Selective immobilization of proteins onto solid supports through split-intein-mediated protein trans-splicing.
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Bacterial Proteins↗
Biomedical subjects
Matthew A Coleman
Publications and source records attributed to Matthew A Coleman.
Candidate protein biodosimeters of human exposure to ionizing radiation.
PURPOSE: To conduct a literature review of candidate protein biomarkers for individual radiation biodosimetry of exposure to ionizing radiation. MATERIALS AND METHODS: Reviewed approximately 300 publications (1973 - April 2006) that reported protein effects in mammalian systems after either in vivo or in vitro radiation exposure. RESULTS: We found 261 radiation-responsive proteins including 173 human proteins. Most of the studies used high doses of ionizing radiation (>4 Gy) and had no information on dose- or time-responses. The majority of the proteins showed increased amounts or changes in phosphorylation states within 24 h after exposure (range: 1.5- to 10-fold). Of the 47 proteins that are responsive at doses of 1 Gy and below, 6 showed phosphorylation changes at doses below 10 cGy. Proteins were assigned to 9 groups based on consistency of response across species, dose- and time-response information and known role in the radiation damage response. CONCLUSIONS: ATM (Ataxia telengiectasia mutated), H2AX (histone 2AX), CDKN1A (Cyclin-dependent kinase inhibitor 1A), and TP53 (tumor protein 53) are top candidate radiation protein biomarkers. Furthermore, we recommend a panel of protein biomarkers, each with different dose and time optima, to improve individual radiation biodosimetry for discriminating between low-, moderate-, and high-dose exposures. Our findings have applications for early triage and follow-up medical assessments.
Biological Assay↗
Whole-genome transcriptional analysis of chemolithoautotrophic thiosulfate oxidation by Thiobacillus denitrificans under aerobic versus denitrifying conditions.
Thiobacillus denitrificans is one of the few known obligate chemolithoautotrophic bacteria capable of energetically coupling thiosulfate oxidation to denitrification as well as aerobic respiration. As very little is known about the differential expression of genes associated with key chemolithoautotrophic functions (such as sulfur compound oxidation and CO2 fixation) under aerobic versus denitrifying conditions, we conducted whole-genome, cDNA microarray studies to explore this topic systematically. The microarrays identified 277 genes (approximately 10% of the genome) as differentially expressed using RMA (robust multiarray average) statistical analysis and a twofold cutoff. Genes upregulated (ca. 6- to 150-fold) under aerobic conditions included a cluster of genes associated with iron acquisition (e.g., siderophore-related genes), a cluster of cytochrome cbb3 oxidase genes, cbbL and cbbS (encoding the large and small subunits of form I ribulose 1,5-bisphosphate carboxylase/oxygenase, or RubisCO), and multiple molecular chaperone genes. Genes upregulated (ca. 4- to 95-fold) under denitrifying conditions included nar, nir, and nor genes (associated, respectively, with nitrate reductase, nitrite reductase, and nitric oxide reductase, which catalyze successive steps of denitrification), cbbM (encoding form II RubisCO), and genes involved with sulfur compound oxidation (including two physically separated but highly similar copies of sulfide:quinone oxidoreductase and of dsrC, associated with dissimilatory sulfite reductase). Among genes associated with denitrification, relative expression levels (i.e., degree of upregulation with nitrate) tended to decrease in the order nar > nir > nor > nos. Reverse transcription-quantitative PCR analysis was used to validate these trends.
Adaptation, Physiological↗
The genome sequence of the obligately chemolithoautotrophic, facultatively anaerobic bacterium Thiobacillus denitrificans.
The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, beta-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by beta-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.
Bacterial Proteins↗
Low-dose irradiation alters the transcript profiles of human lymphoblastoid cells including genes associated with cytogenetic radioadaptive response.
Low-dose ionizing radiation alters the gene expression profiles of mammalian cells, yet there is little understanding of the underlying cellular mechanisms responsible for these changes or of their consequences for genomic stability. We investigated the cytogenetic adaptive response of human lymphoblastoid cell lines exposed to 5 cGy (priming dose) followed by 2 Gy (challenge dose) compared to cells that received a single 2-Gy dose to (a) determine how the priming dose influences subsequent gene transcript expression in reproducibly adapting and non-adapting cell lines, and (b) identify gene transcripts that are associated with reductions in the magnitude of chromosomal damage after the challenge dose. The transcript profiles were evaluated using oligonucleotide arrays and RNA obtained 4 h after the challenge dose. A set of 145 genes (false discovery rate = 5%) with transcripts that were affected by the 5-cGy priming dose fell into two categories: (a) a set of common genes that were similarly modulated by the 5-cGy priming dose irrespective of whether the cells subsequently adapted or not and (b) genes with differential transcription in accordance with the cell lines that showed either adaptive or non-adaptive outcomes. The common priming-dose response genes showed up-regulation for protein synthesis genes and down-regulation of metabolic and signal transduction genes (>10-fold differences). The genes associated with subsequent adaptive and non-adaptive outcomes involved DNA repair, stress response, cell cycle control and apoptosis. Our findings support the importance of TP53-related functions in the control of the low-dose cytogenetic radioadaptive response and suggest that certain low-dose-induced alterations in cellular functions are predictive for the risk of subsequent genomic damage.
Adaptation, Physiological↗
Chemoselective attachment of biologically active proteins to surfaces by expressed protein ligation and its application for "protein chip" fabrication.
The present work describes a general method for the selective attachment of proteins to solid surfaces through their C-termini that can be used for the efficient creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal alpha-thioester and a modified surface containing N-terminal Cys residues. alpha-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing engineered inteins. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain using a protein microarrayer.
Cross-Linking Reagents↗
Differential basal expression of genes associated with stress response, damage control, and DNA repair among mouse tissues.
Efficient recognition and repair of DNA damage is essential for maintaining genomic integrity. Tissues and cell types within tissues appear to vary in both DNA damage susceptibilities and cancer incidences, yet the molecular mechanisms underlying these differences are not well understood. The purpose of this study was to characterize the baseline transcription profiles of selected genes involved in DNA damage recognition and repair processes among several tissues of healthy adult B6C3F1 mice (testis, brain, liver, spleen and heart), which are routinely used by the National Toxicology Program (NTP) to conduct long-term chemical carcinogenicity studies. Stress response, damage control and DNA repair-associated genes were differentially expressed among the tissues examined. Overall, stress response genes exhibited the greatest variation among tissues with the highest expression in liver and heart while DNA repair genes exhibited the least variation. Damage control genes associated with cell cycle regulation and DNA repair genes generally had the highest expression in testis. The expression levels of several genes were rank correlated with the spontaneous cancer incidences among these tissues. Variations in basal expression of DNA damage recognition and repair-associated genes among healthy tissues may contribute to their differential response to genotoxic agents and susceptibility to genetic disease.
Animals↗
Laboratory scale structural genomics.
At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.
Cloning, Molecular↗
Temporal global changes in gene expression during temperature transition in Yersinia pestis.
DNA microarrays encompassing the entire genome of Yersinia pestis were used to characterize global regulatory changes during steady-state vegetative growth occurring after shift from 26 to 37 degrees C in the presence and absence of Ca2+. Transcriptional profiles revealed that 51, 4, and 13 respective genes and open reading frames (ORFs) on pCD, pPCP, and pMT were thermoinduced and that the majority of these genes carried by pCD were downregulated by Ca2+. In contrast, Ca2+ had little effect on chromosomal genes and ORFs, of which 235 were thermally upregulated and 274 were thermally downregulated. The primary consequence of these regulatory events is profligate catabolism of numerous metabolites available in the mammalian host.
Adaptation, Physiological↗
Identification of chromatin-related protein interactions using protein microarrays.
Dynamic structural changes in chromatin are mediated by protein interactions that modulate multiple cellular processes including replication, transcription, recombination and DNA repair. Complexes that recognize chromatin are defined by several distinct groups of proteins that either directly modify histones or interact with histone-DNA complexes. A protein microarray format was used to analyze the interaction of various DNA repair proteins with chromatin components. We applied proteins, antibodies and DNA to functionalized glass slides and interrogated the slides with our proteins of interest to identify novel protein-protein interactions for proteins involved in DNA double-strand break repair. Here we demonstrate that the DNA repair protein RAD51B, and not its cognate partner RAD51C, interacts with histones and not nucleosomes. Nucleosome-specific interactions were demonstrated with the recently identified SWI/SNF protein, SMARCAL1. Unique RAD51B-histone interactions were corroborated using Far Western analysis. This is the first demonstration of an interaction between RAD51B and histone proteins that may be important for the successful repair of DNA double-strand breaks.
Blotting, Far-Western↗
Comparison of multiplexed techniques for detection of bacterial and viral proteins.
Immobilized antibody microarrays were compared to the Luminex flow cytometry system that utilizes suspensions of polystyrene microbeads covalently coupled with capture antibodies. The two immunoassays were performed for comparison of reproducibility, limits of detection and dynamic range. The Luminex system showed lower limits of detection and increased dynamic range among samples whereas the protein microarrays could be more amenable to miniaturization. Both technologies were capable of sensitive multiplexed detection.
Antibodies, Bacterial↗