GenBank

Acrobeloides maximus consistently comprised a set of different microorganisms compared to the microbiome of Caenorhabditis elegans or the metagenome of the soil sample to which nematodes were exposed. Using 16S rRNA gene amplification, the bacterial genera Ochrobactrum, Pedobacter, and Chitinophaga were identified at significant levels within the A. maximus communities only and were found to be consistently present during each of the three sampling periods over six months. We were able to isolate and grow colonies of Ochrobactrum and Pedobacter from the microbiome of the nematode on Lysogeny Broth-Miller (LB) and Yeast Extract (YE) agar plates. We were unable to isolate the associate from the genus Chitinophaga. The results obtained suggest a possible specific and persistent association between A. maximus and the two bacterial isolates we were able to culture.

With fluorescence in situ hybridization (FISH) with probes specific for Ochrobactrum and Pedobacter, we were able to stain these bacterial cells and show that they reside within the intestinal tract of A. maximus. It is possible that these gut bacteria may be mutualistic by providing A. maximus with essential metabolites, by aiding the nematode in the digestion of other bacteria, or by providing it with protection against pathogens.

Using 16S rRNA amplification and sequencing, three bacterial strains belonging to the genera Ochrobactrum, Pedobacter, and Chitinophaga were detected at significant levels within cultured Acrobeloides maximus populations exposed to soil for 24 hours before extraction and re-inoculation on agar. All three strains were found to persist in re-inoculated A. maximus cultures over a period of six months. For each strain, sequences from our A. maximus associated clones along with relevant 16S rRNA sequences from congeners or related genera available in GenBank were subjected to comprehensive phylogenetic analyses. Sequences were aligned using the multiple sequence alignment algorithms implemented in T-Coffee, MAFFT, Kalign, and WebPRANK, and phylogenetic trees were constructed using Bayesian and maximum-likelihood methods. Similarities and differences among the different alignment and tree-building methods suggest tree building methods with T-Coffee performed more poorly while maximum-likelihood methods with MAFFT and Kalign produced more mutually consistent topologies that correspond better to recognized genus boundaries. Inferences are made about the known species that are phylogenetically closest to each of our three strains. Our Ochrobactrum strains appear to be most closely related to Ochrobactrum tritici, while our Chitinophaga strains are most similar to a bacterium previously identified as “Flexibacter cf. sancti.” Our Pedobacter strains seem to be a previously undescribed sphingobacterium within the genus.

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled ...

—Public DNA databases are composed of data from many different taxa, although the taxonomic annotation on sequences is not always complete, which impedes the utilization of mined data for species-level applications. There is much ongoing work on species identification and delineation based on the molecular data itself, although applying species clustering to whole databases requires consolidation of results from numerous undefined gene regions, and introduces significant obstacles in data organization and computational load. In the current paper, we demonstrate an approach for species delineation of a sequence database. All DNA sequences for the insects were obtained and processed. After filtration of duplicated data, delineation of the database into species or molecular operational taxonomic units (MOTUs) followed a three-step process in which (i) the genetic loci L are partitioned, (ii) the species S are delineated within each locus, then (iii) species units are matched across loci to form the matrix L×S, a set of global (multilocus) species units. Partitioning the database into a set of homologous gene fragments was achieved by Markov clustering using edge weights calculated from the amount of overlap between pairs of sequences, then delineation of species units and assignment of species names were performed for the set of genes necessary to capture most of the species diversity. The complexity of computing pairwise similarities for species clustering was substantial at the cytochrome oxidase subunit I locus in particular, but made feasible through the development of software that performs pairwise alignments within the taxonomic framework, while accounting for the different ranks at which sequences are labeled with taxonomic information. Over 24 different homologs, the unidentified sequences numbered approximately 194,000, containing 41,525 species IDs (98.7% of all found in the insect database), and were grouped into 59,173 single-locus MOTUs by hierarchical clustering under parameters optimized independently for each locus. Species units from different loci were matched using a multipartite matching algorithm to form multilocus species units with minimal incongruence between loci. After matching, the insect database as represented by these 24 loci was found to be composed of 78,091 species units in total. 38,574 of these units contained only species labeled data, 34,891 contained only unlabeled data, leaving 4,626 units composed both of labeled and unlabeled sequences. In addition to giving estimates of species diversity of sequence repositories, the protocol developed here will facilitate species-level applications of modern-day sequence data sets. In particular, the L×S matrix represents a post-taxonomic framework that can be used for species-level organization of metagenomic data, and incorporation of these methods into phylogenetic pipelines will yield matrices more representative of species diversity.

Cinnamyl alcohol dehydrogenase (CAD) which catalyses the synthesis of the cinnamyl alcohols, the immediate precursors of lignins, from the corresponding cinnamaldehydes is considered to be a highly specific marker for lignification We have isolated and characterized a CAD genomic clone from eucalyptus, a woody species of economic importance. The full-length promoter (EuCAD, 2.5 kb) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) reporter gene. These constructs were tested in a homologous transient expression system of eucalyptus protoplasts which enabled the identification of several regions involved in transcriptional control. In order to study the spatial and developmental regulation of the CAD gene, the chimeric gene fusion (EuCAD-GUS) was then transferred via Agrobacterium tumefaciens-mediated transformation into poplar, an easily transformable woody angiosperm. Quantitative fluorometric assays conducted on eight independent in vitro transformants showed that GUS activity was highest in roots followed thereafter by stems and leaves. Histochemical staining for GUS activity on both in vitro primary transformants and more mature greenhouse-grown plants indicated a specific expression in the vascular tissues of stems, roots, petioles and leaves. At the onset of xylem differentiation, GUS activity was detected in parenchyma cells differentiating between the xylem-conducting elements. After secondary growth has occurred, GUS activity was localized in xylem ray cells and parenchyma cells surrounding the lignified phloem and sclerenchyma fibers. This first characterization of a woody angiosperm CAD promoter provides functional evidence for the role of CAD in lignification and suggests that parenchyma cells expressing CAD may provide lignin precursors to the adjacent lignified elements (vessels and fibres).

Identifying life stages of species with complex life histories is problematic as species are often only known and/or described from a single stage. DNA barcoding has been touted as an important tool for linking life-history stages of the same species. To test the current efficacy of DNA barcodes for identifying unknown mollusk life stages, 24 marine gastropod egg capsules were collected off the Philippines in deep water and sequenced for partial fragments of the COI, 16S and 12S mitochondrial genes. Two egg capsules of known shallow-water Mediterranean species were used to calibrate the method. These sequences were compared to those available in GenBank and the Barcode of Life Database (BOLD). Using COI sequences alone, only a single Mediterranean egg capsule was identified to species, and a single Philippine egg capsule was identified tentatively to genus; all other COI sequences recovered matches between 76% and 90% with sequences from BOLD and GenBank. Similarity-based identification using all three markers confirmed the Mediterranean specimens’ identifications. A phylogenetic approach was also implemented to confirm similarity-based identifications and provide a higher-taxonomic identification when species-level identifications were not possible. Comparison of available GenBank sequences to the diversity curve of a well-sampled coral reef habitat in New Caledonia highlights the poor taxonomic coverage achieved at present in existing genetic databases, emphasizing the need to develop DNA barcoding projects for megadiverse and often taxonomically challenging groups such as mollusks, to fully realize its potential as an identification and discovery tool.

Resequencing of the chloroplast genome (cpDNA) of Auxenochlorella protothecoides UTEX 25 was completed (GenBank Accession no. KC631634.1), revealing a genome size of 84,576 base pairs and 30.8% GC content, consistent with features reported for the previously sequenced A. protothecoides 0710, (GenBank Accession no. KC843975). The A. protothecoides UTEX 25 cpDNA encoded 78 predicted open reading frames, 32 tRNAs, and 4 rRNAs, making it smaller and more compact than the cpDNA genome of C. variabilis (124,579 bp) and C. vulgaris (150,613 bp). By comparison, the compact genome size of A. protothecoides was attributable primarily to a lower intergenic sequence content. The cpDNA coding regions of all known Chlorella species were found to be organized in conserved colinear blocks, with some rearrangements. The Auxenochlorella and Chlorella species genome structure and composition were similar, and of particular interest were genes influencing photosynthetic efficiency, i.e., chlorophyll sy...

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled ...

This study explored endophytes from three plants found in a temperate rain forest. The purpose of this study was to isolate, identify, and characterize endophytes from X. Simplicissima, L. inflata, and G. pubescens. Based on Strobel's rational for plant selection, we predicted that these Cherokee medicinal plants would harbor bio-active endophytes (Strobel 2003). With the discovery that the isolation of an endophyte producing beneficial bio-active compounds from these plants proved to be possible, these endophytes could be duplicated in mass quantities within a lab environment. This could make the acquirement and production of medicinal compounds more efficient, eco-friendly, and cheaper to obtain for use in agricultural of pharmaceutical industry.

Introduction: MDM2 is a negative regulator of the p53 tumor suppressor protein. The presence of a variety of MDM2 splice variants have been reported in many different types of tumor and some have been shown to be associated with patient prognosis.Materials and methods: In this study, we demonstrated the occurrence of MDM2 splice variants in OSCC tissues. RNA was extracted from 45 OSCC tissues and 17 normal oral mucosa tissues and reverse transcribed into cDNA. Nested PCR was performed to amplify the MDM2 transcripts and the PCR amplicons were cloned into cloning vector pTZ57R/T and sequenced.Results: We demonstrated that 37/45 OSCC and 14/17 normal oral mucosa tissues contained MDM2 splice variants and a total of 34 transcript variants were found. Comparing with the reported splice variants in GenBank, four known variants, MDM2B, MDM2C, MDM2-EU2 and MDM2-PM2 were found in tumor samples with MDM2B being the most common variants found in our samples. None of these known variants were found in normal oral mucosa tissues. The other 30 transcript variants were novel, where 3 of these were present in both tumor and normal tissues, 23 were found only in tumor tissues and 4 were exclusive to normal oral mucosa tissue.Discussion: The common loss among the variants was sequences coding for the p53 binding domain, acidic domain, nuclear localization and nuclear export domains. In general, all 30 novel transcript variants were aberrantly spliced, however, interestingly, 9/23 aberrantly spliced variants in OSCC codes for an in-frame putative protein, suggesting that despite the aberrant splicing, these transcripts may still result in a functional protein and therefore further work to study how these splice variants affect MDM2 function directly or how they may affect other binding partners is needed.