|
| ||
|
|
||
BLAST Searches (blast + search)
Selected AbstractsEfficient recognition of protein fold at low sequence identity by conservative application of Psi-BLAST: validation,JOURNAL OF MOLECULAR RECOGNITION, Issue 2 2005F. J. Stevens Abstract A substantial fraction of protein sequences derived from genomic analyses is currently classified as representing ,hypothetical proteins of unknown function'. In part, this reflects the limitations of methods for comparison of sequences with very low identity. We evaluated the effectiveness of a Psi-BLAST search strategy to identify proteins of similar fold at low sequence identity. Psi-BLAST searches for structurally characterized low-sequence-identity matches were carried out on a set of over 300 proteins of known structure. Searches were conducted in NCBI's non-redundant database and were limited to three rounds. Some 614 potential homologs with 25% or lower sequence identity to 166 members of the search set were obtained. Disregarding the expect value, level of sequence identity and span of alignment, correspondence of fold between the target and potential homolog was found in more than 95% of the Psi-BLAST matches. Restrictions on expect value or span of alignment improved the false positive rate at the expense of eliminating many true homologs. Approximately three-quarters of the putative homologs obtained by three rounds of Psi-BLAST revealed no significant sequence similarity to the target protein upon direct sequence comparison by BLAST, and therefore could not be found by a conventional search. Although three rounds of Psi-BLAST identified many more homologs than a standard BLAST search, most homologs were undetected. It appears that more than 80% of all homologs to a target protein may be characterized by a lack of significant sequence similarity. We suggest that conservative use of Psi-BLAST has the potential to propose experimentally testable functions for the majority of proteins currently annotated as ,hypothetical proteins of unknown function';. Copyright © 2004 John Wiley & Sons, Ltd. [source] Crystal structure of a major secreted protein of Mycobacterium tuberculosis,MPT63 at 1.5-Å resolutionPROTEIN SCIENCE, Issue 12 2002Celia W. Goulding Abstract MPT63 is a small, major secreted protein of unknown function from Mycobacterium tuberculosis that has been shown to have immunogenic properties and has been implicated in virulence. A BLAST search identified that MPT63 has homologs only in other mycobacteria, and is therefore mycobacteria specific. As MPT63 is a secreted protein, mycobacteria specific, and implicated in virulence, MPT63 is an attractive drug target against the deadliest infectious disease, tuberculosis (TB). As part of the TB Structural Genomics Consortium, the X-ray crystal structure of MPT63 was determined to 1.5-Ångstrom resolution with the hope of yielding functional information about MPT63. The structure of MPT63 is an antiparallel ,-sandwich immunoglobulin-like fold, with the unusual feature of the first ,-strand of the protein forming a parallel addition to the small antiparallel ,-sheet. MPT63 has weak structural similarity to many proteins with immunoglobulin folds, in particular, Homo sapiens ,2-adaptin, bovine arrestin, and Yersinia pseudotuberculosis invasin. Although the structure of MPT63 gives no conclusive evidence to its function, structural similarity suggests that MPT63 could be involved in cell-host interactions to facilitate endocytosis/phagocytosis. [source] Sequence polymorphisms in porcine homologs of murine coat colour-related genesANIMAL GENETICS, Issue 2 2010N. Okumura Summary Herein, we report the variability among 57 porcine homologs of murine coat colour-related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F2 family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F2 family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig. [source] Characterization of an I,B-like gene in Cotesia vestalis polydnavirusARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2008Ya-Feng Chen Abstract Cotesia vestalis (Braconidae, Hymenoptera) depends mainly on 3 regulatory factors to manipulate its host's development and immune response, including polydnavirus, venom, and teratocytes, among which polydnavirus plays a key role in suppressing the host immune system. In the present work, we cloned the full sequence of gene CvBV-ank2, encoding an I,B-like protein in C. vestalis polydnavirus (CvBV). The full sequence of CvBV-ank2 is 955 bp, encoding 162 amino acids with a calculated molecular mass of 18,355 Da. CvBV-ank2 shares high similarity with the exon I and exon II of CvBV-ank1, which is on the same segment with CvBV-ank2. This result suggests that gene duplication might occur in CvBV-ank1 and CvBV-ank2. Phylogenetic analysis indicated that CvBV-ank2 and CvBV-ank1, both on segment CvBV-S2, are, respectively, closely related with CcBV,26.3 and CcBV,26.2, both on segment Circle26 of C. congregata polydnavirus (CcBV). BLAST search using the sequence of segment CvBV-S2 as a query revealed that segment CvBV-S2 shares 90% max identity with segment Circle26 of CcBV over 67% query coverage. These results demonstrate that there is not only gene similarity, but also segment similarity between CvBV and CcBV. Transcripts of CvBV-ank2 were detected as early as 0.5 h post-parasitization and continued to be detected for 6 days, indicating that CvBV-ank2 might be involved in the early protection of the parasitoid egg. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] Genomic BLAST: custom-defined virtual databases for complete and unfinished genomesFEMS MICROBIOLOGY LETTERS, Issue 2 2002Leda Cummings Abstract BLAST (Basic Local Alignment Search Tool) searches against DNA and protein sequence databases have become an indispensable tool for biomedical research. The proliferation of the genome sequencing projects is steadily increasing the fraction of genome-derived sequences in the public databases and their importance as a public resource. We report here the availability of Genomic BLAST, a novel graphical tool for simplifying BLAST searches against complete and unfinished genome sequences. This tool allows the user to compare the query sequence against a virtual database of DNA and/or protein sequences from a selected group of organisms with finished or unfinished genomes. The organisms for such a database can be selected using either a graphic taxonomy-based tree or an alphabetical list of organism-specific sequences. The first option is designed to help explore the evolutionary relationships among organisms within a certain taxonomy group when performing BLAST searches. The use of an alphabetical list allows the user to perform a more elaborate set of selections, assembling any given number of organism-specific databases from unfinished or complete genomes. This tool, available at the NCBI web site http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi, currently provides access to over 170 bacterial and archaeal genomes and over 40 eukaryotic genomes. [source] Phenotypic and genotypic characterization of competitive exclusion products for use in poultryJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2003R.D. Wagner Abstract Aims: Phenotypic and genotypic bacteria identification methods were compared for their efficacy in determining the composition of competitive exclusion (CE) products. Methods and Results: Phenotypic methods used for bacterial identification were fatty acid methyl ester profiles, biochemical assays and carbohydrate utilization profiles. Genotypic methods were MicroSeq16S rRNA sequence analysis and BLAST searches of the GenBank sequence database. Agreement between phenotypic and genotypic methods for identification of bacteria isolated from the Preempt CE product was 20%. A defined test mixture of bacteria was identified to the species level 100% by BLAST analysis, 64% by MicroSeq and 36% by phenotypic techniques. Conclusions: The wide range of facultative and obligate anaerobic bacteria present in a CE product are more accurately identified with 16S rRNA sequence analyses than with phenotypic identification techniques. Significance and Impact of the Study: These results will provide guidelines for manufacturers of CE products to submit more reliable product information for market approval by regulatory agencies. [source] Evaluation of PCR primers from putative transcriptional regulator genes for identification of Staphylococcus aureusLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2005D. Liu Abstract Aims:, To examine if PCR primers derived from putative transcriptional regulator genes can be useful for Staphylococcus aureus identification. Methods and Results:,Staphylococcus aureus gene sequences that encode transcriptional regulators were retrieved from GenBank and compared with other DNA sequences via BLAST searches. Two uniquely present, putative transcriptional regulator genes (i.e. Sa0836 and Sa0856) were selected as a consequence and PCR primers (Sa0836F/R and Sa0856F/R) were then designed from these genes for evaluation. A total of 84 bacterial strains/isolates including 23 Staph. aureus, 18 nonaureus Staphylococcus and 43 other common bacterial isolates were examined. The results indicated that PCR primers from Sa0836 and Sa0856 recognized genomic DNA from Staph. aureus only, but not from other non-aureus Staphylococcus or common bacteria. Conclusions:, PCR detection of the putative transcriptional regulator genes Sa0836 and Sa0856 represents a useful means of identifying Staph. aureus from other bacteria. Significance and Impact of the Study:, The existence of species,species transcriptional regulator genes may be a common phenomenon in bacteria. Besides their value as novel diagnostic markers, further investigation on the putative transcriptional regulator genes Sa0836 and Sa0856 and their related products may shed light on the molecular mechanisms of Staph. aureus adaptation and virulence. [source] DNA-based identification of preys from non-destructive, total DNA extractions of predators using arthropod universal primersMOLECULAR ECOLOGY RESOURCES, Issue 3 2006JOAN PONS Abstract Here, I show that prey sequences can be detected from DNA of tiger beetles of the genus Rivacindela using whole specimens, nondestructive methods, and universal cytochrome b primers for arthropods. BLAST searches of the obtained sequences against public databases revealed that the diet of Rivacindela is mostly composed of flies but also termites and other beetles. Accurate determination of order, family and even genus was achieved in most cases but rarely to species level. Results suggest that stored DNA samples extracted from whole predatory specimens could be an alternative to dissected gut contents as starting source for DNA-based dietary studies. [source] A workflow to increase the detection rate of proteins from unsequenced organisms in high-throughput proteomics experimentsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2007Jonas Grossmann Abstract We present and evaluate a strategy for the mass spectrometric identification of proteins from organisms for which no genome sequence information is available that incorporates cross-species information from sequenced organisms. The presented method combines spectrum quality scoring, de novo sequencing and error tolerant BLAST searches and is designed to decrease input data complexity. Spectral quality scoring reduces the number of investigated mass spectra without a loss of information. Stringent quality-based selection and the combination of different de novo sequencing methods substantially increase the catalog of significant peptide alignments. The de novo sequences passing a reliability filter are subsequently submitted to error tolerant BLAST searches and MS-BLAST hits are validated by a sampling technique. With the described workflow, we identified up to 20% more groups of homologous proteins in proteome analyses with organisms whose genome is not sequenced than by state-of-the-art database searches in an Arabidopsis thaliana database. We consider the novel data analysis workflow an excellent screening method to identify those proteins that evade detection in proteomics experiments as a result of database constraints. [source] Development of microsatellite DNA markers and their chromosome assignment in the common marmosetAMERICAN JOURNAL OF PRIMATOLOGY, Issue 11 2009Hideki Katoh Abstract This study was performed to develop microsatellite DNA markers, which are useful for linkage analyses, gene mapping and blood chimera analyses in the common marmoset (Callithrix jacchus). We searched 153 of 295 bacterial artificial clone DNA sequences of the common marmoset that were archived in the NCBI database in 2004. On the basis of the search, we designed 186 PCR primer sets. When tested using 5 unrelated individuals, we successfully detected 154 markers with PCR products, of which 80 (52%) were polymorphic and 74 (48%) were monomorphic. We assigned each of the 154 markers to a human chromosome based on BLAST searches, which was achieved by searching the entire human genome sequences using an ,3,kb section of each forward primer sequence, including ,1.5,kb of the upstream and ,1.5,kb of the downstream sequences. Combining our assignment data and the chromosome painting-assisted karyotype of the common marmoset [Sherlock et al., Genomics 33:214,219, 1996], we prepared a list of 154 microsatellite DNA markers that were assigned to human chromosomes, except for the Y chromosome, which is equivalent to a chromosome map. Using five microsatellite DNA markers, we have established a fragment analysis method with a sequencer, which can be routinely used for blood chimera analysis, parentage diagnosis and individual identification. Am. J. Primatol. 71:912,918, 2009. © 2009 Wiley-Liss, Inc. [source] Strategic Selection of Hyperthermophilic Esterases for Resolution of 2-Arylpropionic EstersBIOTECHNOLOGY PROGRESS, Issue 5 2003Amitabh C. Sehgal Homologues to Carboxylesterase NP and Candida rugosa lipase, used for the chiral separation of racemic mixtures of 2-arylpropionic methyl esters, were identified by BLAST searches of available genome sequences for hyperthermophilic microorganisms. Two potential candidates were identified: a putative lysophospholipase from Pyrococcus furiosus (Pfu-LPL) and a carboxylesterase from Sulfolobussolfataricus P1 (Sso-EST1). Although both enzymes showed hydrolytic preference toward the (S) methyl ester, only Sso-EST1 yielded highly optically pure (S) naproxen (%eep , 90) and was thus further investigated. Changes in pH or reaction time showed little improvement in %eep or E values with Sso-EST1. However, the addition of 25% methanol resulted in a 25% increase in E. The effect of various cosolvents on the enantiomeric ratio showed no correlation with the log P or dielectric constant values of the solvent. However, an inverse relationship between E and the denaturation capacity (DC) of the water miscible cosolvents was observed. This was attributed to an increase in enzyme flexibility with increasing solvent DC values leading to a concomitant reduction in the resolving power of Sso-EST1. The results here show that although bioinformatics tools can be used to select candidate biocatalysts for chiral resolution of 2-arylpropionic esters, biochemical characterization is needed to definitively determine functional characteristics. [source] Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica, in Virus-free and Virus-infected Isogenic IsolatesJOURNAL OF PHYTOPATHOLOGY, Issue 6 2010Laura Rostagno Abstract Cpkk1 and Cpkk2 are two previously characterized Mitogen-activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT-PCR products resulting from the amplification of purified mRNA. TaqMan® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT-Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth. [source] Characterization of de novo transcriptome for waterhemp (Amaranthus tuberculatus) using GS-FLX 454 pyrosequencing and its application for studies of herbicide target-site genesPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 10 2010Chance W Riggins Abstract BACKGROUND: Waterhemp is a model for weed genomics research in part because it possesses many interesting biological characteristics, rapidly evolves resistance to herbicides and has a solid foundation of previous genetics work. To develop further the genomics resources for waterhemp, the transcriptome was sequenced using Roche GS-FLX 454 pyrosequencing technology. RESULTS: Pyrosequencing produced 483 225 raw reads, which, after quality control and assembly, yielded 44 469 unigenes (contigs + singletons). A total of 49% of these unigenes displayed highly significant similarities to Arabidopsis proteins and were subsequently grouped into gene ontology categories. Blast searches against public and custom databases helped in identifying and obtaining preliminary sequence data for all of the major target-site genes for which waterhemp has documented resistance. Moreover, sequence data for two other herbicide targets [4-hydroxyphenylpyruvate dioxygenase (HPPD) and glutamine synthetase], where resistance has not yet been reported in any plant, were also investigated in waterhemp and six related weedy Amaranthus species. CONCLUSION: These results demonstrate the enormous value of 454 sequencing for gene discovery and polymorphism detection in a major weed species and its relatives. Furthermore, the merging of the 454 transcriptome data with results from a previous whole genome 454 sequencing experiment has made it possible to establish a valuable genomic resource for weed science research. Copyright © 2010 Society of Chemical Industry [source] Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L -gulono-1,4-lactone dehydrogenaseFEBS JOURNAL, Issue 19 2006Beata A. Wolucka The last step of the biosynthesis of l -ascorbic acid (vitamin C) in plants and animals is catalyzed by l -gulono-1,4-lactone oxidoreductases, which use both l -gulono-1,4-lactone and l -galactono-1,4-lactone as substrates. l -Gulono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient animals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis. A blast search using the rat l -gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited number of bacterial species, including several pathogens of human lungs, such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepacia and Bacillus anthracis. The genome of M. tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat l -gulono-1,4-lactone oxidase protein. The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized. The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes l -gulono-1,4-lactone (Km 5.5 mm) but not l -galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (Km 4.7 µm) and phenazine methosulfate as exogenous electron acceptors. Molecular oxygen does not serve as a substrate for the Rv1771 protein. Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain. In conclusion, M. tuberculosis produces a novel, highly specific l -gulono-1,4-lactone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C. [source] Dynamic changes in gene expression during vitellogenic stages of the white shrimp: Fenneropenaeus merguiensis de ManAQUACULTURE RESEARCH, Issue 6 2009Monwadee Wonglapsuwan Abstract Ovarian maturation is a crucial step for shrimp brood stock. A suppressive subtractive hybridization was used to identify differentially expressed genes in the ovaries during vitellogenesis of Fenneropenaeus merguiensis. Three- to sevenfold up-regulated genes were selected. A blast search identified nine unique genes. The genes that may be involved in ovarian maturation, namely translationally controlled tumour protein (TCTP), heat shock protein 70 (HSP70), H-L(3)MBT-LIKE, shrimp ovarian peritrophin (SOP), vitellin (Vn), thrombospondin (TSP) and ribosomal protein L10a (RPL10a), were further studied. The transcripts of HSP70, TCTP, SOP and RPL10a in the ovary showed their highest expression in the early stage and declined in the later stages. In contrast, the transcripts of the H-L(3)MBT-LIKE, TSP and Vn genes increased from the early stage to be significantly up-regulated during the late stage. A comparison of gene expression among organs during the vitellogenesis showed that the transcripts of HSP70, SOP, H-L(3)MBT-LIKE and TSP were down-regulated in the brain, intestine, hepatopancreas and lymphoid (except for TSP) when compared with their expression in shrimp with non-developed ovaries. The mRNA of TCTP and RPL10a was significantly over-expressed in the lymphoid and heart, whereas TCTP transcripts were significantly down-regulated in the brain during the vitellogenesis. The molecular behaviour of the transcripts in this study may, in the future, lead to an ability to stimulate the ovarian development in shrimp. [source] Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosiscFEBS JOURNAL, Issue 16 2005Melissa C. Miles Multiple ankyrin repeat motif-containing proteins play an important role in protein,protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s). [source] Exploring the species diversity of Trichoderma in Norwegian drinking water systems by DNA barcodingMOLECULAR ECOLOGY RESOURCES, Issue 6 2008GUNHILD HAGESKAL Abstract A total of 123 Trichoderma strains were isolated from Norwegian surface-sourced drinking water. The water samples included raw water, treated water, and water from private homes and hospital installations. Trichoderma species are difficult to differentiate morphologically, but recent molecular identification tools, including DNA barcoding, successfully distinguish between closely related species. The diversity of Trichoderma spp. was explored by DNA sequencing of internal transcribed spacer (ITS) and translation elongation factor 1 alpha (TEF-1,). Sequence identification was performed in the TrichOKEY version 2.0 barcode program and in the multilocus similarity search database TrichoBLAST, combined with traditional blast searches in the EMBL/GenBank. A total of 11 known Trichoderma/Hypocrea species were identified. In addition, one group of unidentified Trichoderma strains was found to represent a separate, strongly supported subclade within the Pachybasium,A'/Hamatum clade, based on their TEF-1, haplotypes. Trichoderma viride comprised 49% of the identified strains, and was represented by four and eight slightly different ITS and TEF-1, haplotypes, respectively. Approximately 22% of the surface-derived water samples were positive for T. viride, and the species was frequently isolated throughout the surface-sourced drinking water distribution system. The results indicate that a broad range of Trichoderma species are present in Norwegian surface-sourced drinking. Water treatment has minor effect in removing Trichoderma from raw water, and active growth in the water distribution system is likely to occur. [source] MicroRNA expression profiles of porcine skeletal muscleANIMAL GENETICS, Issue 5 2010B. Zhou Summary MicroRNAs (miRNAs) are endogenous non-coding RNAs of ,22 nucleotides in length that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. To evaluate the roles of miRNA in porcine skeletal muscle, miRNA expression profiles were investigated using longissimus muscle tissue from pigs at embryonic day 90 (E90) and postpartum day 120 (PD120). First, we used previously known miRNA sequences from humans and mice to perform blast searches against the porcine expressed sequence tag (EST) database; 98 new miRNA candidates were identified according to a range of filtering criteria. These miRNA candidates and 73 known miRNAs (miRBase 13.0) from pigs were chosen for porcine miRNA microarray analysis. A total of 16 newly identified miRNAs and 31 previously known miRNAs were detected in porcine skeletal muscle tissues. During later foetal development at E90, miR-1826, miR-26a, miR-199b and let-7 were highly expressed, whilst miR-1a, miR-133a, miR-26a and miR-1826 showed highest abundance during the fast growing stage at PD120. Using the 47 miRNAs detected by the microarray assay, we performed further investigations using the publicly available porcine mRNA database from NCBI and computed potential target hits using the software rnahybrid. This study identified 16 new miRNA candidates, computed potential target hits for 18 miRNA families and determined the miRNA expression profiles in porcine skeletal muscle tissues at different developmental stages. These results provide a valuable resource for investigators interested in post-transcriptional gene regulation in pigs and related animals. [source] Transcriptome response of the Pacific oyster (Crassostrea gigas) to infection with Vibrio tubiashii using cDNA AFLP differential displayANIMAL GENETICS, Issue 5 2009N. Taris Summary We used qualitative complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) differential display analysis and real-time, quantitative PCR (RT-qPCR) to identify genes in the Pacific oyster Crassostrea gigas, whose transcription either changes in response to exposure to a pathogenic bacterium (Vibrio tubiashii) or varies between families known to differ in sensitivity to heat stress, before and at 12 and 36 h after bacterial exposure at a temperature of 25 °C. These conditions simulate those associated with summer mortality syndrome, a poorly understood cause of massive mortalities in cultured Pacific oysters in North America, Asia and Europe. Using 32 AFLP primer pairs, we identified 92 transcript-derived fragments that are qualitatively differentially expressed. We then cloned and sequenced 14 of these fragments, designed fragment-specific primers and quantified their transcription patterns using RT-qPCR. Most of the differences in transcription patterns between stress-tolerant and stress-sensitive families were evident before bacterial exposure, and genes that responded to bacterial exposure did so in parallel between stress-sensitive and stress-tolerant families. blast searches of sequence databases revealed that these fragments represent genes involved in immune response as well as genes related to metabolic processes. Our data support the hypothesis that family level differences in resistance to stress in Pacific oysters are largely attributable to constitutive differences in gene transcription or ,general vigour' that are detectable before and maintained after infection, rather than being due to induced responses at the transcriptome level. [source] | |||||||||||||