The combination of high-density transposon-mediated mutagenesis and high-throughput sequencing has led to significant advancements in research on essential genes, resulting in a dramatic increase in the number of identified prokaryotic essential genes under diverse conditions and a revised essential-gene concept that includes all essential genomic elements, rather than focusing on protein-coding genes only. DEG 10, a new release of the Database of Essential Genes (available at http://www.essentialgene.org), has been developed to accommodate these quantitative and qualitative advancements. In addition to increasing the number of bacterial and archaeal essential genes determined by genome-wide gene essentiality screens, DEG 10 also harbors essential noncoding RNAs, promoters, regulatory sequences and replication origins. These essential genomic elements are determined not only in vitro, but also in vivo, under diverse conditions including those for survival, pathogenesis and antibiotic resistance. We have developed customizable BLAST tools that allow users to perform species- and experiment-specific BLAST searches for a single gene, a list of genes, annotated or unannotated genomes. Therefore, DEG 10 includes essential genomic elements under different conditions in three domains of life, with customizable BLAST tools.
To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the “Porteresiae” clade.
BackgroundChromosomal replication is the central event in the bacterial cell cycle. Identification of replication origins (oriCs) is necessary for almost all newly sequenced bacterial genomes. Given the increasing pace of genome sequencing, the current available software for predicting oriCs, however, still leaves much to be desired. Therefore, the increasing availability of genome sequences calls for improved software to identify oriCs in newly sequenced and unannotated bacterial genomes.ResultsWe have developed Ori-Finder, an online system for finding oriCs in bacterial genomes based on an integrated method comprising the analysis of base composition asymmetry using the Z-curve method, distribution of DnaA boxes, and the occurrence of genes frequently close to oriCs. The program can also deal with unannotated genome sequences by integrating the gene-finding program ZCURVE 1.02. Output of the predicted results is exported to an HTML report, which offers convenient views on the results in both graphical and tabular formats.ConclusionA web-based system to predict replication origins of bacterial genomes has been presented here. Based on this system, oriC regions have been predicted for the bacterial genomes available in GenBank currently. It is hoped that Ori-Finder will become a useful tool for the identification and analysis of oriCs in both bacterial and archaeal genomes.
Although mesenchymal stem cells (MSCs) have been implicated as stromal components of several cancers, their ultimate contribution to tumorigenesis and their potential to drive cancer stem cells, particularly in the unique microenvironment of human brain tumors, remains largely undefined. Consequently, using established criteria, we isolated glioma-associated-human MSCs (GA-hMSCs) from fresh human glioma surgical specimens for the first time. We show that these GA-hMSCs are nontumorigenic stromal cells that are phenotypically similar to prototypical bone marrow-MSCs. Low-passage genomic sequencing analyses comparing GA-hMSCs with matched tumor-initiating glioma stem cells (GSCs) suggest that most GA-hMSCs (60%) are normal cells recruited to the tumor (Group 1 GA-hMSCs), although, rarely (10%), GA-hMSCs may differentiate directly from GSCs (Group 2 GA-hMSCs) or display genetic patterns intermediate between these groups (Group 3 GA-hMSCs). Importantly, GA-hMSCs increase proliferation and self-renewal of GSCs in vitro, and enhance GSC tumorigenicity and mesenchymal features in vivo, confirming their functional significance within the GSC niche. These effects are mediated by GA-hMSC-secreted interleukin-6, which activates STAT3 in GSCs. Our results establish GA-hMSCs as a potentially new stromal component of gliomas that drives the aggressiveness of GSCs, and point to GA-hMSCs as a novel therapeutic target within gliomas.
Of the five echinoderm classes, only the modern sea urchins (euechinoids) generate a precociously specified embryonic micromere lineage that ingresses before gastrulation and then secretes the biomineral embryonic skeleton. The gene regulatory network (GRN) underlying the specification and differentiation of this lineage is now known. Many of the same differentiation genes as are used in the biomineralization of the embryo skeleton are also used to make the similar biomineral of the spines and test plates of the adult body. Here, we determine the components of the regulatory state upstream of these differentiation genes that are shared between embryonic and adult skeletogenesis. An abrupt ''break point'' in the micromere GRN is thus revealed, on one side of which most of the regulatory genes are used in both, and on the other side of which the regulatory apparatus is entirely micromerespecific. This reveals the specific linkages of the micromere GRN forged in the evolutionary process by which the skeletogenic gene batteries were caused to be activated in the embryonic micromere lineage. We also show, by comparison with adult skeletogenesis in the sea star, a distant echinoderm outgroup, that the regulatory apparatus responsible for driving the skeletogenic differentiation gene batteries is an ancient pleisiomorphic aspect of the echinoderm-specific regulatory heritage.embryonic and adult skeletogenesis ͉ gene network evolution ͉ sea urchin ͉ sea star
Strategies to increase rice productivity to meet the global demand have been the main concern of breeders around the world. Although a growing number of functional genes related to crop yield have been characterized, our understanding of its associated regulatory pathways is limited. Using rice as a model, we find that blocking miR396 greatly increases grain yield by modulating development of auxiliary branches and spikelets through direct induction of the growth regulating factor 6 (OsGRF6) gene. The upregulation of OsGRF6 results in the coordinated activation of several immediate downstream biological clades, including auxin (IAA) biosynthesis, auxin response factors, and branch and spikelet development-related transcription factors. This study describes a conserved microRNA (miRNA)-dependent regulatory module that integrates inflorescence development, auxin biosynthesis and signalling pathways, and could potentially be used in engineering high-yield crop plants.
INTRODUCTION Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.” RATIONALE Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies. RESULTS We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain. CONCLUSION Synthet...
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