, because the gene-richest/GC-richest bands and the gene-poorest/GC-poorest bands are predominantly located in the distal and proximal regions, respectively, of chromosomes, and because interphase chromosomes are extremely long. While this polar arrangement is evident in the larger chromosomes, it is not displayed by the chicken microchromosomes and by some small human chromosomes, namely by chromosomes that are almost only composed by GC-rich or by GC-poor DNA. (3) The gene-richest chromosomal regions display a much more spread-out conformation compared to the gene-poorest regions in human nuclei. This finding has interesting implications for the formation of GC-rich isochores of warm-blooded vertebrates.     

Alterations in the spatiotemporal expression pattern and function of N-cadherin inhibit cellular condensation and chondrogenesis of limb mesenchymal cells in vitro

Cartilage formation in the embryonic limb is presaged by a cellular condensation phase that is mediated by both cell-cell and cell-matrix interactions. N-Cadherin, a Ca(2+)-dependent cell-cell adhesion molecule, is expressed at higher levels in the condensing mesenchyme, followed by down-regulation upon chondrogenic differentiation, strongly suggesting a functional role in the cellular condensation process. To further examine the role of N-cadherin, we have generated expression constructs of wild type and two deletion mutants (extracellular and intracellular) of N-cadherin in the avian replication-competent, RCAS retrovirus, and transfected primary chick limb mesenchymal cell cultures with these constructs. The effects of altered, sustained expression of N-cadherin and its mutant forms on cellular condensation, on the basis of peanut agglutinin (DNA) staining, and chondrogenesis, based on expression of chondrocyte phenotypic markers, were characterized. Cellular condensation was relatively unchanged in cultures overexpressing wild type N-cadherin, compared to controls on all days in culture. However, expression of either of the deletion mutant forms of N-cadherin resulted in decreased condensation, with the extracellular deletion mutant demonstrating the most severe inhibition, suggesting a requirement for N-cadherin mediated cell-cell adhesion and signaling in cellular condensation. Subsequent chondrogenic differentiation was also affected in all cultures overexpressing the N-cadherin constructs, on the basis of metabolic sulfate incorporation, the presence of the cartilage matrix proteins collagen type II and cartilage proteoglycan link protein, and alcian blue staining of the matrix. The characteristics of the cultures suggest that the N-cadherin mutants disrupt proper cellular condensation and subsequent chondrogenesis, while the cultures overexpressing wild type N-cadherin appear to condense normally, but are unable to proceed toward differentiation, possibly due to the prolonged maintenance of increased cell-cell adhesiveness. Thus, spatiotemporally regulated N-cadherin expression and function, at the level of both homotypic binding and linkage to the cytoskeleton, is required for chondrogenesis of limb mesenchymal cells.     

C-terminal domain of gyrase A is predicted to have a beta-propeller structure

Two different type II topoisomerases are known in bacteria. DNA gyrase (Gyr) introduces negative supercoils into DNA. Topoisomerase IV (Par) relaxes DNA supercoils. GyrA and ParC subunits of bacterial type II topoisomerases are involved in breakage and reunion of DNA. The spatial structure of the C-terminal fragment in GyrA/ParC is not available. We infer homology between the C-terminal domain of GyrA/ParC and a regulator of chromosome condensation (RCC1), a eukaryotic protein that functions as a guanine-nucleotide-exchange factor for the nuclear G protein Ran. This homology, complemented by detection of 6 sequence repeats with 4 predicted beta-strands each in GyrA/ParC sequences, allows us to predict that the GyrA/ParC C-terminal domain folds into a 6-bladed beta-propeller. The prediction rationalizes available experimental data and sheds light on the spatial properties of the largest topoisomerase domain that lacks structural information.