Homology-based Approach Research Articles

Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons

MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 ( Gossypium barbadense) and Acala 1517-99 ( Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.

MiRNA expression patterns of Triticum dicoccoides in response to shock drought stress

Drought is a major environmental stress factor that affects plant growth and development worldwide. Wild emmer wheat (Triticum turgidum ssp. dicoccoides), the ancestor of domesticated durum wheat (Triticum turgidum ssp. durum), has great potential for improving the understanding of the wheat drought response. MicroRNAs (miRNAs) are a recently discovered class of gene expression regulators that have also been linked to several plant stress responses; however, this relationship is just beginning to be understood. miRNA expression patterns of drought-resistant wild emmer wheat in response to drought stress were investigated using a plant miRNA microarray platform. Expression was detected to be 205 miRNAs in control and 438 miRNAs in drought-stressed leaf and root tissues. Of these miRNAs, the following 13 were differentially regulated in response to drought: miR1867, miR896, miR398, miR528, miR474, miR1450, miR396, miR1881, miR894, miR156, miR1432, miR166 and miR171. Regulation of miRNAs upon 4 and 8h drought stress applications observed by qRT-PCR. Target transcripts of differentially regulated miRNAs were computationally predicted. In addition to miRNA microarray study, five new conserved T. turgidum miRNAs were identified through a homology-based approach, and their secondary structures and putative targets were predicted. These findings both computationally and experimentally highlight the presence of miRNAs in T. dicoccoides and further extend the role of miRNAs under shock drought stress conditions.

Two Oxidosqualene Cyclases Responsible for Biosynthesis of Tomato Fruit Cuticular Triterpenoids

The first committed step in triterpenoid biosynthesis is the cyclization of epoxysqualene into various triterpene alcohol isomers, a reaction catalyzed by oxidosqualene cyclases (OSCs). The different OSCs have characteristic product specificities, which are mainly due to differences in the numbers of high-energy intermediates the enzymes can stabilize. The goal of this investigation was to clone and characterize OSCs from tomato (Solanum lycopersicum), a species known to accumulate δ-amyrin in its fruit cuticular wax, in order to gain insights into the enzymatic formation of this particular triterpenoid. We used a homology-based approach to isolate two tomato OSCs and tested their biochemical properties by heterologous expression in yeast as well as overexpression in tomato. One of the enzymes was found to be a product-specific β-amyrin synthase, while the other one was a multifunctional OSC synthesizing 48% δ-amyrin and six other products. The product spectra of both OSCs together account for both the range and the relative amounts of the triterpenoids found in the fruit cuticle. Both enzymes were expressed exclusively in the epidermis of the tomato fruit, indicating that their major function is to form the cuticular triterpenoids. The relative expression levels of both OSC genes, determined by quantitative reverse transcription-polymerase chain reaction, were consistent with product profiles in fruit and leaves of the tomato cultivar MicroTom. However, the transcript ratios were only partially consistent with the differences in amounts of product triterpenoids between the tomato cultivars MicroTom, M82, and Ailsa Craig; thus, transcriptional control of the two OSCs alone cannot explain the fruit triterpenoid profiles of the cultivars.

MEDELLER: homology-based coordinate generation for membrane proteins.

Motivation: Membrane proteins (MPs) are important drug targets but knowledge of their exact structure is limited to relatively few examples. Existing homology-based structure prediction methods are designed for globular, water-soluble proteins. However, we are now beginning to have enough MP structures to justify the development of a homology-based approach specifically for them.Results: We present a MP-specific homology-based coordinate generation method, MEDELLER, which is optimized to build highly reliable core models. The method outperforms the popular structure prediction programme Modeller on MPs. The comparison of the two methods was performed on 616 target–template pairs of MPs, which were classified into four test sets by their sequence identity. Across all targets, MEDELLER gave an average backbone root mean square deviation (RMSD) of 2.62 Å versus 3.16 Å for Modeller. On our ‘easy’ test set, MEDELLER achieves an average accuracy of 0.93 Å backbone RMSD versus 1.56 Å for Modeller.Availability and Implementation: http://medeller.info; Implemented in Python, Bash and Perl CGI for use on Linux systems; Supplementary data are available at http://www.stats.ox.ac.uk/proteins/resources.Contact: kelm@stats.ox.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

Phyloproteomic classification of unsequenced organisms by top‐down identification of bacterial proteins using capLC‐MS/MS on an Orbitrap

Currently, most MS-based proteomic studies of bacteria and archea match experimental data to known amino acid sequences from the target organism. Top-down studies use a protein's molecular weight along with data gathered from MS/MS experiments to identify proteins by database matching. For Erwinia herbicola and Enterobacter cloacae, studied here, the necessary protein sequences are not available in protein sequence repositories. We apply top-down protein fragmentation, but match the experimental data with homologous proteins from related organisms with sequenced genomes, demonstrating considerable shared protein sequence between closely related bacteria. Using this homology-based approach, we are not only able to identify representative proteins, but are also able to place the two target bacteria in their correct phylogeny. Furthermore, we show that the unexpected mass delta between the experimental precursor and matched protein sequence can often be localized and characterized using accurate-mass precursor and fragment ion measurements. Finally, we demonstrate that proteins identified by top-down workflows provide strong experimental evidence for correct, missing, and misannotated bacterial protein sequences, not only in the analyzed organism, but also for homologous proteins in closely related species.

Network-based function prediction and interactomics: The case for metabolic enzymes

As sequencing technologies increase in power, determining the functions of unknown proteins encoded by the DNA sequences so produced becomes a major challenge. Functional annotation is commonly done on the basis of amino-acid sequence similarity alone. Long after sequence similarity becomes undetectable by pair-wise comparison, profile-based identification of homologs can often succeed due to the conservation of position-specific patterns, important for a protein’s three dimensional folding and function. Nevertheless, prediction of protein function from homology-driven approaches is not without problems. Homologous proteins might evolve different functions and the power of homology detection has already started to reach its maximum. Computational methods for inferring protein function, which exploit the context of a protein in cellular networks, have come to be built on top of homology-based approaches. These network-based functional inference techniques provide both a first hand hint into a proteins’ functional role and offer complementary insights to traditional methods for understanding the function of uncharacterized proteins. Most recent network-based approaches aim to integrate diverse kinds of functional interactions to boost both coverage and confidence level. These techniques not only promise to solve the moonlighting aspect of proteins by annotating proteins with multiple functions, but also increase our understanding on the interplay between different functional classes in a cell. In this article we review the state of the art in network-based function prediction and describe some of the underlying difficulties and successes. Given the volume of high-throughput data that is being reported the time is ripe to employ these network-based approaches, which can be used to unravel the functions of the uncharacterized proteins accumulating in the genomic databases.

Combining Machine Learning and Homology-Based Approaches to Accurately Predict Subcellular Localization in Arabidopsis

A complete map of the Arabidopsis (Arabidopsis thaliana) proteome is clearly a major goal for the plant research community in terms of determining the function and regulation of each encoded protein. Developing genome-wide prediction tools such as for localizing gene products at the subcellular level will substantially advance Arabidopsis gene annotation. To this end, we performed a comprehensive study in Arabidopsis and created an integrative support vector machine-based localization predictor called AtSubP (for Arabidopsis subcellular localization predictor) that is based on the combinatorial presence of diverse protein features, such as its amino acid composition, sequence-order effects, terminal information, Position-Specific Scoring Matrix, and similarity search-based Position-Specific Iterated-Basic Local Alignment Search Tool information. When used to predict seven subcellular compartments through a 5-fold cross-validation test, our hybrid-based best classifier achieved an overall sensitivity of 91% with high-confidence precision and Matthews correlation coefficient values of 90.9% and 0.89, respectively. Benchmarking AtSubP on two independent data sets, one from Swiss-Prot and another containing green fluorescent protein- and mass spectrometry-determined proteins, showed a significant improvement in the prediction accuracy of species-specific AtSubP over some widely used "general" tools such as TargetP, LOCtree, PA-SUB, MultiLoc, WoLF PSORT, Plant-PLoc, and our newly created All-Plant method. Cross-comparison of AtSubP on six nontrained eukaryotic organisms (rice [Oryza sativa], soybean [Glycine max], human [Homo sapiens], yeast [Saccharomyces cerevisiae], fruit fly [Drosophila melanogaster], and worm [Caenorhabditis elegans]) revealed inferior predictions. AtSubP significantly outperformed all the prediction tools being currently used for Arabidopsis proteome annotation and, therefore, may serve as a better complement for the plant research community. A supplemental Web site that hosts all the training/testing data sets and whole proteome predictions is available at http://bioinfo3.noble.org/AtSubP/.

Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans.

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell–cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence.

Genome-wide profiling and analysis of Festuca arundinacea miRNAs and transcriptomes in response to foliar glyphosate application

Glyphosate is a broad spectrum herbicide which has been widely used for non-selective weed control in turfgrass management. Festuca arundinacea cv. Falcon was shown to be one of the tolerant turfgrass species in response to varying levels of glyphosate [5% (1.58 mM), 20% (6.32 mM)] recommended for weed control. However, there is a lack of knowledge on the mRNA expression patterns and miRNA, critical regulators of gene expression, in response to varying levels of glyphosate treatments. Here, we investigate the transcriptome and miRNA-guided post-transcriptional networks using plant miRNA microarray and Affymetrix GeneChip Wheat Genome Array platforms. Transcriptome analysis revealed 93 up-regulated and 78 down-regulated genes, whereas a smaller number showed inverse differential expressions. miRNA chip analysis indicated a number of (34 out of the 853) plant miRNAs were differentially regulated in response to glyphosate treatments. Target transcripts of differentially regulated miRNAs were predicted and nine of them were quantified by quantitative real-time PCR (qRT-PCR). Target transcripts of miRNAs validate the expression level change of miRNAs detected by miRNA microarray analysis. Down-regulation of miRNAs upon 5 and 20% glyphosate applications led to the up-regulation of their target observed by qRT-PCR or vice versa. Quantification of F. arundinacea miRNA, homologous of osa-miR1436, revealed the agreement between the Affymetrix and miRNA microarray analyses. In addition to miRNA microarray experiment, 25 conserved F. arundinacea miRNAs were identified through homology-based approach and their secondary structures were predicted. The results presented serve as analyses of genome-wide expression profiling of miRNAs and target mRNAs in response to foliar glyphosate treatment in grass species.

SoyTEdb: a comprehensive database of transposable elements in the soybean genome

BackgroundTransposable elements are the most abundant components of all characterized genomes of higher eukaryotes. It has been documented that these elements not only contribute to the shaping and reshaping of their host genomes, but also play significant roles in regulating gene expression, altering gene function, and creating new genes. Thus, complete identification of transposable elements in sequenced genomes and construction of comprehensive transposable element databases are essential for accurate annotation of genes and other genomic components, for investigation of potential functional interaction between transposable elements and genes, and for study of genome evolution. The recent availability of the soybean genome sequence has provided an unprecedented opportunity for discovery, and structural and functional characterization of transposable elements in this economically important legume crop.DescriptionUsing a combination of structure-based and homology-based approaches, a total of 32,552 retrotransposons (Class I) and 6,029 DNA transposons (Class II) with clear boundaries and insertion sites were structurally annotated and clearly categorized, and a soybean transposable element database, SoyTEdb, was established. These transposable elements have been anchored in and integrated with the soybean physical map and genetic map, and are browsable and visualizable at any scale along the 20 soybean chromosomes, along with predicted genes and other sequence annotations. BLAST search and other infrastracture tools were implemented to facilitate annotation of transposable elements or fragments from soybean and other related legume species. The majority (> 95%) of these elements (particularly a few hundred low-copy-number families) are first described in this study.ConclusionSoyTEdb provides resources and information related to transposable elements in the soybean genome, representing the most comprehensive and the largest manually curated transposable element database for any individual plant genome completely sequenced to date. Transposable elements previously identified in legumes, the third largest family of flowering plants, are relatively scarce. Thus this database will facilitate structural, evolutionary, functional, and epigenetic analyses of transposable elements in soybean and other legume species.

The CAZyome of Phytophthora spp.: A comprehensive analysis of the gene complement coding for carbohydrate-active enzymes in species of the genus Phytophthora

BackgroundEnzymes involved in carbohydrate metabolism include Carbohydrate esterases (CE), Glycoside hydrolases (GH), Glycosyl transferases (GT), and Polysaccharide lyases (PL), commonly referred to as carbohydrate-active enzymes (CAZymes). The CE, GH, and PL superfamilies are also known as cell wall degrading enzymes (CWDE) due to their role in the disintegration of the plant cell wall by bacterial and fungal pathogens. In Phytophthora infestans, penetration of the plant cells occurs through a specialized hyphal structure called appressorium; however, it is likely that members of the genus Phytophthora also use CWDE for invasive growth because hyphal forces are below the level of tensile strength exhibited by the plant cell wall. Because information regarding the frequency and distribution of CAZyme coding genes in Phytophthora is currently unknown, we have scanned the genomes of P. infestans, P. sojae, and P. ramorum for the presence of CAZyme-coding genes using a homology-based approach and compared the gene collinearity in the three genomes. In addition, we have tested the expression of several genes coding for CE in cultures grown in vitro.ResultsWe have found that P. infestans, P. sojae and P. ramorum contain a total of 435, 379, and 310 CAZy homologs; in each genome, most homologs belong to the GH superfamily. Most GH and PL homologs code for enzymes that hydrolyze substances present in the pectin layer forming the middle lamella of the plant cells. In addition, a significant number of CE homologs catalyzing the deacetylation of compounds characteristic of the plant cell cuticle were found. In general, a high degree of gene location conservation was observed, as indicated by the presence of sequential orthologous pairs in the three genomes. Such collinearity was frequently observed among members of the GH superfamily. On the other hand, the CE and PL superfamilies showed less collinearity for some of their putative members. Quantitative PCR experiments revealed that all genes are expressed in P. infestans when this pathogen grown in vitro. However, the levels of expression vary considerably and are lower than the expression levels observed for the constitutive control.ConclusionsIn conclusion, we have identified a highly complex set of CAZy homologs in the genomes of P. infestans, P. sojae, and P. ramorum, a significant number of which could play roles critical for pathogenicity, by participating in the degradation of the plant cell wall.

Glucocorticoid Regulation of Mouse and Human Dual Specificity Phosphatase 1 (DUSP1) Genes: UNUSUAL CIS-ACTING ELEMENTS AND UNEXPECTED EVOLUTIONARY DIVERGENCE

Anti-inflammatory effects of glucocorticoids (GCs) are partly mediated by up-regulation of DUSP1 (dual specificity phosphatase 1), which dephosphorylates and inactivates mitogen-activated protein kinases. We identified putative GC-responsive regions containing GC receptor (GR) binding site consensus sequences that are well conserved between human and mouse DUSP1 loci in position, orientation, and sequence (at least 11 of 15 positions identical) and lie within regions of extended sequence conservation (minimum 65% identity over at least 100 bp). These were located approximately 29, 28, 24, 4.6, and 1.3 kb upstream of the DUSP1 transcription start site. The homology-based approach successfully identified four cis-acting regions that mediated transcriptional responses to dexamethasone. However, there was surprising interspecies divergence in site usage. This could not be explained by variations of the GR binding sites themselves. Instead, variations in flanking sequences appear to have driven the evolutionary divergence in mechanisms of regulation of mouse and human DUSP1 genes. There was a good correlation between the ability of cis-acting elements to respond to GC in transiently transfected reporter constructs and their ability to recruit GR in the context of intact chromatin. We propose that divergence of gene regulation has involved the loss or gain of binding sites for accessory transcription factors that assist in GR recruitment. Finally, a novel GC-responsive region of the human DUSP1 gene contains a highly unusual element, in which three closely spaced GR half-sites are required for potent transcriptional activation by GC.

A Bifunctional Geranyl and Geranylgeranyl Diphosphate Synthase Is Involved in Terpene Oleoresin Formation inPicea abies

The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.

Purification, cDNA cloning and functional expression of NADPH‐cytochrome P450 reductase from Centaurium erythraea cell cultures

Solubilised NADPH-cytochrome P450 reductase (CPR) was purified from the microsomal fraction of centaury (Centaurium erythraea) cell cultures by Q-anion exchange chromatography and affinity chromatography on adenosine 2',5'-diphosphate agarose. SDS-PAGE demonstrated the presence of three CPR isoforms with molecular masses of 77, 79 and 81 kDa. The 79- and 81-kDa isoforms were identified as glycoproteins when blotted following SDS-PAGE and subjected to a sugar detection procedure. A homology-based approach led to the isolation of a CPR cDNA encoding the 77-kDa isoform. The enzyme was a class I CPR, possessing a short N-terminus upstream of the membrane anchor. The amino acid sequence contained a putative N-glycosylation site, indicating that the two major isoforms of 77 and 79 kDa are related through attachment of an oligosaccharide chain. This glycosylation process was also found upon heterologous expression in yeast. When co-expressed in yeast together with centaury coniferyl alcohol 5-hydroxylase, CPR efficiently supported the activity of the P450 enzyme. The genome of C. erythraea was found to contain a second CPR gene. RT-PCR experiments using gene-specific primers revealed differential regulation of the two CPR genes. While CPR 2 mRNA was strongly induced by the addition of methyl jasmonate to the cell cultures, the CPR 1 expression level did not change after this elicitation.

Identification of the Active Site of DS-epimerase 1 and Requirement of N-Glycosylation for Enzyme Function

Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His-205, Tyr-261, and His-450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions, we propose a model where His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction, generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.

Insights into a putative hinge region in elastin using molecular dynamics simulations

The resiliency and elasticity of vertebrate tissues are traced to elastin, a crosslinked protein with extensive hydrophobic regions. There is little discussion in the literature on the structure and dynamics of the alanine-rich crosslinking regions of elastin that comprise a significant part of the native protein. In particular, the region encoded by exons 21 and 23, a contiguous splice form found in all types of human elastin, is believed to be strategically positioned for proper function of the protein, namely, in the reversible elongation and contraction of tissue. Hence, molecular dynamics (MD) calculations on the EX21/23 domain are reported here. This crosslinking domain has been assumed to adopt an architecture in which the putative hinge region links two α-helices. In this paper, we use a homology-based approach to obtain starting structures in the hinge region. The subsequent MD brings new insights into the possibility of fluctuations between “open” and “closed” states, as well as distinguishing structural features of the latter. The significance of these findings towards an enhanced understanding of structure–function relationships in elastin and the elastic fiber is discussed.

The GC and window-averaged DNA curvature profile of secondary metabolite gene cluster in Aspergillus fumigatus genome

An immense variety of complex secondary metabolites is produced by filamentous fungi including Aspergillus fumigatus, a main inducer of invasive aspergillosis. The identification of fungal secondary metabolite gene cluster is essential for the characterization of fungal secondary metabolism in terms of genetics and biochemistry through recombinant technologies such as gene disruption and cloning. Most of the prediction methods for secondary metabolite gene cluster severely depend on homology searches. However, homology-based approach has intrinsic limitation to unknown or novel gene cluster. We analyzed the GC and window-averaged DNA curvature profile of 26 secondary metabolite gene clusters in the A. fumigatus genome to find out potential conserved features of secondary metabolite gene cluster. Fifteen secondary metabolite gene clusters showed a conserved pattern in window-averaged DNA curvature profile, that is, the DNA regions including secondary metabolic signature genes such as polyketide synthase, nonribosomal peptide synthase, and/or dimethylallyl tryptophan synthase consisted of window-averaged DNA curvature values lower than 0.18 and these DNA regions were at least 20 kb. Forty percent of secondary metabolite gene clusters with this conserved pattern were related to severe regulation by a transcription factor, LaeA. Our result could be used for identification of other fungal secondary metabolite gene clusters, especially for secondary metabolite gene cluster that is severely regulated by LaeA or other proteins with similar function to LaeA.

Conserved upstream open reading frames in higher plants

BackgroundUpstream open reading frames (uORFs) can down-regulate the translation of the main open reading frame (mORF) through two broad mechanisms: ribosomal stalling and reducing reinitiation efficiency. In distantly related plants, such as rice and Arabidopsis, it has been found that conserved uORFs are rare in these transcriptomes with approximately 100 loci. It is unclear how prevalent conserved uORFs are in closely related plants.ResultsWe used a homology-based approach to identify conserved uORFs in five cereals (monocots) that could potentially regulate translation. Our approach used a modified reciprocal best hit method to identify putative orthologous sequences that were then analysed by a comparative R-nomics program called uORFSCAN to find conserved uORFs.ConclusionThis research identified new genes that may be controlled at the level of translation by conserved uORFs. We report that conserved uORFs are rare (<150 loci contain them) in cereal transcriptomes, are generally short (less than 100 nt), highly conserved (50% median amino acid sequence similarity), position independent in their 5'-UTRs, and their start codon context and the usage of rare codons for translation does not appear to be important.

Map- vs. homology-based cloning for the recessive gene ol-2 conferring resistance to tomato powdery mildew

The recessive gene ol-2 confers papilla-associated and race-non-specific resistance to tomato powdery mildew caused by Oidium neolycopersici. In order to facilitate marker assisted selection (MAS) in practical breeding programmes, we identified two simple sequence repeat (SSR) markers and one cleaved amplified polymorphic sequence (CAPS) marker which are linked to the resistance locus and co-dominantly inherited. Aiming to provide a base for ol-2 positional cloning, we used a large segregating F2 population to merge these markers with all the ol-2 linked amplified fragment length polymorphism (AFLP®) markers previously identified in an integrated genetic map. By screening a tomato bacterial artificial chromosome (BAC) library, we detected two BAC clones containing two expressed sequence tags (ESTs) homologous to the gene mlo, responsible for powdery mildew resistance in barley, as well as an ol-2-linked marker. Chromosomal mapping by Fluorescence in situ Hybridization (FISH) revealed major signals of the two BAC DNAs in the pericentromeric heterochromatin of the short arm of chromosome 4, in the same region where the ol-2 gene was previously mapped. The genetic and cytogenetic co-localisation between ol-2 and tomato mlo-homologue(s), in addition to the similarity of ol-2 and mlo resistances for both genetic and phytopathological characteristics, suggests that ol-2 is likely a mlo-homologue. Thus, a homology-based cloning approach could be more suitable than positional cloning for ol-2 isolation.

Cloning and characterization of isoprenyl diphosphate synthases with farnesyl diphosphate and geranylgeranyl diphosphate synthase activity from Norway spruce ( Picea abies) and their relation to induced oleoresin formation

The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene ( PaIDS 4) encodes a farnesyl diphosphate synthase and the other two ( PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.