Expression and purification of recombinant human histones

Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.     

Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants

Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer. Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter. In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis. The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination. The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other. In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested. Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site. The transgenic rice obtained in this study can be used for functional genomics of rice.     

A novel beta-glucanase gene from Bacillus halodurans C-125

A novel endo-beta-1,3(4)-D-glucanase gene was found in the complete genome sequence of Bacillus halodurans C-125. The gene was previously annotated as an "unknown" protein and assigned an incorrect open reading frame (ORF). However, determining the biochemical characteristics has elucidated the function and correct ORF of the gene. The gene encodes 231 amino acids, and its calculated molecular mass was estimated to be 26743.16 Da. The amino acid sequence alignment showed that the highest sequence identity was only 28% with that of the beta-1,3-1,4-glucanase from Bacillus subtilis. Moreover, the nucleotide sequence did not match any other known Bacillus beta-glucanase gene. The member of the gene cluster that includes this novel gene was apparently different from that of the gene cluster including the putative beta-glucanase genes (bh3231 and bh3232) from B. halodurans C-125. Therefore, the novel gene is not a copy of either of these genes, and in B. halodurans cells, the putative role of the encoded protein may differ from that of bh3231 and bh3232. To examine the activity of the gene product, the gene was cloned as a His-tagged protein and expressed in Escherichia coli. The purified enzyme showed activity against lichenan, barley beta-glucan, laminarin, and carboxymethyl curdlan. Thin-layer chromatography showed that the enzyme hydrolyzes substrates in an endo-type manner. When beta-glucan was used as a substrate, the pH optimum was between 6 and 8, and the temperature optimum was 60 degrees C. After 2 h incubation at 50 and 60 degrees C, the residual activity remained 100% and 50%, respectively. The enzymatic activity was abolished after 30 min incubation at 70 degrees C. Based on the results, the gene encodes an endo-type beta-1,3(4)-D-glucanase (E.C. 3.2.1.6).     

Suppression of host defense in compatible plant-Pseudomonas syringae interactions

Despite impressive advances in the study of plant resistance to pathogens, little is known about the molecular basis of plant susceptibility to virulent pathogens. Recent progress in susceptible plant-Pseudomonas syringae interactions has provided a glimpse into the battles fought between plants and bacterial pathogens. A key step for pathogenesis appears to be the suppression of host defenses. Suppression of host defenses, including basal defense, gene-for-gene resistance and nonhost resistance, is a key step for pathogenesis. Defense suppression is mediated by bacterial effector proteins, which are secreted through the type III secretion system, and by coronatine, a bacterial toxin that structurally and functionally mimics methyl jasmonate, a plant defense signaling molecule.     

Coexistence of competitive species with a stage-structured life cycle

Ecological theory provides explanations for exclusion or coexistence of competing species. Most theoretical works on competition dynamics that have shaped current perspectives on coexistence assume a simple life cycle. This simplification, however, may omit important realities. We present a simple two-stage structured competition model to investigate the effects of life-history characteristics on coexistence. The achievement and the stability of coexistence depend not only on competition coefficients but also on a set of life-history parameters that reflect the viability of an individual, namely, adult death rate, maturation rate, and birth rate. High individual viability is necessary for a species to persist, but it does not necessarily facilitate coexistence. Intense competition at the juvenile or adult stage may require higher or lower viability, respectively, for stable coexistence to be possible. The stability mechanism can be explained by the refuge effect of the less competitive stage, and the birth performance, which preserves the less competitive stage as a refuge. Coexistence might readily collapse if the life-history characteristics, which together constitute individual viability, change, even though two species have an inherent competitive relation conducive to stable coexistence.     

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.     

Development of an antibody-based assay for determination of baculovirus titers in 10 hours

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.     

Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel

The importance of voltage-dependent Ca2+ channels (VDCCs) in pain transmission has been noticed gradually, as several VDCC blockers have been shown to be effective in inhibiting this process. In particular, the N-type VDCC has attracted attention, because inhibitors of this channel are effective in various aspects of pain-related phenomena. To understand the genuine contribution of the N-type VDCC to the pain transmission system, we generated mice deficient in this channel by gene targeting. We report here that mice lacking N-type VDCCs show suppressed responses to a painful stimulus that induces inflammation and show markedly reduced symptoms of neuropathic pain, which is caused by nerve injury and is known to be difficult to treat by currently available therapeutic methods. This finding clearly demonstrates that the N-type VDCC is essential for development of neuropathic pain and, therefore, controlling the activity of this channel can be of great importance for the management of neuropathic pain.