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Sequence Analysis (sequence + analysis)
Kinds of Sequence Analysis Selected AbstractsTHE SYSTEMATICS OF A SMALL SPINELESS DESMODESMUS SPECIES, D. COSTATO-GRANULATUS (SPHAEROPLEALES, CHLOROPHYCEAE), BASED ON ITS2 rDNA SEQUENCE ANALYSES AND CELL WALL MORPHOLOGY,JOURNAL OF PHYCOLOGY, Issue 2 2007Pieter Vanormelingen Desmodesmus species taxonomy is one of the most long-standing issues in green microalgal systematics due to problems associated with phenotypic plasticity. Whereas more recent species descriptions and identifications are mainly based on cell wall structures and the use of cultures, comparisons with molecular phylogenies are largely lacking. In this study, the phylogenetic relationships between 22 clones identified as Desmodesmus costato-granulatus (Skuja) E. H. Hegew. were assessed using ITS2 rDNA sequence data in combination with cell wall morphology. The unrooted ITS2 phylogeny showed that the clones cluster into five groups, which also differ in their cell wall structures. Therefore, the taxon is split into five species: D. costato-granulatus, D. elegans, D. fennicus, D. regularis, and D. ultrasquamatus. Compared with other Desmodesmus species, intraspecific sequence variation is extensive and may contain additional (pseudo)cryptic diversity. Compensatory base changes were near-absent within the species and varied from one to 11 between species. Relationships among the species were unresolved. Despite this, they clustered together with the two other Desmodesmus species having a combination of small and large warts in a well-supported lineage. Remarkably, ITS2 sequence variation in this lineage is as high as between all other included Desmodesmus species, even though the morphology of its members is rather uniform. [source] RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS,JOURNAL OF PHYCOLOGY, Issue 6 2006Holly A. Bowers Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species. [source] SEQUENCE ANALYSIS OF A NOVEL INSECT PHOSPHOGLYCERATE MUTASE GENE FROM THE CHINESE HONEYBEE, APIS CERANA,INSECT SCIENCE, Issue 4 2003Jiang-hong Li Abstract A clone inserted with 1 104 bp fragment containing a 765bp Open Reading Frame(ORF), encoding a putative 2,3-bisphosphoglycerate(2,3BPG) dependent Phosphoglycerate mutase(dPGAM) that catalyzes the transfer of a phosphate group from the C3 carbon atom to the C2 carbon atom of phosphoglycerate, was screened by mass sequencing from the cDNA library of the venom glands of Apis cerana. The deduced amino acid sequence shared high similarities (39% - 88%)with the dPGAM of 7 other organisms, but the similarities with the iPGAM of 4 other organisms were low (10% - 12%). Moreover, the alignment of Ac-PGAM with the dPGAMs from 7 other organisms showed that all the active site amino acid residues were conserved. This result shows that Ac-PGAM is a typical dPGAM. Thus, this is the second PGAM gene reported in Insecta. Furthermore, phylogenetic analysis showed that the evolutionary tree of PGAMs reflects the systematic relationship of species. [source] SEQUENCE ANALYSIS AND TRANSCRIPTIONAL REGULATION OF IRON ACQUISITION GENES IN TWO MARINE DIATOMS,JOURNAL OF PHYCOLOGY, Issue 4 2007Adam B. Kustka The centric diatom Thalassiosira pseudonana Hasle et Heimdal and the pennate diatom Phaeodactylum tricornutum Bohlin possess genes with translated sequences homologous to high-affinity ferric reductases present in model organisms. Thalassiosira pseudonana also possesses putative genes for membrane-bound ferroxidase (TpFET3) and two highly similar iron (Fe) permeases (TpFTR1 and TpFTR2), as well as a divalent metal (M2+) transporter belonging to the NRAMP superfamily (TpNRAMP). In baker's yeast, the ferroxidase,permease complex transports Fe(II) produced by reductases. We investigated transcript abundances of these genes as a function of Fe quota (QFe). Ferric reductase transcripts are abundant in both species (15%,60% of actin) under low QFe and are down-regulated by 5- to 35-fold at high QFe, suggesting Fe(III) reduction is a common, inducible strategy for Fe acquisition in marine diatoms. Permease transcript abundance was regulated by Fe status in T. pseudonana, but we did not detect significant differences in expression of the copper (Cu)-containing ferroxidase. TpNRAMP showed the most dramatic regulation by QFe, suggesting a role in cellular Fe transport in either cell-surface uptake or vacuolar mobilization. We could not identify ferroxidase or permease homologues in the P. tricornutum genome. The up-regulation of genes in T. pseudonana that appear to be missing altogether from P. tricornutum as well as the finding that P. tricornutum seems to have an efficient system to acquire Fe,, suggest that diverse (and uncharacterized) Fe-uptake systems may be at play within diatom assemblages. Different uptake systems among diatoms may provide a mechanistic basis for niche differentiation with respect to Fe availability in the ocean. [source] 16S rDNA Sequence Analysis of Bacterial Isolates from Die-back Affected Sissoo Trees (Dalbergia sissoo Roxb.) in BangladeshJOURNAL OF PHYTOPATHOLOGY, Issue 9 2005H. Tantau Abstract A new form of disease called ,die-back' has been established in Dalbergia sissoo trees. This disease has reached epidemic proportions in Bangladesh as well as in other countries of South Asia and is characterized by browning of the leaves, signs of wilting, and trunk lesions with gum flow. The trees die within a few months. In order to investigate the causes of this die-back disease, samples were taken for a first trial in the Rajshahi division at two sites around Sherpur. For the isolation of bacteria, surface-sterilized plant material (leaves, twigs and trunk bark) from diseased trees was transferred to LB medium and incubated. After isolation of single colonies, various bacteria species could be identified by polymerase chain reaction analysis with two primers specific for highly conserved sequence regions in the bacterial 16S rDNA and by sequencing. First indications for the presence of bacteria with phytopathogenic potential were found. [source] Comparative Sequence Analysis of Coat Protein Gene of Iranian Citrus tristeza virus IsolatesJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2005A. Barzegar Abstract Twenty-two isolates of Citrus tristeza virus (CTV) collected from two different geographical regions of Iran were characterized based on coat protein (CP) gene sequences. Thirteen virus isolates were collected from northern parts of Iran with high distribution of CTV infection and nine isolates were obtained from southern regions where the presence and aphid transmission of CTV was previously reported. All isolates were recovered from field trees showing varied CTV symptoms such as decline in most citrus varieties on sour orange rootstock, inverse pitting on some sour orange rootstocks below bud union, mild-to-moderate stem pitting on the trunk of some sweet orange. Isolates F, G, MB1, MB7, MB2, MB8, MB9, MB11 and MB17 were recovered from healthy looking Miyagawa Satsuma on trifoliate rootstock originally infected with CTV imported from Japan in late-1960s. The presence of virus in citrus samples was confirmed using polyclonal as well as monoclonal antibody. The CTV CP gene of all isolates was amplified by reverse transcriptase polymerase chain reaction (RT,PCR) using CP gene-specific primers yielding 672 bp amplicon. The restriction fragment length polymorphism (RFLP) profile, nucleotide and deduced amino acid sequences were analysed and compared with each other and also with some other exotic CP gene sequences of CTV isolates available in GenBank. Analysis of our data revealed that Iranian isolates have high similarity to California SY568 severe stem pitting and Japanese NUagA seedling yellows strains (up to 97%). The dendrogram generated from the deduced amino acid sequence could separate MB1, MB2, MB8, MB9, MB11 and MB17 isolates from others. However, no major dissociation between the isolates from northern and southern region could be obtained. [source] The Blasticidin S Biosynthesis Gene Cluster from Streptomyces griseochromogenes: Sequence Analysis, Organization, and Initial CharacterizationCHEMBIOCHEM, Issue 9 2003Martha C. Cone Abstract Blasticidin S is a potent antifungal and cytotoxic peptidyl nucleoside antibiotic from Streptomyces griseochromogenes. The mixed biosynthesis of the compound is evident from the three distinct structural components: a cytosine base, an amino deoxyglucuronic acid, and N -methyl , -arginine. The blasticidin S biosynthesis gene cluster was cloned from S. griseochromogenes and the pathway heterologously expressed in S. lividans from a cosmid harboring a 36.7-kb fragment of S. griseochromogenes DNA. The complete DNA sequence of this insert has now been determined and evidence suggests a contiguous 20-kb section defines the blasticidin S biosynthesis cluster. The predicted functions of several open reading frames are consistent with the expected biochemistry and include an arginine 2,3-aminomutase, a cytosylglucuronic acid synthase, and a guanidino N -methyltransferase. Insight into other steps in the assembly of blasticidin S was evident from sequence homology with proteins of known function and heterologous expression of fragments of the cluster. Additionally, the gene that directs the production of free cytosine, blsM, was subcloned and expressed in Escherichia coli. Characterization of BlsM revealed that cytidine monophosphate serves as the precursor to cytosine. [source] Differentiation trapping screen in live culture for genes expressed in cardiovascular lineagesDEVELOPMENTAL DYNAMICS, Issue 2 2004Weisheng V. Chen Abstract We have developed a gene trap vector that transduces an EGFP-neo fusion gene (Eno) to monitor the expression of trapped genes in living cells and embryos. Upon in vitro differentiation, most gene-trapped embryonic stem (ES) cell clones exhibited detectable green fluorescence in various specialized cell types, which can be followed in the live culture in real time. Populations of ES cell-derived cardiomyocytes, smooth muscle cells, vascular endothelial cells, and hematopoietic cells were readily recognized by their distinctive morphologies coupled with unique activities, allowing efficient screening for clones with trapped genes expressed in cardiovascular lineages. Applying G418 selection in parallel differentiation cultures further increased detection sensitivity and screening throughput by enriching reporter-expressing cells with intensified green fluorescent protein signals. Sequence analyses and chimera studies demonstrated that the expression of trapped genes in vivo closely correlated with the observed lineage specificity in vitro. This provides a strategy to identify and mutate genes expressed in lineages of interest for further functional studies. Developmental Dynamics 229:319,327, 2004. © 2004 Wiley-Liss, Inc. [source] Culture-independent evidence for the persistent presence and genetic diversity of microcystin-producing Anabaena (Cyanobacteria) in the Gulf of FinlandENVIRONMENTAL MICROBIOLOGY, Issue 4 2009David P. Fewer Summary The late summer mass occurrences of cyanobacteria in the Baltic Sea are among the largest in the world. These blooms are rarely monotypic and are often composed of a diverse assemblage of cyanobacteria. The toxicity of the blooms is attributed to Nodularia spumigena through the production of the hepatotoxic nodularin. However, the microcystin hepatotoxins have also been reported from the Baltic Sea on a number of occasions. Recent evidence links microcystin production in the Gulf of Finland directly to the genus Anabaena. Here we developed a denaturing gradient gel electrophoresis (DGGE) method based on the mcyE microcystin synthetase gene and ndaF nodularin synthetase gene that allows the culture-independent discrimination of microcystin- and nodularin-producing cyanobacteria directly from environmental samples. We PCR-amplified microcystin and nodularin synthetase genes from environmental samples taken from the Gulf of Finland and separated them on a denaturing gradient gel using optimized conditions. Sequence analyses demonstrate that uncultured microcystin-producing Anabaena strains are genetically more diverse than previously demonstrated from cultured strains. Furthermore, our data show that microcystin-producing Anabaena are widespread in the open Gulf of Finland. Non-parametric statistical analysis suggested that salinity plays an important role in defining the distribution of microcystin-producing Anabaena. Our results indicate that microcystin-producing blooms are a persistent phenomenon in the Gulf of Finland. [source] PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES,JOURNAL OF PHYCOLOGY, Issue 4 2003Ingo Busse Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well-known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group. [source] Affinities of the freshwater red alga Audouinella macrospora (Florideophyceae, Rhodophyta) and related forms based on ssu rrna gene sequence analysis and pit plug ultrastructureJOURNAL OF PHYCOLOGY, Issue 2 2000Curt M. Pueschel Small subunit rDNA sequencing and transmission electron microscopy were performed to clarify the ordinal affinities of Audouinella macrospora (Wood) Sheath et Burkholder isolates 3394, 3395, and 3603, as well as Chantransia sp. isolate 3585. Culture 3603 is known to produce thalli of Batrachospermum -like morphology under certain culture conditions. Sequence analyses unequivocally placed the three Audouinella macrospora isolates in a clade with Batrachospermum macrosporum Montagne of the Batrachospermales, and Chantransia sp. was found to have affinities with B. louisianae Skuja and B. virgato-decaisneanum Sirodot. The pit plugs of the Audouinella macrospora cultures 3394 and 3395 were nearly identical in size and structure, having thickened plug caps and no cap membranes. Both of these features agree with those of the Batrachospermaceae, with the latter feature showing batrachospermacean rather than acrochaetioid affinities. Pit plugs in the chantransia phase of 3603 were similar, but the plug caps were less well developed. The Batrachospermum phase generated from 3603 had pit plugs that were variable in diameter, according to location in the thallus, thus reflecting the more variable cell size in this phase. Dome-like outer caps, considered typical of Batrachospermum, were present between cells of the determinate lateral filaments. The pit plugs of Chantransia sp. had prominent, dome-like outer caps, but the plug cores were strikingly and consistently smaller in diameter than those of the A. macrospora chantransia cultures, suggesting that plug diameter may be of systematic value in some contexts. [source] Characterization of Phytoplasmas Associated With Almond Diseases in IranJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2009L. Zirak Abstract In recent years, almond witches'-broom disease has been prevalent in almond growing areas in the centre and south of Iran. Furthermore, almond trees showing different symptoms of phytoplasma diseases such as little leaf, leaf rolling, dieback of branches, rosette and yellowing were observed in the central regions of Iran. DNA isolated from symptomatic almond trees was used to amplify 16S rDNA and 16S-23S rDNA intergenic spacer (IS) fragments by nested polymerase chain reaction (PCR) using phytoplasma universal primer pairs (P1/P7, R16F2/R2, PA2F/R and NPA2F/R). Phytoplasmas were detected in symptomatic almonds in two major almond-growing regions, Isfahan and ChaharMahal-O-Bakhtiari. Restriction fragment length polymorphism analyses of nested PCR products using endonuclease enzymes HpaII and TaqI revealed that phytoplasmas associated with infected almonds are genetically different. Sequence analyses of amplified fragments of 16S rDNA and IS region indicated that the almond phytoplasmas in Iran are closely related to ,Candidatus (Ca.) Phytoplasma aurantifolia', ,Ca. Phytoplasma phoenicium', ,Ca. Phytoplasma solani' and ,Ca. Phytoplasma trifolii'. The phytoplasmas related to ,Ca. Phytoplasma aurantifolia' were more prevalent than other phytoplasmas in the central regions of Iran. [source] Sex Identification of the Black Swan (Cygnus atratus) using the Locus-specific PCR and Implications for its ReproductionREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2005P-J He Contents Over the last 4,5 years the small captive population of black swans (Cygnus atratus) has consistently failed to reproduce at the Chengdu Research Base of Giant Panda Breeding. The probable cause was hypothesized to be an abnormal sex distribution of the population. The black swan is an example of a sexually monomorphic species. The locus-specific polymerase chain reaction (PCR) approach based on the chromo-helicase-DNA-binding 1 (CHD1) gene, was adopted for the sex determination of the black swans. For this purpose, F1, F2 and R primers were designed using the primerselect software for amplification of the CHD1 gene region. DNA agarose gel electrophoresis showed that the female control displayed two bands, whereas only a single band was found in the male control. Sequence analyses of all seven unknown sex black swans demonstrated the sex-specific DNA band for female. Therefore, it was inferred that all the individuals of the black swan population are females, which has resulted in unfertilized eggs and reproduction failure. This method can be extended to the sexing of other monomorphic avian species and will assist in the design of breeding projects. [source] The maize (Zea mays L.) RTCS gene encodes a LOB domain protein that is a key regulator of embryonic seminal and post-embryonic shoot-borne root initiationTHE PLANT JOURNAL, Issue 4 2007Graziana Taramino Summary Maize has a complex root system composed of different root types formed during different stages of development. The rtcs (rootless concerning crown and seminal roots) mutant is impaired in the initiation of the embryonic seminal roots and the post-embryonic shoot-borne root system. The primary root of the mutant shows a reduced gravitropic response, while its elongation, lateral root density and reaction to exogenously applied auxin is not affected. We report here the map-based cloning of the RTCS gene which encodes a 25.5 kDa LOB domain protein located on chromosome 1S. The RTCS gene has been duplicated during evolution. The RTCS-LIKE (RTCL) gene displays 72% sequence identity on the protein level. Both genes are preferentially expressed in roots. Expression of RTCS in coleoptilar nodes is confined to emerging shoot-borne root primordia. Sequence analyses of the RTCS and RTCL upstream genomic regions identified auxin response elements. Reverse transcriptase-PCR revealed that both genes are auxin induced. Microsynteny analyses between maize and rice genomes revealed co-linearity of 14 genes in the RTCS region. We conclude from our data that RTCS and RTCL are auxin-responsive genes involved in the early events that lead to the initiation and maintenance of seminal and shoot-borne root primordia formation. [source] Effect of Thymus vulgaris essential oil on intestinal bacterial microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum) and bacterial isolatesAQUACULTURE RESEARCH, Issue 10 2010Paola Navarrete Abstract The application of natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics. We evaluated the effect of diet supplementation with Thymus vulgaris essential oil (TVEO) on the allochthonous microbial composition of rainbow trout. DNA was extracted directly from the intestinal contents, and the V3-V4 regions of the 16S rRNA genes were amplified by PCR. The bacterial composition was analysed using temporal temperature gradient electrophoresis (TTGE). No significant changes (P>0.05) were detected in the TTGE profiles of TVEO-treated trout compared with the controls. The Dice similarity index revealed a high stability (Cs >70%) of the intestinal microbiota in both groups during the 5-week period. Sequence analyses of the TTGE bands revealed the same bacterial composition in both groups, with most bacteria belonging to the Proteobacteria and Firmicutes phyla. The in vitro antibacterial activity of TVEO was assessed using a range of normal intestinal isolates and fish pathogens. The inhibitory concentrations for all the tested bacteria were higher than the TVEO levels used in trout, which may explain the in vivo results. [source] New developments in the production and use of stereoselective antibodiesCHIRALITY, Issue S1 2005Heike Hofstetter Abstract This article describes the production of stereoselective antibodies using both classical immunological and modern molecular biological techniques. Stereoselective antibodies against ,-hydroxy acids were raised in rabbits and mice and compared with previously produced anti-,-amino acid antibodies. It was found that both types of antibodies combine stereoselectivity with class-specificity. Sequence analyses revealed that antibodies with opposing stereoselectivities can be formed during the affinity maturation process from a common progenitor or independently using nonhomologous binding sites. For the first time, phage display was employed to obtain stereoselective antibody fragments. The versatility of stereoselective antibodies as chiral selectors was demonstrated by applying them in several immunosensors and in chiral chromatography. A simple, membrane-based optical sensor allowed detection of enantiomeric impurities at the 1/2,000 level (99.9% ee). Silica-based antibody chiral stationary phases could be used for enantiomer separation of aliphatic amino acids in standard-sized columns, while miniaturized columns allowed interfacing with an MS-detector. Chirality 17:S9,S18, 2005. © 2004 Wiley-Liss, Inc. [source] Genes involved in the RNA interference pathway are differentially expressed during sea urchin developmentDEVELOPMENTAL DYNAMICS, Issue 11 2007Jia L. Song Abstract RNA-mediated interference (RNAi) is a conserved gene silencing mechanism that involves double-stranded RNA as a signal to trigger the sequence-specific degradation of target mRNA, resulting in posttranscriptional silencing and/or translational repression. Bioinformatic searches in the sea urchin genome database identified homologs of Drosha, DGCR5, Dicer, TRBP, Exportin-5, and Argonautes. Quantitative, real-time polymerase chain reaction indicated that all mRNA accumulate in eggs and in variable levels throughout early development. Whole-mount in situ RNA hybridization showed that all of the important players of the RNAi silencing pathway have abundant mRNA accumulation in oocytes and eggs, but have distinct spatial and temporal expression patterns throughout development. Sequence analysis revealed that each of the four Argonautes examined contain conserved residues important for RNAseH activity within the Piwi domain. This study elucidated that genes involved in the RNAi silencing pathway have dynamic expression and, thus, may have regulatory roles during germ cell development and embryogenesis. Developmental Dynamics 236:3180,3190, 2007. © 2007 Wiley-Liss, Inc. [source] Electrophoretic variants of cardiac myosin heavy chain-, in Sprague Dawley ratsELECTROPHORESIS, Issue 3 2004Peter J. Reiser Abstract Analysis of cardiac myosin revealed differences in gel electrophoretic migration patterns of the ,-isoform of myosin heavy chain, but not the ,-isoform, in Sprague Dawley rats. No differences in the migration patterns of the ,-or ,-isoforms were observed in other rat strains. Three electrophoretic migration patterns of the ,-isoforms were observed in individual rats: a slower migrating isoform alone (4% of all rats tested), a faster migrating isoform alone (55%), and both isoforms (41%). The isoform expression pattern was identical in all myocardial regions in each rat. Frequency of expression patterns suggests multiple gene sequences for ,-cardiac myosin heavy chain in Sprague Dawley rats. Sequence analysis of amplified regions of the Sprague Dawley and Brown Norway rat ,-myosin genes, specifically the 5'-untranslated region, exons 1,3, and associated introns, showed numerous single nucleotide polymorphisms in coding and noncoding regions, including putative regulatory sites in Sprague Dawley rats, but not in Brown Norway rats. All Sprague Dawley rats varied from Brown Norway rats and no heterogeneity was observed in Brown Norway rats. Several deletions and dimorphic positions were also observed. Dimorphic positions were evident on automated sequencing comparisons. The data indicate that at least two ,-myosin heavy chain isoforms exist in Sprague Dawley rats and these rats exhibit sequence diversity within that portion of the ,-myosin heavy chain gene reported in this study. [source] Molecular analysis of the phosphorus starvation response in Trichodesmium spp.ENVIRONMENTAL MICROBIOLOGY, Issue 9 2009Elizabeth D. Orchard Summary The marine diazotroph Trichodesmium is a major contributor to primary production and nitrogen fixation in the tropical and subtropical oceans. These regions are often characterized by low phosphorus (P) concentrations, and P starvation of Trichodesmium could limit growth, and potentially constrain nitrogen fixation. To better understand how this genus responds to P starvation we examined four genes involved in P acquisition: two copies of a high-affinity phosphate binding protein (pstS and sphX) and two putative alkaline phosphatases (phoA and phoX). Sequence analysis of these genes among cultured species of Trichodesmium (T. tenue, T. erythraeum, T. thiebautii and T. spiralis) showed that they all are present and conserved within the genus. In T. erythraeum IMS101, the expression of sphX, phoA and phoX were sensitive to P supply whereas pstS was not. The induction of alkaline phosphatase activity corresponded with phoA and phoX expression, but enzyme activity persisted after the expression of these genes returned to basal levels. Additionally, nifH (nitrogenase reductase; involved in nitrogen fixation) expression was downregulated under P starvation conditions. These data highlight molecular level responses to low P and lay a foundation for better understanding the dynamics of Trichodesmium P physiology in low-P environments. [source] Application of nr-DNA ITS sequence for identification of Fusarium culmorum isolates,EPPO BULLETIN, Issue 3-4 2000P. K. Mishra Variation within the internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal DNA region of 60 Fusarium culmorum isolates (section Discolor), representing different hosts and diverse geographical origins was examined by polymerase chain reaction (PCR), coupled with sequencing. Phylogenetic relationships of these F. culmorum isolates were estimated in relation to Fusarium spp. from this and other sections of the form-genus, using sequences available from Genbank. The amplified ITS region was approximately 570 bp long in 56 isolates and approximately 585 bp in four other isolates. The inferred phylogeny distinguished clearly four isolates supplied as F. culmorum. These isolates differed in both morphology and sequence from the remaining F. culmorum material. Sequence analysis revealed that the remaining 56 isolates were divided into three ITS types, within which the divergence was extremely low. ITS sequence comparison among the Fusarium isolates showed two major clades, one comprising sections Discolor, Sporotrichiella and Gibbosum and the other comprising Elegans, Liseola, Martiella and Roseum. These results demonstrate the use of the ITS region to resolve the identification and taxonomic problems of Fusarium spp. especially at sectional level but demonstrate the need to develop some other molecular markers for identification at the level of species or race. [source] Pathologic expression of MHC class,II is driven by mitogen-activated protein kinasesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2007Isabelle Martins Abstract The class,II transactivator (CIITA) is the master regulator of MHC class,II molecules (MHC,II). In melanoma, the MHC,II are constitutively expressed due to an abnormal transcription of CIITA from its promoter,III (pIII), and requires the presence of a 1-kb enhancer located upstream from this latter. Since mitogen-activated protein kinases (MAPK) have been shown to be activated in most melanomas, we sought to analyze their possible involvement in CIITA expression. Using chemical inhibitors and dominant-negative constructs of MAPK-ERK kinase (Mek1) and MAPK-JNK, we evidenced the inhibition of MHC,II and CIITA expression in melanoma cell lines displaying activated MAPK. Transcriptional regulation by MAPK is known to involve the AP-1 transcription factor family. Sequence analysis revealed an AP-1-responsive motif in the enhancer of CIITA pIII at ,5954/,5947 from the site of transcription initiation. Its mutagenesis reduced CIITA expression four- to fivefold in melanoma cell lines and alleviated the effect of dominant-negative constructs of the MAPK pathway. Together, our findings demonstrate that MAPK-ERK and MAPK-JNK are regulators of CIITA transcription in melanoma, and pinpoint an AP-1-responsive site in the CIITA gene pIII. This should have considerable impact on our understanding of the physio-pathologic expression of MHC,II. [source] Marginal zone B cell enrichment and strong follicular B cell reduction correlate with a delayed IgG response in a light chain diversity restricted mouse modelEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2004Yacine Abstract Recently developed B6.,,,SEG mice (by crossing ,, and C57BL/6 mice congenic for the wild Mus spretus SEG strain , locus lacking genes coding for ,1 and ,3) have a very reduced light chain diversity. B6.,,,SEG mice produce only ,2 and ,x light chains. Regardless of their Igh haplotype, B6.,,,SEG mice show a restricted B cell distribution by light chain subtype with ,x dominance in all peripheral compartments except peritoneal cavity where ,2 is dominant. This distribution suggests that selection mechanisms act differently in different B cell compartments on ,2 and ,x bearing B cells. Sequence analysis before or following immunization did not reveal unusual mechanisms of diversification. B6.,,,SEG mice still respond to various challenging antigens using new Ab patterns. In particular, regardless of Igha or Ighb haplotypes, the anti-2,4-dinitrophenyl response is characterized by a restricted diversity for both heavy and light chains and a delayed IgG response when compared to B6 and B6.,, mice. We suggest that the delayed IgG response is due to the expansion of marginal zone B cells whereas follicular B cells are strongly reduced. [source] Expression of Hoxa-11 and Hoxa-13 in the pectoral fin of a basal ray-finned fish, Polyodon spathula: implications for the origin of tetrapod limbsEVOLUTION AND DEVELOPMENT, Issue 3 2005Brian D. Metscher Summary Paleontological and anatomical evidence suggests that the autopodium (hand or foot) is a novel feature that distinguishes limbs from fins, while the upper and lower limb (stylopod and zeugopod) are homologous to parts of the sarcopterygian paired fins. In tetrapod limb development Hoxa-11 plays a key role in differentiating the lower limb and Hoxa-13 plays a key role in differentiating the autopodium. It is thus important to determine the ancestral functions of these genes in order to understand the developmental genetic changes that led to the origin of the tetrapod autopodium. In particular it is important to understand which features of gene expression are derived in tetrapods and which are ancestral in bony fishes. To address these questions we cloned and sequenced the Hoxa-11 and Hoxa-13 genes from the North American paddlefish, Polyodon spathula, a basal ray-finned fish that has a pectoral fin morphology resembling that of primitive bony fishes ancestral to the tetrapod lineage. Sequence analysis of these genes shows that they are not orthologous to the duplicated zebrafish and fugu genes. This implies that the paddlefish has not duplicated its HoxA cluster, unlike zebrafish and fugu. The expression of Hoxa-11 and Hoxa-13 in the pectoral fins shows two main phases: an early phase in which Hoxa-11 is expressed proximally and Hoxa-13 is expressed distally, and a later phase in which Hoxa-11 and Hoxa-13 broadly overlap in the distal mesenchyme of the fin bud but are absent in the proximal fin bud. Hence the distal polarity of Hoxa-13 expression seen in tetrapods is likely to be an ancestral feature of paired appendage development. The main difference in HoxA gene expression between fin and limb development is that in tetrapods (with the exception of newts) Hoxa-11 expression is suppressed by Hoxa-13 in the distal limb bud mesenchyme. There is, however, a short period of limb bud development where Hoxa-11 and Hoxa-13 overlap similarly to the late expression seen in zebrafish and paddlefish. We conclude that the early expression pattern in tetrapods is similar to that seen in late fin development and that the local exclusion by Hoxa-13 of Hoxa-11 from the distal limb bud is a derived feature of limb developmental regulation. [source] Genetic analysis of three important genes in pigmentation and melanoma susceptibility: CDKN2A, MC1R and HERC2/OCA2EXPERIMENTAL DERMATOLOGY, Issue 9 2010Maider Ibarrola-Villava Please cite this paper as: Genetic analysis of three important genes in pigmentation and melanoma susceptibility: CDKN2A, MC1R and HERC2/OCA2. Experimental Dermatology 2010; 19: 836,844. Abstract:, The CDKN2A gene is regarded as the major familial malignant melanoma (MM) susceptibility gene. Human pigmentation is one of the main modulators of individual risk of developing MM. Therefore, the genes involved in the determination of skin colour and tanning response are potentially implicated in MM predisposition and may be useful predictors of MM risk in the general population. The human melanocortin-1 receptor gene (MC1R) plays a crucial role in pigmentation and also appears to be important in MM. The OCA2 gene has emerged as a new and significant determinant of human iris colour variation. We present a case,control study in Spanish population including 390 consecutive patients with melanoma and 254 control subjects. Sequence analysis of the entire coding region and genotyping of 5 tag-SNPs in the genomic region of MC1R was performed. We identified 27 variants, two reaching statistical significance [R160W (OR: 4.18, 95% CI: 1.24,14.04, P = 0.02) and D294H (OR: 3.10, 95% CI: 1.37,7.01, P = 0.01)] and we detected two novel non-synonymous changes: V92L and T308M. Odds ratio for carrying two functional variants was 4.25 (95% CI: 2.30,7.84, P = 3.63 × 10,6). Haplotypes of the entire MC1R region have been established, and we observed an enrichment of a rare European haplotype similar to African values carrying variants V92M and I155T. In addition, three potentially functional SNPs were selected in p16/CDKN2A and in the promoter region of OCA2/HERC2. Our data for CDKN2A gene did not reach statistically significant results for any of the two studied alleles. We found that the variant allele A > G of OCA2/HERC2 (rs12913832) was associated with pigmentation features: eye, hair and skin colour; P -values = 1.8 × 10,29, 9.2 × 10,16, 1.1 × 10,3, respectively, validating previous results. [source] Genomic structure and expression analysis of the RNase , family ortholog gene in the insect Ceratitis capitataFEBS JOURNAL, Issue 24 2008Theodoros N. Rampias Cc RNase is the founding member of the recently identified RNase , family, which is represented by a single ortholog in a wide range of animal taxonomic groups. Although the precise biological role of this protein is still unknown, it has been shown that the recombinant proteins isolated so far from the insect Ceratitis capitata and from human exhibit ribonucleolytic activity. In this work, we report the genomic organization and molecular evolution of the RNase , gene from various animal species, as well as expression analysis of the ortholog gene in C. capitata. The high degree of amino acid sequence similarity, in combination with the fact that exon sizes and intronic positions are extremely conserved among RNase , orthologs in 15 diverse genomes from sea anemone to human, imply a very significant biological function for this enzyme. In C. capitata, two forms of RNase , mRNA (0.9 and 1.5 kb) with various lengths of 3, UTR were identified as alternative products of a single gene, resulting from the use of different polyadenylation signals. Both transcripts are expressed in all insect tissues and developmental stages. Sequence analysis of the extended region of the longer transcript revealed the existence of three mRNA instability motifs (AUUUA) and five poly(U) tracts, whose functional importance in RNase , mRNA decay remains to be explored. [source] Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptideFEBS JOURNAL, Issue 6 2008Adam Frederick To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChET subunit, which should be able to produce all three globular forms of AChE: monomers (G1), dimers (G2), and tetramers (G4), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G1a) and non-amphiphilic tetramers (G4na) are found. Amphiphilic dimers (G2a) and non-amphiphilic tetramers (G4na) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A12 form of the enzyme. Collagenase digestion of the A12 AChE produces a lytic G4 form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates. [source] Molecular cloning, expression and characterization of cDNA encoding cis -prenyltransferases from Hevea brasiliensisFEBS JOURNAL, Issue 23 2003A key factor participating in natural rubber biosynthesis Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis -configuration. The enzyme responsible for the cis -1,4-polymerization of isoprene units has been idengified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis -prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis -prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis -prenyltransferase. Northern blot analysis indicated that the transcript(s) of the Hevea cis -prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 × 103,1 × 104 Da. Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased. These results suggest that the Hevea cis -prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber. [source] Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum)FEBS JOURNAL, Issue 18 2003Susan E. Douglas Antimicrobial peptides form one of the first lines of defense against invading pathogens by killing the microorganisms and/or mobilizing the host innate immune system. Although over 800 antimicrobial peptides have been isolated from many different species, especially insects, few have been reported from marine fish. Sequence analysis of two genomic clones (15.6 and 12.5 kb) from the winter flounder, Pseudopleuronectes americanus (Walbaum) resulted in the identification of multiple clustered genes for novel pleurocidin-like antimicrobial peptides. Four genes and three pseudogenes (,) are encoded in these clusters, all of which have similar intron/exon boundaries but specify putative antimicrobial peptides differing in sequence. Pseudogenes are easily detectable but have incorrect initiator codons (ACG) and often contain a frameshift(s). Potential promoters and binding sites for transcription factors implicated in regulation of expression of immune-related genes have been identified in upstream regions by comparative genomics. Using reverse transcription-PCR assays, we have shown for the first time that each gene is expressed in a tissue-specific and developmental stage-specific manner. In addition, synthetic peptides based on the sequences of both genes and pseudogenes have been produced and tested for antimicrobial activity. These data can be used as a basis for prediction of antimicrobial peptide candidates for both human and nonhuman therapeutants from genomic sequences and will aid in understanding the evolution and transcriptional regulation of expression of these peptides. [source] Interallelic complementation provides genetic evidence for the multimeric organization of the Phycomyces blakesleeanus phytoene dehydrogenaseFEBS JOURNAL, Issue 3 2002Catalina Sanz The Phycomyces blakesleeanus wild-type is yellow, because it accumulates ,-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, ,-carotene and neurosporene, in decreasing amounts, and traces of ,-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase. [source] Cellulose-binding modules from extracellular matrix proteins of Dictyostelium discoideum stalk and sheathFEBS JOURNAL, Issue 15 2001Yingzi Wang Cellulose-binding modules (CBMs) of two extracellular matrix proteins, St15 and ShD, from the slime mold Dictyostelium discoideum were expressed in Escherichia coli. The expressed proteins were purified to >,98% purity by extracting inclusion bodies at pH 11.5 and refolding proteins at pH 7.5. The two refolded CBMs bound tightly to amorphous phosphoric acid swollen cellulose (PASC), but had a low affinity toward xylan. Neither protein exhibited cellulase activity. St15, the stalk-specific protein, had fourfold higher binding affinity toward microcrystalline cellulose (Avicel) than the sheath-specific ShD CBM. St15 is unusual in that it consists of a solitary CBM homologous to family IIa CBMs. Sequence analysis of ShD reveals three putative domains containing: (a) a C-terminal CBM homologous to family IIb CBMs; (b) a Pro/Thr-rich linker domain; and (c) a N-terminal Cys-rich domain. The biological functions and potential role of St15 and ShD in building extracellular matrices during D. discoideum development are discussed. [source] | |||||||||||||||||||||||||||||||||||||||||||