Project description:To fully characterize the extent of genomic changes in senescent silk, we performed a time-course tissue harvesting. Material was collected at four time points: 3-, 7-, 11- and 15 days after silk emergence (DASE). Maize inbred line B104; 2 cm of basal part of silk from rings 6-10
Project description:Spider silk proteins are synthesized in the silk-producing glands, where the spidroins are produced, stored and processed into a solid fiber from a crystalline liquid solution. Despite great interest in the spider silk properties, that make this material suitable for biomedical and biotechnological applications, the mechanism of formation and spinning of the silk fibers has not been fully elucidated; and no combination of proteomic and transcriptomic study has been carried out so far in the spider silk-producing glands. Nephila clavipes is an attractive orb-web spider to investigate the spinning process of silk production, given the properties of strength, elasticity and biocompatibility of their silk fibers. Thus, considering that the combination of proteomic and transcriptomic analysis may reveal an extensive repertoire of novel proteins involved in the silk spinning process, and in order to facilitate and enable proteomics in this non-model organism, the current study aims to construct a high quality reference mRNA-derived protein database that could be used to identify tissue specific expression patterns in spider silk glands. Next-generation sequencing has offered a powerful and cost-efficient technique for the generation of transcriptomic datasets in non-model species using diverse platforms such as the Illumina HiSeq, Roche 454, Pacific Biosystems, and Applied Biosystems SOLiD; In the current study, the Illumina HiSeq 2000 platform will be used to generate a N. clavipes spider silk glands transcriptome-based protein database. The transcriptome data generated in this study will provide a comprehensive and valuable genomic resource for future research of the group of spider silk-producing glands, in order to improve our understanding of the overall mechanism of action involved in production, secretion, storage, transport, protection and conformational changes of spidroins during the spinning process, and prey capture; and the results may be relevant for scientists in material Science, biology, biochemistry, and environmental scientists.
Project description: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.
Project description:Yersinia pestis, the etiologic agent of plague, emerged as a flea-borne pathogen only within the last 6,000 years. Just five simple genetic changes in the Yersinia pseudotuberculosis progenitor, which served to eliminate toxicity to fleas and to enhance survival and biofilm formation in the flea digestive tract, were key to the transition to the arthropod-borne transmission route. To gain a deeper understanding of the genetic basis for the development of a transmissible biofilm infection in the flea foregut, we evaluated additional gene differences and performed in vivo transcriptional profiling of Y. pestis, Y. pseudotuberculosis wild-type (unable to form biofilm in the flea foregut), and a Y. pseudotuberculosis mutant strain (able to produce foregut-blocking biofilm in fleas) recovered from fleas 1 day and 14 days after an infectious bloodmeal. Surprisingly, the Y. pseudotuberculosis mutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change expression levels of the hms genes responsible for the synthesis and export of the extracellular polysaccharide matrix required for mature biofilm formation. The Y. pseudotuberculosis mutant uniquely expressed much higher levels of one of the Yersinia Type VI secretion systems (T6SS-4) in the flea, and this locus was required for flea blockage by Y. pseudotuberculosis, but not by Y. pestis. Significant differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing related genes, transcriptional regulators, and stress response genes were evident during flea infection. The results provide insights into how Y. pestis has adapted to life in its flea vector and point to evolutionary changes in the regulation of biofilm development pathways in these two closely related species
Project description:A microarray was developed to screen rodent samples for pathogens of zoonotic importance In the work described here, a homologue to Yersinia pestis was found in rodent samples after screening with the microarray A number of rodent samples from the UK and Canada were identified as carrying a homologue to a Yersinia pestis gene
Project description:Microarrays were used to determine the transcriptional profile of Y. pestis that is growing inside macrophages. J774A.1 macrophage-like cells were infected with Y. pestis KIM5 and incubated in the presence of gentamicin in tissue culture media. RNA was isolated from intracellular bacteria at various time points post infection. Control bacteria were grown for 4 hours in tissue culture medium under the same conditions without macrophages or gentamicin. The transcriptional profiles of intracellular Y. pestis at different time points were compared to those of control Y. pestis using the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute (Y. pestis microarray version 2). RNA from samples of Yersinia pestis KIM5 at 3 time points in were compared to 12 Yersinia pestis in DMEM for 4 hours.
Project description:The silk gland (SG) of the domesticated silkworm Bombyx mori, an economically important insect that has been used for silk production for over 5000 years, is a remarkable organ that produces vast amounts of silk with exceptional properties . Little is known about which SG cells execute silk protein synthesis and its precise spatiotemporal control. Here, we used single-cell RNA-seq to build a comprehensive cell atlas of the B. mori SG, consisting of 14,972 high-quality cells representing 10 distinct cell types, in three early developmental stages. We annotated all 10 cell types and determined their distributions in each region of the SG, decoded their developmental trajectory and gene-switch status, and discovered marker genes involved in the regulation of SG development and silk protein synthesis. Our study reveals the high heterogeneity of B. mori SG cells and their gene expression dynamics for the first time, affording a deeper understanding of silk-producing organs at the single-cell level .