Expression and Subcellular Localization of Arabidopsis thaliana Auxin-Binding Protein 1 (ABP1)

Expression of Arabidopsis thaliana ABP1 (AUXIN-BINDING PROTEIN 1) was studied using a promoter:GUS approach. Two promoter regions were analyzed. The 1585-bp promoter region upstream of the translation start site (P _ABP1 ) showed different activity compared to the promoter region that included, in addition, the first two introns and three exons of the transcribed ABP1 sequence (P _ABP1i1,2), indicating that cis elements were present downstream of the start codon. P _ABP1i1,2-driven β-glucuronidase activity was highest in growing leaves, in the root meristem, in vascular tissues, and in hydathodes. ABP1 promoter activities overlapped largely but not completely with that of DR5, which is a marker for the ARF-AuxRE-dependent auxin response. Subcellular ABP1 localization was studied using a constitutively overexpressed EGFP-ABP1 fusion protein. Results confirmed predominant localization to the endoplasmic reticulum as was concluded previously.     

拟南芥STN7和STN8蛋白激酶的结构预测和功能分析

STN7和STN8蛋白激酶分别参与了LHCⅡ蛋白和PSⅡ核心蛋白的磷酸化.作者用折叠识别的方法建立了拟南芥蛋白激酶STN7和STN8核心结构域的三维结构,同时结合其他生物信息学方法对STN7和STN8蛋白的结构和作用机制进行了探讨,选择特异位点合成多肽,并制备抗体.结果表明STN7和STN8蛋白激酶活性区域的电荷和形状互补性决定了两者作用底物蛋白的差异,蛋白印迹结果显示分别制备得到了STN7特异抗体和STN8特异抗体,证实结构预测的正确.     

An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.     

Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana

The X-ray crystal structure of the At5g18200.1 protein has been determined to a nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyl transfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself; thus, the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of (14)C between ADP-[(14)C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping-pong bi-bi kinetic mechanism, with a k(cat) of 4.1 s(-)(1) and K(m) values of 1.4 and 83 microM for ADP-[(14)C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 degrees C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping-pong bi-bi steady state kinetics, with a k(cat) of 2.7 s(-)(1) and K(m) values of 6.9 and 90 microM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 degrees C. The kinetics are consistent with a double-displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was determined to 1.83 A resolution and shows the AMP group bonded to His(186). The value of K(eq) in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 degrees C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl(2).     

Isolation and Characterization of a Ferredoxin Gene from Arabidopsis thaliana

We report the cloning and characterization of an Arabidopsis thaliana (L.) Heynh. (Columbia ecotype) ferredoxin gene (Fed A). Sequence analysis of a genomic clone shows an intron-free, 444-base pair open reading frame which encodes a 96 amino acid mature ferredoxin polypeptide preceded by a 52 amino acid transit peptide. Comparison with other plant ferredoxin proteins suggests that Fed A encodes a leaf ferredoxin. Genomic Southern blot analysis indicates the presence of a second, weakly related gene, consistent with other reports of at least two ferredoxins in plants. The Fed A gene promoter contains two regions, ACGCCACGTGGTAGATAGGATT (G-I box) and CCACGCCATTTCCACAAGC (CCAC box), which are strongly conserved in both sequence and position between the Arabidopsis and pea ferredoxin genes. Similarities with other better characterized plant promoter elements are also discussed.     

Molecular Cloning and Characterization of a Calmodulin Gene from Arabidopsis thaliana

The calcium binding protein, calmodulin Is involved in regulating various cellular and biochemical processes. A gene tor calmodulin (CaM) has been Isolated from a genomic library of Arabidopsis thaliana constructed in ; EMBL-4 using a heterologous cDNA probe from electric eel. One of the positive clones was characterized and the region containing the calmodulin gene sequences was Identified, excised using appropriate restriction enzymes and subcloned Into a plasmid vector. The genomic clone contains a complete copy of the calmodulin gene. A comparison of the nucleotide sequence of the part of the clone with those of the other plant and animal systems confirms that the clone In fact contains the calmodulin gene sequences. Southern hybridization ulling the calmodulin gene sequences as a probe reveals the presence of more than one copy of the calmodulin gene. The results of this investigation taken together with those Iff the other. indicate that the calmodulin gene belongs to a small mutigene family consisting of atieast four member. In the Arabidopsis genome.     

Structure and evolution of the 7SL RNA component of the signal recognition particle

We have cloned and characterized cDNA copies of larval and adult Drosophila 7SL RNA. The Drosophila 7SL sequence shares 66.3% homology with that of human 7SL RNA. The homology is not evenly distributed along the sequence, but is concentrated in blocks in the central part of the molecule. We have analysed the secondary structure of Drosophila and human 7SL RNA free in solution by digestion with single and double strand specific nucleases. Similar experiments with the 7SL RNA bound to proteins within the signal recognition particle show essentially the same digestion pattern. A model of the secondary structure common to Drosophila and human 7SL RNA is presented.     

Cloning and Characterization of an Arabidopsis thaliana Topoisomerase I Gene

cDNA and genomic clones encoding DNA topoisomerase I were isolated from Arabidopsis thaliana λgt11 and λFix libraries by low stringency hybridization with a Saccharomyces cerevisiae TOP1 probe. The cDNA clones include a 2748-base pair open reading frame predicting an amino acid sequence that is highly homologous to sequences encoded by TOP1 from yeast and human sources. The sequence of the upstream genomic region reveals two putative TATA-like elements and a purine-rich region, but no other obvious controlling elements. Southern blot analysis shows that the gene is present as a single copy in the Arabidopsis genome. When expressed in a S. cerevisiae top1 mutant under the control of the GAL1 promoter, the gene complements the phenotype caused by loss of topoisomerase activity and directs the expression of a protein that cross-reacts with a human anti-topoisomerase I antibody.     

Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.     

Purification and Properties of an Auxin-Binding Protein from the Shoot Apex of Peach Tree

A soluble auxin-binding protein was purified from the shootapices of peach trees by chromatography on columns of CM-Toyopearl,Sephacryl S-200, 2,4-D-linked-Sepharose 4B and ConA-Sepharose.The molecular mass of the purified protein was estimated tobe about 100 kDa. After electrophoresis on a denaturing gel,the protein gave a single band with a molecular mass of 20 kDa.From Scatchard analyses, the dissociation constant for 2,4-Dwas calculated to be 4.1 10^–5 M and the specific bindingof 2,4-D at saturating concentration was 42 nmol (mg protein)^–1.The binding of [¹⁴C]-2,4-D to the protein was reversible andwas inhibited by IAA, 1-naphthylacetic acid and p-chlorophenoxyisobutyricacid. (Received June 25, 1992; Accepted October 20, 1992)     

Dicer-dependent biogenesis of small RNAs derived from 7SL RNA

It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down. However, Alu-derived small RNAs showed different characteristics from miRNAs and siRNAs, the classic Dicer-processed products. Interestingly, we found that small RNAs derived from 7SL RNA accounted for 3.1% of the total cellular small RNAs in the control cells, and the abundance dropped about 3.4 folds in Dicer knockdown cells. Dicer-dependent biogenesis of 7SL RNA-derived small RNAs was validated by northern blotting. In vitro cleavage assay using the recombinant human Dicer protein also showed that synthetic 7SL RNA was processed by Dicer into fragments of different lengths. Further functional analysis suggested that 7SL RNA-derived small RNAs do not function like miRNAs, neither do they regulate the expression of 7SL RNA. In conclusion, the current study demonstrated that Dicer can process 7SL RNA, however, the biological significance remains to be elucidated.     

The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana

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