Metallothioneins 1 and 2 Have Distinct but Overlapping Expression Patterns in Arabidopsis1

The spatial and temporal expression patterns of metallothionein (MT) isoforms MT1a and MT2a were investigated in vegetative and reproductive tissues of untreated and copper-treated Arabidopsis by in situ hybridization and by northern blotting. In control plants, MT1a mRNA was localized in leaf trichomes and in the vascular tissue in leaves, roots, flowers, and germinating embryos. In copper-treated plants, MT1a expression was also observed in the leaf mesophyll and in vascular tissue of developing siliques and seeds. In contrast, MT2a was expressed primarily in the trichomes of both untreated and copper-treated plants. In copper-treated plants, MT2a mRNA was also expressed in siliques. Northern-hybridization studies performed on developing seedlings and leaves showed temporal variations of MT1a gene expression but not of MT2a expression. The possible implications of these findings for the cellular roles of MTs in plants are discussed.

Applying semantic knowledge to the automatic processing of temporal expressions and events in natural language

This paper addresses the problem of the automatic recognition and classification of temporal expressions and events in human language. Efficacy in these tasks is crucial if the broader task of temporal information processing is to be successfully performed. We analyze whether the application of semantic knowledge to these tasks improves the performance of current approaches. We therefore present and evaluate a data-driven approach as part of a system: TIPSem. Our approach uses lexical semantics and semantic roles as additional information to extend classical approaches which are principally based on morphosyntax. The results obtained for English show that semantic knowledge aids in temporal expression and event recognition, achieving an error reduction of 59% and 21%, while in classification the contribution is limited. From the analysis of the results it may be concluded that the application of semantic knowledge leads to more general models and aids in the recognition of temporal entities that are ambiguous at shallower language analysis levels. We also discovered that lexical semantics and semantic roles have complementary advantages, and that it is useful to combine them. Finally, we carried out the same analysis for Spanish. The results obtained show comparable advantages. This supports the hypothesis that applying the proposed semantic knowledge may be useful for different languages.

Gene expression during early ascidian metamorphosis requires signaling by Hemps, an EGF-like protein

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.

Temporal and spatial transcriptional programs in murine kidney development

We have performed a systematic temporal and spatial expression profiling of the developing mouse kidney using Compugen long-oligonucleotide microarrays. The activity of 18,000 genes was monitored at 24-h intervals from 10.5-day-postcoitum (dpc) metanephric mesenchyme (MM) through to neonatal kidney, and a cohort of 3,600 dynamically expressed genes was identified. Early metanephric development was further surveyed by directly comparing RNA from 10.5 vs. 11.5 vs. 13.5dpc kidneys. These data showed high concordance with the previously published dynamic profile of rat kidney development (Stuart RO, Bush KT, and Nigam SK. Proc Natl Acad Sci USA 98: 5649-5654, 2001) and our own temporal data. Cluster analyses were used to identify gene ontological terms, functional annotations, and pathways associated with temporal expression profiles. Genetic network analysis was also used to identify biological networks that have maximal transcriptional activity during early metanephric development, highlighting the involvement of proliferation and differentiation. Differential gene expression was validated using whole mount and section in situ hybridization of staged embryonic kidneys. Two spatial profiling experiments were also undertaken. MM (10.5dpc) was compared with adjacent intermediate mesenchyme to further define metanephric commitment. To define the genes involved in branching and in the induction of nephrogenesis, expression profiling was performed on ureteric bud (GFP+) FACS sorted from HoxB7-GFP transgenic mice at 15.5dpc vs. the GFP- mesenchymal derivatives. Comparisons between temporal and spatial data enhanced the ability to predict function for genes and networks. This study provides the most comprehensive temporal and spatial survey of kidney development to date, and the compilation of these transcriptional surveys provides important insights into metanephric development that can now be functionally tested.

Investigation of molecular mechanisms regulating biomineralization of pearl oyster Pinctada maxima

Biomineralization is a process encompassing all mineral containing tissues produced within an organism. The most dynamic example of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this remarkable architecture. Subsequently, for the past decade considerable research have been undertaken to identify and characterize the protein components involved in biomineralization. Despite these efforts the general understanding of the process remains ambiguous. This study employs a novel molecular approach to further the elucidation of the shell biomineralization. A microarray platform has been custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from the mantle, an organ involved in shell formation. This microarray has been used as the primary tool for three separate investigations in an effort to associate transcriptional gene expression from P. maxima to the process of shell biomineralization. The first investigation analyzes the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and each analyzed for gene expression with PmaxArray 1.0. Over 2000 ESTs were differentially expressed among the tissue sections, identifying five major expression regions. Three of these regions have been proposed to have shell formation functions belonging to nacre, prismatic calcite and periostracum. The spatial gene expression map was confirmed by in situ hybridization, localizing a subset of ESTs from each expression region to the same mantle area. Comparative sequence analysis of ESTs expressed in the proposed shell formation regions with the BLAST tool, revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell formation genes. The second investigation correlates temporal EST expression during P. maxima larval ontogeny with transitions in shell mineralization during the same period. A timeline documenting the morphologicat microstructural and mineralogical shell characteristics of P. maxima throughout larval ontogeny has been established. Three different shell types were noted based on the physical characters and termed, prodissoconch I, prodissoconch 11 and dissoconch. PmaxArray 1.0 analyzed ESTs expression of animals throughout the larval development of P. maxima, noting up-regulation of 359 ESTs in association with the shell transitions from prodissoconch 1 to prodissoconch 11 to dissoconch. Comparative sequence analysis of these ESTs indicates a number of the transcripts are novel as well as showing significant sequence similarities between ESTs and known shell matrix associated genes and proteins. These ESTs are discussed in relation to the shell characters associated with their temporal expression. The third investigation uses PmaxArray 1.0 to analyze gene expression in the mantle tissue of P. maxima specimens exposed to sub-lethal concentrations of a shell-deforming toxin, tributyltin (TBT). The shell specific effects of TBT are used in this investigation to interpret differential expression of ESTs with respect to shell formation functions. A lethal and sublethal TBT concentration range was established for P. maxima, noting a concentration of 50 ng L- 1 TBT as sub-lethal over a 21 day period. Mantle tissue from P. maxima animals treated with 50 ng L- 1 TBT was assessed for differential EST expression with untreated control animals. A total of 102 ESTs were identified as differentially expressed in association with TBT exposure, comparative sequence identities included an up-regulation of immunity and detoxification related genes and down-regulation of several shell matrix genes. A number of transcripts encoding novel peptides were additionally identified. The potential actions of these genes are discussed with reference to TBT toxicity and shell biomineralization. This thesis has used a microarray platform to analyze gene expression in spatial, temporal and toxicity investigations, revealing the involvement of numerous gene transcripts in specific shell formation functions. Investigation of thousands of transcripts simultaneously has provided a holistic interpretation of the organic components regulating shell biomineralization.

Enhanced processing of SPOT multispectral satellite imagery for environmental monitoring and modelling

The Taita Hills in southeastern Kenya form the northernmost part of Africa’s Eastern Arc Mountains, which have been identified by Conservation International as one of the top ten biodiversity hotspots on Earth. As with many areas of the developing world, over recent decades the Taita Hills have experienced significant population growth leading to associated major changes in land use and land cover (LULC), as well as escalating land degradation, particularly soil erosion. Multi-temporal medium resolution multispectral optical satellite data, such as imagery from the SPOT HRV, HRVIR, and HRG sensors, provides a valuable source of information for environmental monitoring and modelling at a landscape level at local and regional scales. However, utilization of multi-temporal SPOT data in quantitative remote sensing studies requires the removal of atmospheric effects and the derivation of surface reflectance factor. Furthermore, for areas of rugged terrain, such as the Taita Hills, topographic correction is necessary to derive comparable reflectance throughout a SPOT scene. Reliable monitoring of LULC change over time and modelling of land degradation and human population distribution and abundance are of crucial importance to sustainable development, natural resource management, biodiversity conservation, and understanding and mitigating climate change and its impacts. The main purpose of this thesis was to develop and validate enhanced processing of SPOT satellite imagery for use in environmental monitoring and modelling at a landscape level, in regions of the developing world with limited ancillary data availability. The Taita Hills formed the application study site, whilst the Helsinki metropolitan region was used as a control site for validation and assessment of the applied atmospheric correction techniques, where multiangular reflectance field measurements were taken and where horizontal visibility meteorological data concurrent with image acquisition were available. The proposed historical empirical line method (HELM) for absolute atmospheric correction was found to be the only applied technique that could derive surface reflectance factor within an RMSE of < 0.02 ps in the SPOT visible and near-infrared bands; an accuracy level identified as a benchmark for successful atmospheric correction. A multi-scale segmentation/object relationship modelling (MSS/ORM) approach was applied to map LULC in the Taita Hills from the multi-temporal SPOT imagery. This object-based procedure was shown to derive significant improvements over a uni-scale maximum-likelihood technique. The derived LULC data was used in combination with low cost GIS geospatial layers describing elevation, rainfall and soil type, to model degradation in the Taita Hills in the form of potential soil loss, utilizing the simple universal soil loss equation (USLE). Furthermore, human population distribution and abundance were modelled with satisfactory results using only SPOT and GIS derived data and non-Gaussian predictive modelling techniques. The SPOT derived LULC data was found to be unnecessary as a predictor because the first and second order image texture measurements had greater power to explain variation in dwelling unit occurrence and abundance. The ability of the procedures to be implemented locally in the developing world using low-cost or freely available data and software was considered. The techniques discussed in this thesis are considered equally applicable to other medium- and high-resolution optical satellite imagery, as well the utilized SPOT data.

Transcription factor erect wing (EWG) is involved in indirect flight muscle patterning, development and maintenance in Drosophila

Muscle development is a multistep process which includes myoblast diversification, proliferation, migration, fusion, differentiation and growth. A hierarchical exhibition of myogenic factors is important for dexterous execution of progressive events in muscle formation. EWG (erect wing) is a transcription factor known to have a role in indirect flight muscle development (IFM) in Drosophila. We marked out the precise spatio-temporal expression profile of EWG in the myoblasts, and in the developing muscles. Mutant adult flies null for EWG in myoblasts show variable number of IFM, suggesting that EWG is required for patterning of the IFM. The remnant muscle found in the EWG null flies show proper assembly of the structural proteins, which implies that some myoblasts manage to fuse, develop and differentiate normally indicating that EWG is not required for differentiation program per se. However, when EWG expression is extended beyond its expression window in a wild type background, muscle thinning is observed implying EWG function in protein synthesis inhibition. Mis-expression studies in wing disc myoblasts hinted at its role in myoblast proliferation. We thus conclude that EWG is important for regulating fusion events which in turn decides the IFM pattern. Also IFM in EWG null mutants show clumps containing broken fibres and an altered mitochondrial morphology. The vertebrate homolog of EWG is nuclear respiratory factor1 (NRF1) which is known to have a function in mitochondrial biogenesis and protection against oxidative stress. Gene expression for inner mitochondrial membrane protein, Opa1-like was found to be absent in these mutants. Also, these flies were more sensitive to oxidative stress, indicating a compromised mitochondrial functioning. Our results therefore demonstrate that EWG functions in maintaining muscles’ structural integrity by ensuing proper mitochondrial activity.

Pattern formation during Caenorhabditis Elegans vulval development

Pattern formation during animal development involves at least three processes: establishment of the competence of precursor cells to respond to intercellular signals, formation of a pattern of different cell fates adopted by precursor cells, and execution of the cell fate by generating a pattern of distinct descendants from precursor cells. I have analyzed the fundamental mechanisms of pattern formation by studying the development of Caenorhabditis elegans vulva.

In C. elegans, six multipotential vulval precursor cells (VPCs) are competent to respond to an inductive signal LIN-3 (EGF) mediated by LET- 23 (RTK) and a lateral signal via LIN-12 (Notch) to form a fixed pattern of 3°-3°-2°-1°-2°-3°. Results from expressing LIN-3 as a function of time in animals lacking endogenous LIN-3 indicate that both VPCs and VPC daughters are competent to respond to LIN-3. Although the daughters of VPCs specified to be 2° or 3° can be redirected to adopt the 1°fate, the decision to adopt the 1° fate is irreversible. Coupling of VPC competence to cell cycle progression reveals that VPC competence may be periodic during each cell cycle and involve LIN-39 (HOM-C). These mechanisms are essential to ensure a bias towards the 1° fate, while preventing an excessive response.

After adopting the 1° fate, the VPC executes its fate by dividing three rounds to form a fixed pattern of four inner vulF and four outer vulE descendants. These two types of descendants can be distinguished by a molecular marker zmp-1::GFP. A short-range signal from the anchor cell (AC), along with signaling between the inner and outer 1° VPC descendants and intrinsic polarity of 1° VPC daughters, patterns the 1° lineage. The Ras and the Wnt signaling pathways may be involved in these mechanisms.

The temporal expression pattern of egl-17::GFP, another marker ofthe 1° fate, correlates with three different steps of 1° fate execution: the commitment to the 1° fate, as well as later steps before and after establishment of the uterine-vulval connection. Six transcription factors, including LIN-1(ETS), LIN-39 (HOM-C), LIN-11(LIM), LIN-29 (zinc finger), COG-1 (homeobox) and EGL-38 (PAX2/5/8), are involved in different steps during 1° fate execution.

Characterization of an early gene encoding for dUTPase in Rana grylio virus

dUTPase (DUT) is a ubiquitous and important enzyme responsible for regulating levels of dUTP. Here, an iridovirus DUT was identified and characterized from Rana grylio virus (RGV) which is a pathogen agent in pig frog. The DUT encodes a protein of 164aa with a predicted molecular mass of 17.4 kDa, and its transcriptional initiation site was determined by 5'RACE to start from the nucleotide A at 15 nt upstream of the initiation codon ATG. Sequence comparisons and multiple alignments suggested that RGV DUT was quite similar to other identified DUTs that function as homotrimers. Phylogenetic analysis implied that DUT horizontal transfers might have occurred between the vertebrate hosts and iridoviruses. Furthermore, its temporal expression pattern during RGV infection course was characterized by RT-PCR and Western blot analysis. It begins to transcribe and translate as early as 4 h postinfection (p.i.), and remains detectable at 48 h p.i. DUT-EGFP fusion protein was observed in the cytoplasm of pEGFP-N3-Dut transfected EPC cells. Immunofluorescence also confirmed DUT cytoplasm localization in RGV-infected cells. Using drug inhibition analysis by a de novo protein synthesis inhibitor (cycloheximide) and a viral DNA replication inhibitor (cytosine arabinofuranoside), RGV DUT was classified as an early (E) viral gene during the in vitro infection. Moreover, RGV DUT overexpression was shown that there was no effect on RGV replication by viral replication kinetics assay. (c) 2006 Published by Elsevier B.V.

青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

Two thymosin-repeated molecules with structural and functional diversity coexist in Chinese mitten crab Eriocheir sinensis

Recently, beta-thymosin-like proteins with multiple thymosin domains (defined as thymosin-repeated proteins) have been identified from invertebrate. In the present study, the cDNAs of two thymosin-repeated proteins (designated EsTRP1 and EsTRP2) were cloned from Chinese mitten crab by expressed sequence tags (EST) techniques. BLAST analysis presented three and two thymosin domains in EsTRP1 and EsTRP2, respectively, with the identities amongst the five domains varying from 47% to 100%. Both EsTRP1 and EsTRP2 shared high similarities with previously identified vertebrate beta-thynnosins and invertebrate thymosin-repeated proteins (TRPs) with the identities ranging from 43% to 78%, indicating that EsTRPs were new members of the beta-thymosin family. Real-time RT-PCR assay was adopted to determine the tissue distribution of EsTRPs and their temporal expression profile in hemocytes after pathogen stimulation and injury challenge. The expression of EsTRP1 transcript was predominantly detectable in the tissues of hemocytes, hepatopancreas and gonad with the highest expression in hemocytes, while the highest expression level of EsTRP2 was found in heart. EsTRP1 mRNA expression in hemocytes significantly increased at 3 and 48 h after Listonella anguillarum challenge, but there was no significant variation in EsTRP2 temporal expression profile. The injury challenge reduced the mRNA expression of EsTRPs, with the down-regulation of EsTRP2 expression occurred earlier than that of EsTRP1. The cDNA fragments encoding their mature peptides of EsTRP1 and EsTRP2 were recombined and expressed in Escherichia coli. The activities of recombinant proteins (rEsTRP1 and rEsTRP2) were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and lysoplate assay. rEsTRP2 could significantly accelerate the growth of human hepatocellular carcinoma cell line, but there was no significant effect of rEsTRP1 on the tumor cell proliferation. Both rEsTRP1 and rEsTRP2 did not possess the ability of killing Micrococcus luteus and L. anguillarum. The differences in the tissue distribution of mRNA transcripts, the response to pathogen stimulation and injury challenge, and the effect of recombinant proteins on human cell proliferation, indicated that there were functional diversity between the two structurally different molecules, EsTRP1 and EsTRP2. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of peroxiredoxin 6 from Chinese mitten crab Eriocheir sinensis

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). In this study, the full-length cDNA encoding peroxiredoxin 6 (designated EsPrx6) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5' untranslated region (UTR) of 69 bp, a 3' UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 in various tissues and its temporal expression in haemocytes of crabs challenged with Listonella anguillarum. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12 h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

A cyclophilin A inducible expressed in gonad of zhikong scallop Chlamys farreri

Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A (CsA), is a cis-trans peptidyl-prolyl isomerase (PPIase) which accelerates the cis-trans isomerization of prolyl-peptide bonds, interacts with a variety of proteins and therefore regulates their activities. One CypA (designated CfCypA) cDNA was cloned from Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CfCypA consisted of 1,248 nucleotides with a canonical polyadenylation signal sequence AATAAA, a poly (A) tail, and an open reading frame (ORF) of 495 nucleotides encoding a polypeptide of 164 amino acids. The deduced amino acid sequence shared high similarity with CypA from the other species, indicating that CfCypA should be a new member of the CypA family. Quantitative real-time (RT) PCR was employed to assess the mRNA expression of CfCypA in various tissues and its temporal expression in haemocytes and gonad of scallops challenged with Vibrio anguillarum. The mRNA transcripts of CfCypA could be detected in all the examined tissues with highest expression level in gonad. After bacterial challenge, the expression level of CfCypA was almost unchanged in haemocytes, but up-regulated in gonad and increased to the peak (22.59-fold; P < 0.05) at 4 h post-injection, and then dropped to the original level at 8 h post-injection. These results indicated that CfCypA was constitutive expressed in haemocytes, but could be induced in gonad, and perhaps played a critical role in response to the bacterial challenge in gonad.

A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like domains. Many members of this family play important roles as pattern recognition receptors in innate immune responses. The cDNA of bay scallop Argopecten irradians FREP (designated as AiFREP) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiFREP was of 990 bp. The open reading frame encoded a polypeptide of 251 amino acids, including a signal sequence and a 213 amino acids fibrinogen-like domain. The fibrinogen-like domain of AiFREP was highly similar to those of mammalian ficolins and other FREPs. The temporal expression of AiFREP mRNA in hemolymph was examined by fluorescent quantitative real-time PCR. The mRNA level of scallops challenged by Listonella anguillarum was significantly up-regulated, peaked to 9.39-fold at 9 h after stimulation, then dropped back to 4.37-fold at 12 h, while there was no significant change in the Micrococcus luteus challenged group in all periods of treatment. The function of AiFREP was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiFREP (rAiFREP) agglutinated chicken erythrocytes and human A, B, O-type erythrocytes. The agglutinating activities were calcium-dependent and could be inhibited by acetyl group-containing carbohydrates. rAiFREP also agglutinated Gram-negative bacteria E. coli JM109, L anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions. These results collectively suggested that AiFREP functions as a pattern recognition receptor in the immune response of bay scallop and contributed to nonself recognition in invertebrates, which would also provide clues for elucidating the evolution of the lectin pathway of the complement system. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of a C-type lectin from the bay scallop Argopecten irradians

C-type lectins are Ca2+ dependent carbohydrate-recognition proteins that play crucial roles in the invertebrate innate immunity, such as nonself recognition, activation of proPO system, antibacterial activity, promotion of phagocytosis and nodule formation. In this study, a novel C-type lectin of bay scallops Argopecten irradians (Ai Lec) was identified using expressed sequence tag (EST) and RACE techniques. The Ai Lec cDNA encoded a polypeptide of 171 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 150 amino acids. The deduced amino acid sequence of Ai Lec was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 131 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. The expression of Ai Lec transcript was dominantly detected in the hepatopancreas and slightly detected in the haemocytes of normal scallops. 6 h after Vibrio anguillarum-challenge and 8 h after Micrococcus luteus-challenge, the temporal expression of Ai Lec mRNA in hemocytes was increased by 4.4- and 3.6-folds, respectively. The results suggested that Ai Lec was a constitutive and inducible acute-phase protein and might be involved in immune response to Gram-negative and Gram-positive microbial infection in bay scallop A. irradians.