Proteins encoded by an auxin-regulated gene family of tobacco share limited but significant homology with glutathione S-transferases and one member indeed shows in vitro GST activity

A number of cDNAs corresponding to auxin-regulated mRNAs have been isolated from tobacco and found to be encoded by a multigene family consisting of three subfamilies. Homologous proteins have been isolated independently from soybean and potato. Here we report that the encoded proteins show a limited but significant homology to both plant and animal glutathione S-transferases (GST, EC 2.5.1.18). For the protein NT103, encoded by a member of the Nt103 subfamily, we demonstrate an in vitro GST activity. This is the first time a function is attributed to a member of this group of auxin-induced proteins or any of its homologues. The implications of this finding and the possible relationships between auxins and GSTs are discussed.     

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.     

Cloning, expression and purification of DNA-binding protein Mvo10b from Methanococcus voltae

Mvo10b from the mesophilic archaeon Methanococcus voltae is a member of the Sac10b family which may play an important role in the organization and accessibility of genetic information in Archaea. Since Mvo10b is a DNA-binding protein as the other member in the Sac10b family, to obtain a recombinant Mvo10b requires an efficient and inexpensive expression and purification system for producing the protein free of nucleic acid contamination. Previously, the hyperthermophilic archaeal Ssh10b of the Sac10b family was successfully purified. However, the protocol adopted to purify Ssh10b is not appropriate for purifying the mesophilic Mvo10b. This study describes the successful expression and purification of the recombinant Mvo10b. The expression of recombinant Mvo10b was carried out in Escherichia coli, and the target protein was expressed in the soluble form. The protein was purified by polyethyleneimine (PEI) precipitation followed by nickel ion metal affinity chromatography. The purity of Mvo10b was checked to insure being free of nucleic acid contamination. The final protein yield is about 30 mg/l of LB culture. The ensemble of NMR and far-UV CD data shows that the purified Mvo10b has abundant regular secondary structures and is correctly folded, which may have similar 3D structure as its hyperthermophilic counterpart [P62A]Ssh10b. The developed protocol has potential application in the production of the other thermophilic and mesophilic proteins in the Sac10b family.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.     

An Arabidopsis gene with homology to glutathione S-transferases is regulated by ethylene

A cDNA clone obtained from Arabidopsis leaf RNA encodes a 24 kDa protein with homology to glutathione S-transferases (GST). It is most homologous with a tobacco GST (57% identity). In Arabidopsis, expression of GST mRNA is regulated by ethylene. Exposure of plants to ethylene increased the abundance of GST mRNA, while treatment with norbornadiene had the reverse effect. Ethylene had no effect on the mRNA level in ethylene-insensitive etr1 plants. The abundance of this mRNA increased with the age of plants. DNA hybridizations indicate that GSTs are encoded by a large multigene family in Arabidopsis.     

Further evidence that rat liver microsomal glutathione transferase 1 is not a cellular protein target for S-nitrosylation

By adopting biotin switch method, we recently reported that liver microsomal glutathione transferase 1 (MGST1) might not be a protein target for S-nitrosylation in rat microsomes or in vivo. However, alternative analytic methods are needed to confirm this observation, as a single biotin switch method in judging specific protein S-nitrosylation in biological samples is increasingly recognized as insufficient, or even unreliable. Besides, only MGST1 localized on endoplasmic reticulum (ER), but not mitochondria which favors protein S-nitrosylation was examined in the previous report. Present study was therefore carried out to address these issues. Primary cultured hepatocytes were used. A physiological existing nitric oxide (NO) donor S-nitrosoglutathione (GSNO) was adopted to trigger protein S-nitrosylation. MGST1 was immunoprecipitated and its S-nitrosothiol content was measured by the NO probe 2,3-diaminonaphthalene. In parallel, S-nitrosylated proteins were immunoprecipitated by a monoclonal anti-S-nitrosocysteine antibody and probed with an anti-MGST1 antibody. In hepatocytes, neither ER nor mitochondria were found to contain S-nitrosylated MGST1 after GSNO treatment, showing that differently distributed MGST1 was consistently un-nitrosylable in the cellular environment. But under broken cell conditions, when samples were incubated directly with GSNO, MGST1 S-nitrosylation was indeed detectable in both the microsomal and mitochondrial proteins, indicating that previous failure in detecting MGST1 S-nitrosylation in microsomes is due to the limitations of biotin switch method. These results clearly, if not definitely, demonstrate that MGST1 is not a ready candidate for S-nitrosylation in the cellular content, despite its susceptibility to S-nitrosylation under broken cell conditions.     

毛白杨PtoGSTF4基因克隆及相关特性鉴定

该研究从毛白杨中克隆到1个Phi类谷胱苷肽S-转移酶（GST）基因（PtoGSTF4）,编码213个氨基酸。表达模式分析发现,PtoGSTF4在正常生长、H₂O₂和莠去津处理后的茎、叶以及茎的韧皮部均表达,属于组成型表达基因。在大肠杆菌中表达并纯化了PtoGSTF4重组蛋白,酶学性质分析表明PtoGSTF4对CDNB、NBD-Cl、NBC和Cum-OOH等4种底物均有活性。动力学分析发现,PtoGSTF4对GSH具有较高的亲和力,而对CDNB的亲和力相对较低。在不同pH及温度条件下对PtoGSTF4蛋白进行活性检测,发现PtoGSTF4在pH 7.5~10.5范围内或30 ℃~60 ℃温度范围内有较高的活性。研究推测,PtoGSTF4可能在毛白杨的抗逆生理中发挥重要作用。     

Coordinated activation of as-1-type elements and a tobacco glutathione S-transferase gene by auxins, salicylic acid, methyl-jasmonate and hydrogen peroxide

The molecular mechanism of signal transduction pathways which mediate the action of phytohormones are poorly understood. Recently, we and others have shown that the as-1 type cis-acting elements can respond to auxin and salicylic acid, two well-characterized signaling molecules in plants. In the present work, we have examined a comprehensive set of physiological and abiotic agents and found that auxin, salicylic acid and methyl-jasmonate are three effective inducers of the as-1-type elements in transgenic tobacco. Using a cell suspension culture containing a synthetic promoter-GUS fusion, we demonstrated rapid and sensitive induction of the as-1-type element by these phytohormones. Furthermore, a tobacco glutathione S-transferase gene, GNT35, that contains an as-1-type binding site in its promoter is also inducible by auxin, salicylic acid and methyl-jasmonate with similar kinetics. As Ulmasov et al. have recently reported, we found that the as-1-type elements can also respond to weak/inactive analogues of auxin and salicylic acid. In addition, we show that hydrogen peroxide can also effectively activate the expression of GNT35 as well as the as-1-type element in a cell suspension culture, but not with whole seedlings. These results are discussed with respect to the possible mechanism(s) through which a single cis element may respond to a diverse array of molecules.     

OsBP-73, a rice gene,encodes a novel DNA-binding protein with a SAP-like domain and its genetic interference by double-stranded RNA inhibits rice growth

The SAP domain is a recently defined DNA binding domain that forms a helix-extended-helix structure. SAP proteins have been implicated in nuclear architecture and/or RNA metabolism. In this paper, we describe the cloning and characterization of a rice gene, OsBP-73, encoding a 375 amino acid protein with a SAP-like domain. We identified the binding sequence of OsBP-73 by gel retardation assays and southwestern blotting. Northern blot analysis demonstrated that OsBP-73 is weakly expressed in root, leaf and immature seed. OsBP-73 gene expression was also examined by histochemical studies of transgenic rice plants carrying an OsBP-73 5/GUS reporter gene. The reporter gene is mainly expressed in the tissues with high cell division activities, such as root tip, stem node, panicle and immature seed. Genetic interference of OsBP-73 gene expression by double-stranded RNA strikingly inhibits the whole plant growth but does not affect the passage from the juvenile to adult phase. These results suggest that OsBP-73 may play an important role in the regulation of cell proliferation.     

LaTBP1: a Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP.