Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Microsatellite markers uncover cryptic species of Odontotermes (Termitoidae: Termitidae) from Peninsular Malaysia

Termites from the genus Odontotermes are known to contain numerous species complexes that are difficult to tell apart morphologically or with mitochondrial DNA sequences. We developed markers for one such cryptic species complex, that is, Odontotermes srinakarinensis sp. nov. from Maxwell Hill Forest Reserve (Perak, Malaysia), and characterised them using a sample of 41 termite workers from three voucher samples from the same area. We then genotyped 150 termite individuals from 23 voucher samples/colonies of this species complex from several sites in Peninsular Malaysia. We analysed their population by constructing dendograms from the proportion of shared-alleles between individuals and genetic distances between colonies; additionally, we examined the Bayesian clustering pattern of their genotype data. All methods of analysis indicated that there were two distinct clusters within our data set. After the morphologies of specimens from each cluster were reexamined, we were able to separate the two species morphologically and found that a single diagnostic character found on the mandibles of its soldiers could be used to separate the two species quite accurately. The additional species in the clade was identified as Odontotermes denticulatus after it was matched to type specimens at the NHM London and Cambridge Museum of Zoology.     

Regulation of cell proliferation and migration in gallbladder cancer by zinc finger X-chromosomal protein

Gallbladder carcinoma (GBC) is one of the mostly aggressive and fatal malignancies. However, little is known about the oncogenic genes that contributed to the development of GBC. Zinc finger X-chromosomal protein (ZFX) was a novel member of the Krueppel C2H2-type zinc-finger protein family and its down-regulation led to impaired cell growth in human laryngeal squamous cell carcinoma. Here, we aim to investigate the function of ZFX in GBC cell proliferation and migration. Loss of function analysis was performed on GBC cell line (GBC-SD) using lentivirus-mediated siRNA against ZFX. The proliferation, in vitro tumorigenesis (colony-formation) ability as well as cell migration was significantly suppressed after GBC-SD cells which were infected with ZFX-siRNA-expressing lentivirus (Lv-shZFX). Our finding suggested that ZFX promoted the growth and migration of GBC cells and could present a potential molecular target for gene therapy of GBC.     

DNA-damage response in chromatin of ribosomal genes and the surrounding genome

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1β, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

‘金州报春’兜兰的非共生萌发与快速繁殖

收集授粉后不同时期报春兜兰栽培品种‘金州报春’的种子,观察种子形态特征和萌发过程,建立了高效快繁体系。试验结果表明种子萌发的最佳培养基RE＋O-3nag·L～NAA＋0．1mg·L～6-BA＋100mL·L-1椰子乳（体积比）;授粉162d后的种子萌发率较高并且无白化现象,授粉203d的种子播种后萌发率可达94-33％;分化后的原球茎在壮苗培养基上培养4个月后可得到有5～6片叶、高3～5cm的健壮试管苗。     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

A diterpenoid derivate compound targets selenocysteine of thioredoxin reductases and induces Bax/Bak-independent apoptosis

We have previously shown that the natural diterpenoid derivative S3 induced Bim upregulation and apoptosis in a Bax/Bak-independent manner. However, the exact molecular target(s) of S3 and the mechanism controlling Bim upregulation are still not clear. Here, we identify that S3 targets the selenoproteins TrxR1 and TrxR2 at the selenocysteine residue of the reactive center of the enzymes and inhibits their antioxidant activities. Consequently, cellular ROS is elevated, leading to the activation of FOXO3a, which contributes to Bim upregulation in Bax/Bak-deficient cells. Moreover, S3 retards tumor growth in subcutaneous xenograft tumors by inhibiting TrxR activity in vivo. Our studies delineate the signaling pathway controlling Bim upregulation, which results in Bax/Bak-independent apoptosis and provide evidence that the compounds can act as anticancer agents based on mammalian TrxRs inhibition.     

Characterisation of a thermo-alkali-stable lipase from oil-contaminated soil using a metagenomic approach

Lipases are widely used for a variety of biotechnological applications. Screening these industrial enzymes directly from environmental microorganisms is a more efficient and practical approach than conventional cultivation-dependent methods. Combined with activity-based functional screening, six clones with lipase activity were detected and a gene (termed lipZ01) isolated from a target clone with the highest lipase activity was cloned from an oil-contaminated soil-derived metagenomic library and then sequenced. Gene lipZ01 was expressed in Pichia pastoris GS115 and the molecular weight of the recombinant lipase LipZ01 was estimated by electrophoresis analysis to be approximately 50 kDa. The maximum activity of the purified lipase was 42 U/mL, and the optimum reaction temperature and pH value were 45 °C and 8.0, respectively. The enzyme was highly stable in the temperature range 35–60 °C and under alkaline conditions (pH 7–10). The presence of Ca²⁺ and Mn²⁺ ions could significantly enhance the activity of the lipase. The purified lipase preferentially hydrolysed triacylglycerols with acyl chain lengths ≥8 carbon atoms, and the conversion degree of biodiesel production was nearly 92% in a transesterification reaction using olive oil and methanol. Some attractive properties suggested that the recombinant lipase may be valuable in industrial applications.