Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.     

Characterization of Borna disease virus p56 protein, a surface glycoprotein involved in virus entry.

Borna disease virus (BDV) is a nonsegmented negative-stranded (NNS) RNA virus, prototype of a new taxon in the Mononegavirales order. BDV causes neurologic disease manifested by behavioral abnormalities in several animal species, and evidence suggests that it may be a human pathogen. To improve our knowledge about the biology of this novel virus, we have identified and characterized the product of BDV open reading frame IV (BVp56). Based on sequence features, BVp56 encodes a virus surface glycoprotein. Glycoproteins play essential roles in the biology of NNS RNA viruses. Expression of BVp56 resulted in the generation of two polypeptides with molecular masses of about 84 and 43 kDa (GP-84 and GP-43). GP-84 and GP-43 likely correspond to the full-length BVp56 gene and to its C terminus, respectively. Endoglycosidase studies demonstrated that both products were glycosylated and that this process was required for the stabilization of newly synthesized products. Moreover, our results suggested that GP-43 is generated by cleavage of GP-84 by a cellular protease. Subcellular localization studies demonstrated that GP-84 accumulates in the ER, whereas GP-43 reaches the cell surface. Both BVp56 products were found to be associated with infectious virions, and antibodies to BVp56 had neutralizing activity. Our findings suggest that BVp56 exhibits a novel form of processing for an animal NNS RNA virus surface glycoprotein, which might influence the assembly and budding of BDV.     

The contribution of TRPV4-mediated calcium signaling to calcium homeostasis in endothelial cells

A large variety of cation transport systems are involved in the regulation of calcium homeostasis in endothelial cells. The focus of the present study is to determine the contribution of nonselective cation channels from the TRP (transient receptor potential) family to cellular calcium homeostasis of porcine aortic endothelial cells (PAEC). One member of the TRPV (vanniloid) subfamily, TRPV4, has previously been shown to be involved in cation transport induced by a large variety of stimulations including osmolarity, temperature, mechanical stress, and phosphorylation. Here, we demonstrate the existence of several TRP proteins, including TRPV4, in PAEC using RT-PCR. To test whether this channel is functional, we performed FURA-2 calcium measurements and whole-cell patch-clamp experiments. We observed the induction of large calcium signals following mechanical stress, altered extracellular temperature, and the selective TRPV4 activator 4-alpha -PDD. These effects were diminished in the presence of the TRPV4 inhibitor miconazole, suggesting the involvement of this channel in mediating endothelial calcium signals. The large amounts of transported calcium and the short signaling ways suggest a potentially important role of this channel in many physiological processes.     

Overlapping expression patterns among the genes encoding Arabidopsis chromosomal high mobility group (HMG) proteins

High mobility group (HMG) proteins are usually considered ubiquitous components of the eukaryotic chromatin. Using HMG gene promoter-GUS reporter gene fusions we have examined the expression of the reporter gene in transgenic Arabidopsis plants. These experiments have revealed that the different HMGA and HMGB promoters display overlapping patterns of activity, but they also show tissue- and developmental stage-specific differences. Moreover, leader introns that are present in some of the HMGB genes can modulate reporter gene expression. The differential HMG gene expression supports the view that the various HMG proteins serve partially different architectural functions in plant chromatin.     

The relative contributions of polymer annealing and subunit exchange to microtubule dynamics in vitro

Microtubules that are free of microtubule-associated protein undergo dynamic changes at steady state, becoming longer but fewer in number with time through a process which was previously assumed to be based entirely on mechanisms of subunit exchange at polymer ends. However, we recently demonstrated that brain and erythrocyte microtubules are capable of joining end-to-end and suggested that polymer annealing may also affect the dynamic behavior of microtubules in vitro (Rothwell, S. W., W. A. Grasser, and D. B. Murphy, 1986, J. Cell Biol. 102:619-627). In the present study, we first show that annealing is a general property of cytoplasmic microtubules and is not a specialized characteristic of erythrocyte microtubules by documenting annealing between tryosinolated and detyrosinolated brain microtubules. We then examine the contributions of polymer annealing and subunit exchange to microtubule dynamics by analyzing the composition and length of individual polymers in a mixture of brain and erythrocyte microtubules by immunoelectron microscopy. In concentrated preparations of short-length microtubules at polymer-mass steady state, annealing was observed to be the principal factor responsible for the increase in polymer length, whereas annealing and subunit exchange contributed about equally to the reduction in microtubule number.     

Multiple proteins bind to the P2 promoter region of the zein gene pMS1 of maize

Summary A 216 by promoter fragment of the 19 kDa protein zein gene pMS1, containing the CCAAT and TATA boxes, was analysed by a variety of techniques for in vitro interactions with nuclear proteins from endosperm tissue. HMG proteins were found to form stable complexes with these A/T-rich promoter sequences and several specific DNA-binding proteins appear to be involved in the formation of DNA-protein complexes with this fragment. A 29 bp region spanning the two CCAAT boxes was protected from DNase I digestion in footprinting experiments.     

End-to-end annealing of microtubules in vitro

Mixtures of pre-formed microtubules, polymerized from chicken erythrocyte and brain tubulin, rapidly anneal end-to-end in vitro in standard microtubule assembly buffer. The erythrocyte tubulin segments in annealed heteropolymers can be distinguished by an immunoelectron microscopic assay that uses an antibody specific for chicken erythrocyte beta-tubulin. An annealing process is consistent with the following observations: (a) Microtubule number decreases while the polymer mass remains constant. (b) As the total number of microtubules declines, the number of heteropolymers, and the number of segments contained in each heteropolymer, increases. (c) The size of the segments determined after annealing and antibody labeling is the same as the original microtubule polymers. (d) Points of discontinuity in the annealing heteropolymers can be observed directly by electron microscopy, and correspond to type-specific polymer domains. The junctions probably represent initial contact points during the annealing process. Microtubule annealing occurs rapidly in vitro and may be significant for determining properties of microtubule dynamics in vivo.     

中国环境管理分区:方法与方案

我国生态环境可持续性及其影响因素的区域差异显著,各地区环境管理面临的主要挑战和需要优先解决的生态环境问题不同。进行环境管理分区,根据各地区生态环境特征及其影响因素的差异性,制定有针对性的环境管理政策,将有效促进我国区域生态环境的整体优化。采取定性和定量分析相结合的方法进行我国环境管理分区。首先,在我国3大自然区的基础上,根据我国的自然地理格局和已有的相关区划成果,把我国划分为4个环境管理大区,包括:南部季风区、北部季风区、西北干旱区和青藏高寒区。其次,通过建立的包含13个指标的环境管理分区指标体系,采用一维化欧式距离法分析各环境管理大区下相邻省级行政区环境特征的相似性,把环境特征相似性大的相邻地区划分到同一分区,得到以省级行政区为基本单元的我国环境管理分区方案。然后,结合地区间历史渊源和区域未来发展趋势分析,对基于相似性分析的初步分区方案进行调整,把我国划分为8个以省级行政区为基本单元环境管理区。最后,根据相关调整原则和方法,对以省级行政区为基本单元的分区方案的边界线进行调整,得到以地级行政区为基本单元的分区方案,把我国划分为东北地区、华北平原区、华北山地与高原区、东南沿海地区、长江流域中游地区、西南地区、西北干旱区和青藏高寒区8个环境管理区。     

Plant chromosomal HMGB proteins efficiently promote the bacterial site-specific beta-mediated recombination in vitro and in vivo

In the presence of an accessory DNA bending protein, the bacterial site-specific beta recombinase catalyzes resolution and DNA inversion. Five different maize high mobility group B (HMGB) proteins were examined for their potential to facilitate beta recombination in vitro using DNA substrates with different intervening distances (73-913 bp) between two directly oriented recombination (six) sites. All analyzed HMGB proteins (HMGB1 to HMGB5) could promote beta recombination, but depending on the DNA substrate with different efficiencies. The HMGB1 protein displayed an activity comparable to that of the natural promoting protein Hbsu, whereas the other HMGB proteins were less effective. Phosphorylation of the HMGB1 protein resulted in an increased efficiency of HMGB1 to promote beta recombination. Analyses of DNA substrates with closely spaced six sites demonstrated that in the presence of HMGB1 the recombination rate was correlated to the distance between the six sites, but independent of the helical orientation of the six sites. Using a Bacillus subtilis strain defective in Hbsu, the coexpression of beta recombinase and HMGB1 (or a truncated HMGB1 derivative) revealed that a plant HMG-box domain protein is sufficient for assisting beta to catalyze recombination in vivo. Our results using beta recombination as a model system suggest that the various plant HMGB proteins (and their posttranslationally modified versions) have the potential of forming a repertoire of different DNA structures, which is compatible with the idea that the HMGB proteins can act as architectural factors in a variety of nucleoprotein structures.