Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Bombyx mori Posterior and Median Silk Gland SAGE transcriptome

ABSTRACT: To identify functions that distinguish the posterior and median cells producing fibroin and sericin in the silk gland of Bombyx mori, serial analysis of gene expression (SAGE) profiles from both silk gland regions were analyzed and compared. The construction of a B. mori reference tag collection extracted from a set of 38000 Bombyx EST sequenced from the 3’ side, helped annotating the SAGE libraries. Most of the tags appeared at similar relative concentration in the two libraries except for those corresponding to silk proteins that were found region-specific and highly abundant. Strikingly, besides tags from silk protein mRNAs, 19 tags were found in the class of high abundance in the median cell library, which were absent in the posterior cell tag collection. Except tags from SP1 mRNA, no PSG specific tags were found in the same class of abundance. The analysis of MSG-specific different transcripts led to suggest that middle silk gland cell realizes more diversified functions as those already known, of synthesis and secretion of the silk sericins. EST libraries from 11 silkworm tissues were 3’-sequenced to ensure the identification of the most terminal tag. 37,920 sequences were analyzed on ABI 3700 or 3730XL sequencers. Electrophoregrams were processed with KB Basecaller (3730XL traces) or with PHRED (3700 traces) to obtain the .phd files from which were extracted text sequences and their corresponding quality files in Fasta format (Phd2Fasta, Green and Ewing, 1995; 2002). Vector sequences and bad quality regions were removed with an home made software after identification by Lucy (Chou and Holmes, 2001). Chimera were removed by an home made software and retrotransposon sequences were masked by RepeatMasker (http://repeatmasker.org). The cleaned sequences were clusterized with TGICL package (TIGR) and assembled into contigs with CAP3 (Huang and Madan, 1999). Contigs were identified with Blastn and Blastx on GenBank and SwissProt/TREMBL, respectively. Some clusters, splitted by CAP3 procedure, have been regrouped on the basis of Blastx identity. Identitag (Keime et al., 2004) was used to extract all possible tags (forward and reverse) to create a reference database. Moreover, a quality index is attached to each tag depending to the presense of poly-A, polyadenylation signal and its proximity to 3-prime extremity in the original mRNA sequence. This database was supplemented with the tags extracted from public B. mori sequences (GenBank, Silkbase) with the same software. Tags were extracted from MSG and PSG libraries (from 2304 and 3072 sequenced inserts respectively) with the Sagenhaft software (Beissbarth et al., 2004) then identified and compared with Identitag. The assessment of significant differences among the two libraries was performed by using the Z-test used for comparison of SAGE libraries of different size (Kal et al., 1999). For graphic purpose and to avoid division by zero we used a tag value of 1 for tags that were not detected in MSG or PSG libraries. Since the two SAGE libraries showed up different TAG number, we used the Z-test for comparing the two mRNA populations.

ORGANISM(S): Bombyx mori

SUBMITTER: Pierre Couble

PROVIDER: E-GEOD-23098 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis.

Royer Corinne C Briolay Jérôme J Garel Annie A Brouilly Patrick P Sasanuma Shun-ichi S Sasanuma Motoe M Shimomura Michihiko M Keime Céline C Gandrillon Olivier O Huang Yongping Y Chavancy Gérard G Mita Kazuei K Couble Pierre P

Insect biochemistry and molecular biology 20101113 2

Serial analysis of gene expression (SAGE) profiles, from posterior and median cells of the silk gland of Bombyx mori, were analyzed and compared, so as to identify their respective distinguishing functions. The annotation of the SAGE libraries was performed with a B. mori reference tag collection, which was extracted from a novel set of Bombyx ESTs, sequenced from the 3' side. Most of the tags appeared at similar relative concentration within the two libraries, and corresponded with region-speci ...[more]

PMID: 21078388

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Project description:The analysis of differentially expressed genes is a powerful approach to elucidate the genetic mechanisms underlying the morphological and evolutionary diversity among serially homologous structures, both within the same organism (e.g., hand vs. foot) and between different species (e.g., hand vs. wing). In the developing embryo, limb-specific expression of Pitx1, Tbx4, and Tbx5 regulates the determination of limb identity. However, numerous lines of evidence, including the fact that these three genes encode transcription factors, indicate that additional genes are involved in the Pitx1-Tbx hierarchy. To examine the molecular distinctions coded for by these factors, and to identify novel genes involved in the determination of limb identity, we have used Serial Analysis of Gene Expression (SAGE) to generate comprehensive gene expression profiles from intact, developing mouse forelimbs and hindlimbs. To minimize the extraction of erroneous SAGE tags from low-quality sequence data, we used a new algorithm to extract tags from -analyzed sequence data and obtained 68,406 and 68,450 SAGE tags from forelimb and hindlimb SAGE libraries, respectively. We also developed an improved method for determining the identity of SAGE tags that increases the specificity of and provides additional information about the confidence of the tag-UniGene cluster match. The most differentially expressed gene between our SAGE libraries was Pitx1. The differential expression of Tbx4, Tbx5, and several limb-specific Hox genes was also detected; however, their abundances in the SAGE libraries were low. Because numerous other tags were differentially expressed at this low level, we performed a 'virtual' subtraction with 362,344 tags from six additional nonlimb SAGE libraries to further refine this set of candidate genes. This subtraction reduced the number of candidate genes by 74%, yet preserved the previously identified regulators of limb identity. This study presents the gene expression complexity of the developing limb and identifies candidate genes involved in the regulation of limb identity. We propose that our computational tools and the overall strategy used here are broadly applicable to other SAGE-based studies in a variety of organisms. Keywords: other

Project description:The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment by SAGE analysis. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unabl e to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g., an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modeling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors. SAGE analysis of C. neoformans cells isolated from macrophages The murine macrophage-like cell line J774A.1 was maintained at 37M-BM-0C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 M-BM-5g mL-1 penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against capsule (1 M-BM-5g mL-1), and macrophages were treated with recombinant mouse gamma interferon (IFN-gamma) (50 U mL-1) and lipopolysaccharide (LPS) (0.3 M-BM-5g mL-1) prior to co-incubation at a multiplicity of infection (MOI) of 1:1. Macrophages were inoculated with H99 cells and washed after 1 h of inoculation to remove unattached, extracellular fungal cells. After 6 h of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells (4 x 107) were harvested by centrifugation. H99 control cells were prepared by growth under the same condition but without macrophages, and 108 cell s were harvested. SAGE library construction, sequencing and analysis SAGE library construction, sequencing and analysis were as previously described (Hu et al., 2007; Hu et al., 2008; Steen et al., 2002; Steen et al., 2003). Briefly, RNA was isolated from lyophilized cells by vortexing with glass beads (3.0 mm, acid-washed and RNase-free) for 15 min in 15 mL of TRIZOL extraction buffer (Invitrogen, Carlsbad, CA, USA). The mixture was incubated for 15 min at room temperature and total RNA was isolated according to the manufacturer's instructions. Total RNA was used directly for SAGE library construction as described by Velculescu et al. (1995) using the I-Long SAGE kit (Invitrogen). The tagging enzyme for cDNA digestion was NlaIII and 29 PCR cycles were performed to amplify the ditags during library construction. Colonies were screened by PCR (M13F and M13R primers) to assess the average clone insert size and the percentage of recombinants. Clones from the libraries were sequenced by BigDye primer cycle sequencing on an ABI PRISM 3700 DNA analy zer. Sequence chromatograms were processed using PHRED (Ewing et al., 1998), and vector sequence was detected using Cross_match (Gordon et al., 1998). Fourteen-base-pair tags were extracted from the vector-clipped sequence, and an overall quality score for each tag was derived based on the cumulative PHRED score. Duplicate ditags and linker sequences were removed as described previously (Steen et al., 2002). Only tags with a predicted accuracy of 99% were used, and statistical differences between tag abundance in different libraries were determined as described (Audic and Claverie, 1997). The libraries yielded 23,904 tags from cells grown in media without macrophages for 6hr and 38,5444 tags from cells grown in media with macrophages for 6hr. An overview of the abundance classes for both SAGE libraries is presented in Table S1 in the supplementary material, with both the number of different tag sequences and the total number of tags present in each class for the cells from th e 6h infection and the control. All libraries were normalized to 23,904 to allow direct comparisons, and the tags that appeared less than once in any given library were removed. The EST database available for strain H99 at the University of Oklahoma's Advanced Center for Genome Technology http://www.genome.ou.edu/cneo.html) was used for the preliminary assignment of tags to genes. When an EST sequence could not be identified for a particular tag, the genomic sequence for H99 at the Duke University Center for Genome Technology (http://cgt.genetics.duke.edu/data/index.html) and the Broad Institute (http://cneo.genetics.duke.edu/) was used to identify contigs with unambiguous tag assignments. Note that a limitation of the SAGE approach is that some transcripts are not detected because of low abundance and/or the absence of an NlaIII site for transcript processing. 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Dataset Information

Bombyx mori Posterior and Median Silk Gland SAGE transcriptome

Publications

Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis.

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