Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.Soybean (Glycine max) and numerous other legumes can establish a symbiosis with rhizobia, resulting in the formation of root nodules comprising specialized plant and bacterial cells (for review, see Mylona et al., 1995). Rhizobia reduce atmospheric nitrogen to ammonia within nodules, which is assimilated by the plant host to fulfill its nutritional nitrogen requirement. The high energy requirement for nitrogen fixation necessitates efficient respiration by the prokaryote within the microaerobic milieu of the nodule. The plant host synthesizes a nodule-specific hemoglobin (leghemoglobin) that serves to facilitate oxygen diffusion to the bacterial endosymbiont and to buffer the free oxygen concentration at a low tension (for review, see Appleby, 1992). Both of these functions require that the hemoglobin concentration be high, and, indeed, it exceeds 1 mm in soybean nodules (Appleby, 1984) and is the predominant plant protein in that organ. Once thought to be confined to legume nodules, hemoglobins are found throughout the plant kingdom, and leghemoglobin likely represents a specialization of a general plant phenomenon (for review, see Hardison, 1996). A gene encoding a nonsymbiotic hemoglobin has been identified in soybean and other legumes (Andersson et al., 1996); therefore, expression in nodules involves the specific activation of a subset of genes within a gene family. Leghemoglobin genes may have arisen from gene duplication, followed by specialization (Andersson et al., 1996).Hemes and chlorophyll are tetrapyrroles synthesized from common precursors; chlorophyll is quantitatively the major tetrapyrrole in plants, with heme and other tetrapyrroles being present in minor amounts. Legume root nodules represent an exception, in which heme is synthesized in high quantity in the absence of chlorophyll, thus requiring the activity of enzymes not normally expressed highly in nonphotosynthetic tissues. Heme is synthesized from the universal tetrapyrrole precursor ALA by seven successive enzymatic steps; chlorophyll formation diverges after the synthesis of protoporphyrin, the immediate heme precursor (for review, see O''Brian, 1996). Biochemical and genetic evidence shows that soybean heme biosynthesis genes are strongly induced in root nodules (Sangwan and O''Brian, 1991, 1992, 1993; Madsen et al., 1993; Kaczor et al., 1994; Frustaci et al., 1995; Santana et al., 1998), and immunohistochemical studies demonstrate that induction is concentrated in infected nodule cells (Santana et al., 1998).ALA is synthesized from Glu in plants by a three-step mechanism called the C₅ pathway (Fig. ​(Fig.1);1); the latter two steps are committed to ALA synthesis and are catalyzed by GTR and GSAT, respectively (for review, see Beale and Weinstein, 1990; Jahn et al., 1991). Plant cDNA or genes encoding GTR (Gtr, also called HemA) and GSAT (Gsa) have been identified in several plant species (Grimm, 1990; Sangwan and O''Brian, 1993; Hofgen et al., 1994; Ilag et al., 1994; Frustaci et al., 1995; Wenzlau and Berry-Lowe, 1995; Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). Two genes for each enzyme have been described, and some genes are reported to be specific to a tissue, tetrapyrrole, or light regimen (Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). However, soybean Gsa1 is highly expressed in both leaves and nodules and contains a cis-acting element in its promoter that binds to a nuclear factor found in both tissues. (Frustaci et al., 1995). In this study we isolated soybean Gtr1 and characterized the genetic basis of GTR expression in root nodules. Figure 1C₅ pathway for ALA synthesis. The committed steps for ALA synthesis catalyzed by GTR and GSAT are boxed. Glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA^Glu also participate in protein synthesis. The gene designations in plants are shown in parentheses ...     

Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO₄, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.     

同源盒基因A10编码蛋白的靶调节基因的筛选与鉴定

目的：筛选和鉴定同源盒基因A10编码蛋白（HOXA10）的靶调节基因。方法：以人子宫内膜细胞系AN3CA为实验对象,采用染色质免疫沉淀方法筛选HOXA10靶基因;采用平端克隆方法构建HOXA10靶基因库;采用DNA序列分析结合生物信息学方法鉴定HOXA10靶基因。结果：共获得含有HOXA10结合片段的克隆197个,选取插入片段大于100bp的质粒67个进行DNA序列分析,其中含有HOXA10结合序列TTAT的基因16个。结论：初步筛选出16个HOXA10候选靶基因,为进一步研究HOXA10的基因调节机理提供了新的思路。     

Identification of a Lifespan Extending Mutation in the Schizosaccharomyces pombe Cyclin Gene clg1 + by Direct Selection of Long-Lived Mutants

Model organisms such as budding yeast, worms and flies have proven instrumental in the discovery of genetic determinants of aging, and the fission yeast Schizosaccharomyces pombe is a promising new system for these studies. We devised an approach to directly select for long-lived S. pombe mutants from a random DNA insertion library. Each insertion mutation bears a unique sequence tag called a bar code that allows one to determine the proportion of an individual mutant in a culture containing thousands of different mutants. Aging these mutants in culture allowed identification of a long-lived mutant bearing an insertion mutation in the cyclin gene clg1 ⁺. Clg1p, like Pas1p, physically associates with the cyclin-dependent kinase Pef1p. We identified a third Pef1p cyclin, Psl1p, and found that only loss of Clg1p or Pef1p extended lifespan. Genetic and co-immunoprecipitation results indicate that Pef1p controls lifespan through the downstream protein kinase Cek1p. While Pef1p is conserved as Pho85p in Saccharomyces cerevisiae, and as cdk5 in humans, genome-wide searches for lifespan regulators in S. cerevisiae have never identified Pho85p. Thus, the S. pombe system can be used to identify novel, evolutionarily conserved lifespan extending mutations, and our results suggest a potential role for mammalian cdk5 as a lifespan regulator.     

Identification and targeting of G-quadruplex structures in MALAT1 long non-coding RNA

RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. While a number of rG4 examples have been reported in coding messenger RNAs (mRNA), so far only limited works have studied rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs) that are of emerging interest and significance in biology. Herein, we report that MALAT1 lncRNA contains conserved rG4 motifs, forming thermostable rG4 structures with parallel topology. We also show that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in nuclear cell lysate, and we provide cellular data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by the rG4-specific small molecule, peptide, and L-aptamer, leading to the dissociation of MALAT1 rG4-NONO protein interaction. Altogether, this study uncovers new and important rG4s in MALAT1 lncRNAs, reveals their specific interactions with NONO protein, offers multiple strategies for targeting MALAT1 and its RNA–protein complex via its rG4 structure and illustrates the prevalence and significance of rG4s in ncRNAs.     

Identification and Characterization of a PutAMT1;1 Gene from Puccinellia tenuiflora

Nitrogen is one of the most important limiting factors for plant growth. However, as ammonium is readily converted into ammonia (NH₃) when soil pH rises above 8.0, this activity depletes the availability of ammonium (NH₄ ⁺) in alkaline soils, consequently preventing the growth of most plant species. The perennial wild grass Puccinellia tenuiflora is one of a few plants able to grow in soils with extremely high salt and alkaline pH (>9.0) levels. Here, we assessed how this species responds to ammonium under such conditions by isolating and analyzing the functions of a putative ammonium transporter (PutAMT1;1). PutAMT1;1 is the first member of the AMT1 (ammonium transporter) family that has been identified in P. tenuiflora. This gene (1) functionally complemented a yeast mutant deficient in ammonium uptake (2), is preferentially expressed in the anther of P. tenuiflora, and (3) is significantly upregulated by ammonium ions in both the shoot and roots. The PutAMT1;1 protein is localized in the plasma membrane and around the nuclear periphery in yeast cells and P. tenuiflora suspension cells. Immunoelectron microscopy analysis also indicated that PutAMT1;1 is localized in the endomembrane. The overexpression of PutAMT1;1 in A. thaliana enhanced plant growth, and increased plant susceptibility to toxic methylammonium (MeA). Here, we confirmed that PutAMT1;1 is an ammonium-inducible ammonium transporter in P. tenuiflora. On the basis of the results of PutAMT1;1 overexpression in A. thaliana, this gene might be useful for improving the root to shoot mobilization of MeA (or NH₄ ⁺).     

Biochemical Characterization of Two Thermostable Xylanolytic Enzymes Encoded by a Gene Cluster of Caldicellulosiruptor owensensis

The xylanolytic extremely thermophilic bacterium Caldicellulosiruptor owensensis provides a promising platform for xylan utilization. In the present study, two novel xylanolytic enzymes, GH10 endo-β-1,4-xylanase (Coxyn A) and GH39 β-1,4-xylosidase (Coxyl A) encoded in one gene cluster of C.owensensis were heterogeneously expressed and biochemically characterized. The optimum temperature of the two xylanlytic enzymes was 75°C, and the respective optimum pH for Coxyn A and Coxyl A was 7.0 and 5.0. The difference of Coxyn A and Coxyl A in solution was existing as monomer and homodimer respectively, it was also observed in predicted secondary structure. Under optimum condition, the catalytic efficiency (k _cat/K _m) of Coxyn A was 366 mg ml⁻¹ s⁻¹ on beechwood xylan, and the catalytic efficiency (k _cat/K _m) of Coxyl A was 2253 mM⁻¹ s⁻¹ on pNP-β-D-xylopyranoside. Coxyn A degraded xylan to oligosaccharides, which were converted to monomer by Coxyl A. The two intracellular enzymes might be responsible for xylooligosaccharides utilization in C.owensensis, also provide a potential way for xylan degradation in vitro.     

Identification and Manipulation of the Caprazamycin Gene Cluster Lead to New Simplified Liponucleoside Antibiotics and Give Insights into the Biosynthetic Pathway

Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2 and belong to the translocase I inhibitor family. Their complex structure is derived from 5′-(β-O-aminoribosyl)-glycyluridine and comprises a unique N-methyldiazepanone ring. The biosynthetic gene cluster has been identified, cloned, and sequenced, representing the first gene cluster of a translocase I inhibitor. Sequence analysis revealed the presence of 23 open reading frames putatively involved in export, resistance, regulation, and biosynthesis of the caprazamycins. Heterologous expression of the gene cluster in Streptomyces coelicolor M512 led to the production of non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions validated the boundaries of the cluster and inactivation of cpz21 resulted in the accumulation of novel simplified liponucleoside antibiotics that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as an acyltransferase in caprazamycin biosynthesis. In vivo and in silico analysis of the caprazamycin biosynthetic gene cluster allows a first proposal of the biosynthetic pathway and provides insights into the biosynthesis of related uridyl-antibiotics.Caprazamycins (CPZs)² (Fig. 1, 1) are liponucleoside antibiotics isolated from a fermentation broth of Streptomyces sp. MK730-62F2 (1, 2). They show excellent activity in vitro against Gram-positive bacteria, in particular against the genus Mycobacterium including Mycobacterium intracellulare, Mycobacterium avium, and Mycobacterium tuberculosis (3). In a pulmonary mouse model with M. tuberculosis H37Rv, administration of caprazamycin B exhibited a therapeutic effect but no significant toxicity (4). Structural elucidation (2) revealed a complex and unique composition of elements the CPZs share only with the closely related liposidomycins (LPMs, 2) (5). The core skeleton is the (+)-caprazol (5) composed of an N-alkylated 5′-(β-O-aminoribosyl)-glycyluridine, also known from FR-900493 (6) (6) and the muraymycins (7) (7), which is cyclized to form a rare diazepanone ring. Attached to the 3′″-OH are β-hydroxy fatty acids of different chain length resulting in CPZs A–G (1). They differ from the LPMs in the absence of a sulfate group at the 2″-position of the aminoribose and the presence of a permethylated l-rhamnose β-glycosidically linked to the 3-methylglutaryl (3-MG) moiety.Open in a separate windowFIGURE 1.Nucleoside antibiotics of the translocase I inhibitor family.The LPMs have been shown to inhibit biosynthesis of the bacterial cell wall by targeting the formation of lipid I (8). The CPZs are expected to act in the same way and are assigned to the growing number of translocase I inhibitors that include other nucleoside antibiotics, like the tunicamycins and mureidomycins (9). During peptidoglycan formation, translocase I catalyzes the transfer of UDP-MurNAc-pentapeptide to the undecaprenyl phosphate carrier to generate lipid I (10). This reaction is considered an unexploited and promising target for new anti-infective drugs (11).Recent investigations indicate that the 3″-OH group (12), the amino group of the aminoribosyl-glycyluridine, and an intact uracil moiety (13) are essential for the inhibition of the Escherichia coli translocase I MraY. The chemical synthesis of the (+)-caprazol (5) was recently accomplished (14), however, this compound only shows weak antibacterial activity. In contrast, the acylated compounds 3 and 4 exhibit strong growth inhibition of mycobacteria, suggesting a potential role of the fatty acid side chain in penetration of the bacterial cell (15, 16). Apparently, the acyl-caprazols (4) represent the most simplified antibiotically active liponucleosides and a good starting point for further optimization of this class of potential therapeutics.Although chemical synthesis and biological activity of CPZs and LPMs has been studied in some detail, their biosynthesis remains speculative and only few data exists about the formation of other translocase I inhibitors (17, 18). Nevertheless, we assume that the CPZ biosynthetic pathway is partially similar to that of LPMs, FR-90043 (6), and muraymycins (7) and presents a model for the comprehension and manipulation of liponucleoside formation. Considering the unique structural features of the CPZs we also expect some unusual biotransformations to be involved in the formation of, e.g. the (+)-caprazol.Here we report the identification and analysis of the CPZ gene cluster, the first cluster of a translocase I inhibitor. A set of gene disruption experiments provide insights into the biosynthetic origin of the CPZs and moreover, heterologous expression of the gene cluster allows the generation of novel bioactive derivatives by pathway engineering.     

Gene variation and genetic differentiation among populations of the solitary mud dauber wasp Trypoxylon (Trypargilum) albitarse Fabricius 1804 (Hymenoptera, Crabronidae)

Trypoxylon is a genus of solitary crabronid wasps whose populationgenetics is poorly known. The purpose of the present study was to investigate thegenetic variation and differentiation among five populations of Trypoxylonalbitarse, a species widely distributed throughout the Neotropics, withrecords from Panama to northern Argentina. Eight species-specific microsatellite lociwere used for genotyping 96 adult wasps (one female per nest) sampled at five sitesin Brazil. The analysis of allelic richness and private alleles indicated highgenetic diversity in the populations sampled. Pairwise comparisons using theF_st and D_est indices revealed significant differentiation for all, but one pair ofpopulations. F_st, D_est, AMOVA and assignment test values pointed to inter-population differentiation.Additionally, the analysis of population structure using Bayesian and PCA methodscharacterized two alternative genetic groups. The Mantel test indicated nocorrelation between genetic and geographic distances. Despite evidence ofconsiderable dispersal capacity for T. albitarse, the data indicatelow to moderate population structuring in this species.     

Criconematina (Nematoda: Tylenchida) from the Aleutian Islands

A new genus (Cerchnotocriconenta) and three new species (C. psephinum, Hemicycliophora amchitkaensis, and Paratylenchns amundseni) are described from Adak and Amchitka Islands in the Aleutian chain. The new genus differs from all other criconematid genera in having irregular, convex sculpturing consisting of small, oval plates on the anterior and posterior regions of each annule, with the mid-annular region minutely punctate or dentate. H. arnchitkaensis n. sp. resembles H. similis Thorne and H. zuckermani Brzeski, but has only one head annule, instead of two. P. antundseni n. sp., which has a stylet 17-19 μm long, is similar to P. tateae Wu &Townshend and P. labiosus Anderson &Kimpinski, but differs by the presence of males and the possession of conoid-truncate lip region, functional spermatheca, and long male tail (c = 8.5-9.5). Seriespinula seymouri Wu (Mehta &Raski), Nothocrieonema longulum (Gunhold) De Grisse &Loof, and Macroposthonia xenoplax (Raski) De Grisse &Loof are also reported from the islands.     

Asymptomatic infection in individuals from the municipality of Barcelos (Brazilian Amazon) is not associated with the anti-Plasmodium falciparum glycosylphosphatidylinositol antibody response

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-₁₉) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-₁₉may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.