Jason Steel

DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research

The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743–D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease.

PSI:Biology-materials repository: a biologist's resource for protein expression plasmids.

The Protein Structure Initiative:Biology-Materials Repository (PSI:Biology-MR; MR; http://psimr.asu.edu) sequence-verifies, annotates, stores, and distributes the protein expression plasmids and vectors created by the Protein Structure Initiative (PSI). The MR has developed an informatics and sample processing pipeline that manages this process for thousands of samples per month from nearly a dozen PSI centers. DNASU (http://dnasu.asu.edu), a freely searchable database, stores the plasmid annotations, which include the full-length sequence, vector information, and associated publications for over 130,000 plasmids created by our laboratory, by the PSI and other consortia, and by individual laboratories for distribution to researchers worldwide. Each plasmid links to external resources, including the PSI Structural Biology Knowledgebase (http://sbkb.org), which facilitates cross-referencing of a particular plasmid to additional protein annotations and experimental data. To expedite and simplify plasmid requests, the MR uses an expedited material transfer agreement (EP-MTA) network, where researchers from network institutions can order and receive PSI plasmids without institutional delays. As of March 2011, over 39,000 protein expression plasmids and 78 empty vectors from the PSI are available upon request from DNASU. Overall, the MR’s repository of expression-ready plasmids, its automated pipeline, and the rapid process for receiving and distributing these plasmids more effectively allows the research community to dissect the biological function of proteins whose structures have been studied by the PSI.

The ORFeome Collaboration: a genome-scale human ORF-clone resource

To the Editor: Here we describe the ORFeome Collaboration (OC) open reading frame (ORF) clone collection, created by the OC (http://www.orfeomecollaboration.org/), an international collaboration of academic and commercial groups committed to providing genome-scale clone resources for human genes via worldwide commercial and academic clone distributors. Proteins are the predominant functional modules determining the fate of cells, tissues and organisms. An encyclopedic understanding of cellular physiology requires protein expression for proteinprotein interaction screening, cellular functional screening, validation of knockout and knockdown phenotypes, and numerous other approaches. Performing such studies on individual proteins or at the proteome scale requires a comprehensive collection of human protein expression clones. Our collection comprises ORF clones (Supplementary Note) and covers 17,154 RefSeq and Ensembl genes, nearly 73% of human RefSeq genes (http://www.ncbi.nlm.nih.gov/refseq/rsg/) and 79% of the highly curated Consensus Coding DNA Sequence Project (CCDS) human genes (http://www.ncbi.nlm.nih.gov/CCDS/ CcdsBrowse.cgi) (Fig. 1a and Supplementary Data). The collection includes clones of transcript variants for 6,304 (37%) of those genes. All major functional categories of human genes are substantially represented (Fig. 1b). All clones are provided in the Gateway vector format (Life Technologies), permitting high-throughput, precise and directional transfer of ORFs to a large variety of vectors for protein expression in biological systems such as Escherichia coli, yeast and mammals or using cell-free protein expression1 (Supplementary Note). OC clones were generated primarily by PCR amplification of the ORF from full-length, sequence-verified human cDNA clones of the Mammalian Gene Collection2 or the German cDNA Consortium3; ORFs were also prepared by directed RT-PCR cloning4 or DNA synthesis2. All 5′ and 3′ untranslated regions were excluded, permitting direct expression of ORFs as fusions to aminoor carboxy-terminal polypeptides, or as native protein, after transfer to a Gatewayexpression vector1. The clones are designed to maintain the correct reading frame for both aminoand carboxy-fusion proteins. Among all genes represented in the OC collection, 64% of clones are without stop codons, 5% have stop codons, and 31% are present in both versions. Each OC clone was isolated from a single colony and is fully sequenced. Individual clone sequences have been deposited in the GenBank, EMBL and DDBJ databases. The OC website provides a searchable database with annotation of all OC clones, their respective genes, and clone confidence levels based on CCDS and RefSeq annotations (Supplementary Note) along with links to the UCSC and RIKEN browsers (http://genome.ucsc.edu/cgi-bin/hgGateway and http://fantom.gsc.riken.jp/zenbu/gLyphs/#config), which provide graphical representations of the gene structures and transcripts. OC clones are distributed via a good faith agreement, giving unrestricted clone access to all scientists worldwide. The OC website lists OC clone distributors. The value of the OC resource has been demonstrated in numerous studies covering a broad range of applications. These include large-scale binary protein-protein interaction mapping5, production of recombinant human proteins6, mapping of co-complex associations, fluorescent protein tagging for human protein localization in mammalian cells and microscopy-based functional screening of proteins, development of disease-specific protein interaction Figure 1 | RefSeq and Ensembl genes and functional gene categories represented in the OC. (a) Numbers of protein-coding genes represented in the OC collection from RefSeq (blue) and Ensembl (green) gene catalogs. The table summarizes these numbers, together with OC coverage for RefSeq-only and Ensembl-only genes. (b) Numbers of human RefSeq genes represented in the OC collection versus in the human genome, compared in nine functional categories; percentages of genes in the OC are presented above the bars. The methods used to calculate the gene numbers in each category are explained in the Supplementary Note and contrasted to the standard Gene Ontology categories. An expanded list of the top Gene Ontology categories is also provided in the Supplementary Note. The data underlying the graphs are provided as Supplementary Data. a

A versatile protein microarray platform enabling antibody profiling against denatured proteins

We aim to develop a protein microarray platform capable of presenting both natural and denatured forms of proteins for antibody biomarker discovery. We will further optimize plasma screening protocols to improve detection.

High-throughput cloning and expression library creation for functional proteomics.

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single‐gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high‐throughput proteomics platforms, such as protein microarrays and cell‐based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high‐throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and CreatorTM DNA Cloning System) and compare them side‐by‐side. We also report an example of high‐throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).

Immunoproteomic Profiling of Antiviral Antibodies in New-Onset Type 1 Diabetes Using Protein Arrays

The rapid rise in the incidence of type 1 diabetes (T1D) suggests the involvement of environmental factors including viral infections. We evaluated the association between viral infections and T1D by profiling antiviral antibodies using a high-throughput immunoproteomics approach in patients with new-onset T1D. We constructed a viral protein array comprising the complete proteomes of seven viruses associated with T1D and open reading frames from other common viruses. Antibody responses to 646 viral antigens were assessed in 42 patients with T1D and 42 age- and sex-matched healthy control subjects (mean age 12.7 years, 50% males). Prevalence of antiviral antibodies agreed with known infection rates for the corresponding virus based on epidemiological studies. Antibody responses to Epstein-Barr virus (EBV) were significantly higher in case than control subjects (odds ratio 6.6; 95% CI 2.0–25.7), whereas the other viruses showed no differences. The EBV and T1D association was significant in both sex and age subgroups (≤12 and >12 years), and there was a trend toward early EBV infections among the case subjects. These results suggest a potential role for EBV in T1D development. We believe our innovative immunoproteomics platform is useful for understanding the role of viral infections in T1D and other disorders where associations between viral infection and disease are unclear.

Exploration of panviral proteome: high-throughput cloning and functional implications in virus-host interactions.

Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies.

Draft Genome Sequence of the Filamentous Cyanobacterium Leptolyngbya sp. Strain Heron Island J, Exhibiting Chromatic Acclimation.

ABSTRACT Leptolyngbya sp. strain Heron Island is a cyanobacterium exhibiting chromatic acclimation. However, this strain has strong interactions with other bacteria, making it impossible to obtain axenic cultures for sequencing. A protocol involving an analysis of tetranucleotide frequencies, G+C content, and BLAST searches has been described for separating the cyanobacterial scaffolds from those of its cooccurring bacteria.

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins.

Response of skeletal muscle UCP2-expression during metabolic adaptation to caloric restriction

Background/objectivesSpendthrift vs. thrifty individuals expend more energy and experience greater weight loss during caloric restriction (CR). Adaptive mechanisms in skeletal muscle, adipose tissue, and on hormone level modulate energy expenditure (EE) during weight loss. Metabolic mechanisms underlying the variability in EE during CR are unclear. The present study explored whether during long-term CR (i) gene expression changes in skeletal muscle and adipose tissue relate with the individual EE response and weight loss, and (ii) altered catecholamine and FGF21-concentrations are associated with measures of metabolic adaptation.Subjects/methodsIn a 10-week inpatient study, 24-h EE was measured before and after 6 weeks of 50% CR in 12 subjects using whole-room indirect calorimetry. Weight loss was assessed and repeated hormone measurements performed. Muscle and adipose tissue biopsies were taken before and after CR, and gene expression was assessed (RNA-Seq). Genes showing the most significant changes after CR were tested for association with EE and followed-up for further association with metabolic measures in a separate phenotyping study (nu2009=u2009103).ResultsMuscle UCP2 showed the strongest change after CR (log2-fold changeu2009=u2009−1.57, false discovery rateu2009=u20090.10) and was considered the best gene for exploration of metabolic adaptive processes. A greater decrease in UCP2-expression was associated with less weight loss (Pu2009=u20090.03, ru2009=u20090.77) and relatively lower 24-h EE after CR (Pu2009=u20090.001, ru2009=u2009−0.96). Post-CR changes in FGF21-plasma concentrations correlated with UCP2-expression change (Pu2009=u20090.02, ru2009=u2009−0.89) and weight loss (Pu2009=u20090.003, ru2009=u2009−0.83). In a separate metabolic phenotyping study, muscle UCP2-expression correlated with respiratory quotient and macronutrient oxidation. In adipose tissue, no candidate genes for metabolic exploration were found.ConclusionsChanges in muscle UCP2-expression reflect an inter-individual metabolic response to long-term CR and may influence EE and weight loss via modulation of substrate oxidation.