RESUMEN
BACKGROUND: Genomic information is increasingly used in medical practice giving rise to the need for efficient analysis methodology able to cope with thousands of individuals and millions of variants. The widely used Hadoop MapReduce architecture and associated machine learning library, Mahout, provide the means for tackling computationally challenging tasks. However, many genomic analyses do not fit the Map-Reduce paradigm. We therefore utilise the recently developed SPARK engine, along with its associated machine learning library, MLlib, which offers more flexibility in the parallelisation of population-scale bioinformatics tasks. The resulting tool, VARIANTSPARK provides an interface from MLlib to the standard variant format (VCF), offers seamless genome-wide sampling of variants and provides a pipeline for visualising results. RESULTS: To demonstrate the capabilities of VARIANTSPARK, we clustered more than 3,000 individuals with 80 Million variants each to determine the population structure in the dataset. VARIANTSPARK is 80 % faster than the SPARK-based genome clustering approach, ADAM, the comparable implementation using Hadoop/Mahout, as well as ADMIXTURE, a commonly used tool for determining individual ancestries. It is over 90 % faster than traditional implementations using R and Python. CONCLUSION: The benefits of speed, resource consumption and scalability enables VARIANTSPARK to open up the usage of advanced, efficient machine learning algorithms to genomic data.
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Biología Computacional/métodos , Genotipo , Algoritmos , Análisis por Conglomerados , Humanos , Polimorfismo de Nucleótido Simple , Programas InformáticosRESUMEN
BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.
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Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética/métodos , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/metabolismo , Células HCT116 , Células HT29 , HumanosRESUMEN
Many cellular processes are carried out by large macromolecular assemblies. We systematically analyzed large macromolecular assemblies in the cytoplasm of mouse macrophages (RAW264.7 cell line), cells with crucial roles in immunity and inflammation. Fractionation of the cytoplasmic fraction was performed using sucrose density gradient centrifugation, and individual fractions were subjected in parallel to (i) identification of constituent proteins by mass spectrometry and (ii) structural visualization by electron microscopy. Macromolecular assemblies present in the fractions were analyzed by integrating available data using bioinformatic approaches. We identified 368 unique proteins in our sample. Among these are components of some well-characterized assemblies involved in diverse cellular processes and structures including translation, proteolysis, protein folding, metabolism, and the cytoskeleton, as well as less characterized proteins that may correspond to additional components of known assemblies or other homo- or hetero-oligomeric structures. Single-particle analysis of electron micrographs of negatively stained samples allowed the identification of clearly distinguishable two-dimensional projections of discrete protein assemblies. Among these, we can identify small ribosomal subunits and preribosomal particles, the 26S proteasome complex and small ringlike structures resembling the molecular chaperone complexes. In addition, a broad range of discrete and different complexes were seen at size ranges between 11 to 38 nm in diameter. Our procedure selects the assemblies on the basis of abundance and ease of isolation, and therefore provides an immediately useful starting point for further study of structure and function of large assemblies. Our results will also contribute toward building a molecular cell atlas.
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Citoplasma/metabolismo , Macrófagos/metabolismo , Proteoma/metabolismo , Animales , Línea Celular , Citoplasma/ultraestructura , Análisis de Fourier , Perfilación de la Expresión Génica , Sustancias Macromoleculares/aislamiento & purificación , Sustancias Macromoleculares/metabolismo , Macrófagos/ultraestructura , Ratones , Microscopía Electrónica de Transmisión , Peso Molecular , Análisis Multivariante , Conformación Proteica , Proteoma/química , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismoRESUMEN
Although proteins are translated on cytoplasmic ribosomes, many of these proteins play essential roles in the nucleus, mediating key cellular processes including but not limited to DNA replication and repair as well as transcription and RNA processing. Thus, understanding how these critical nuclear proteins are accurately targeted to the nucleus is of paramount importance in biology. Interaction and structural studies in the recent years have jointly revealed some general rules on the specificity determinants of the recognition of nuclear targeting signals by their specific receptors, at least for two nuclear import pathways: (i) the classical pathway, which involves the classical nuclear localization sequences (cNLSs) and the receptors importin-α/karyopherin-α and importin-ß/karyopherin-ß1; and (ii) the karyopherin-ß2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-ß2. The understanding of specificity rules allows the prediction of protein nuclear localization. We review the current understanding of the molecular determinants of the specificity of nuclear import, focusing on the importin-αâ¢cargo recognition, as well as the currently available databases and predictive tools relevant to nuclear localization. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.
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Transporte Activo de Núcleo Celular/fisiología , Señales de Localización Nuclear/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bases de Datos de Proteínas , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Proteína Relacionada con la Hormona Paratiroidea/química , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal/fisiología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/química , Proteína 2 de Unión a Elementos Reguladores de Esteroles/fisiología , alfa Carioferinas/química , alfa Carioferinas/fisiología , beta Carioferinas/química , beta Carioferinas/fisiologíaRESUMEN
The Predikin webserver allows users to predict substrates of protein kinases. The Predikin system is built from three components: a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. The Predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate-determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. The web interface provides user-friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. The server is available at http://predikin.biosci.uq.edu.au.
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Péptidos/química , Proteínas Quinasas/química , Programas Informáticos , Dominio Catalítico , Internet , Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/clasificación , Proteínas Quinasas/metabolismo , Análisis de Secuencia de Proteína , Especificidad por SustratoRESUMEN
BIOLOGY IS ENCODED IN MOLECULAR SEQUENCES: deciphering this encoding remains a grand scientific challenge. Functional regions of DNA, RNA, and protein sequences often exhibit characteristic but subtle motifs; thus, computational discovery of motifs in sequences is a fundamental and much-studied problem. However, most current algorithms do not allow for insertions or deletions (indels) within motifs, and the few that do have other limitations. We present a method, GLAM2 (Gapped Local Alignment of Motifs), for discovering motifs allowing indels in a fully general manner, and a companion method GLAM2SCAN for searching sequence databases using such motifs. glam2 is a generalization of the gapless Gibbs sampling algorithm. It re-discovers variable-width protein motifs from the PROSITE database significantly more accurately than the alternative methods PRATT and SAM-T2K. Furthermore, it usefully refines protein motifs from the ELM database: in some cases, the refined motifs make orders of magnitude fewer overpredictions than the original ELM regular expressions. GLAM2 performs respectably on the BAliBASE multiple alignment benchmark, and may be superior to leading multiple alignment methods for "motif-like" alignments with N- and C-terminal extensions. Finally, we demonstrate the use of GLAM2 to discover protein kinase substrate motifs and a gapped DNA motif for the LIM-only transcriptional regulatory complex: using GLAM2SCAN, we identify promising targets for the latter. GLAM2 is especially promising for short protein motifs, and it should improve our ability to identify the protein cleavage sites, interaction sites, post-translational modification attachment sites, etc., that underlie much of biology. It may be equally useful for arbitrarily gapped motifs in DNA and RNA, although fewer examples of such motifs are known at present. GLAM2 is public domain software, available for download at http://bioinformatics.org.au/glam2.
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Algoritmos , Secuencias de Aminoácidos , Mapeo Cromosómico/métodos , Elementos Transponibles de ADN/genética , Eliminación de Gen , Alineación de Secuencia/métodos , Análisis de Secuencia/métodos , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
BACKGROUND: We have previously described an approach to predicting the substrate specificity of serine-threonine protein kinases. The method, named Predikin, identifies key conserved substrate-determining residues in the kinase catalytic domain that contact the substrate in the region of the phosphorylation site and so determine the sequence surrounding the phosphorylation site. Predikin was implemented originally as a web application written in Javascript. RESULTS: Here, we describe a new version of Predikin, completely revised and rewritten as a modular framework that provides multiple enhancements compared with the original. Predikin now consists of two components: (i) PredikinDB, a database of phosphorylation sites that links substrates to kinase sequences and (ii) a Perl module, which provides methods to classify protein kinases, reliably identify substrate-determining residues, generate scoring matrices and score putative phosphorylation sites in query sequences. The performance of Predikin as measured using receiver operator characteristic (ROC) graph analysis equals or surpasses that of existing comparable methods. The Predikin website has been redesigned to incorporate the new features. CONCLUSION: New features in Predikin include the use of SQL queries to PredikinDB to generate predictions, scoring of predictions, more reliable identification of substrate-determining residues and putative phosphorylation sites, extended options to handle protein kinase and substrate data and an improved web interface. The new features significantly enhance the ability of Predikin to analyse protein kinases and their substrates. Predikin is available at http://predikin.biosci.uq.edu.au.
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Dominio Catalítico , Proteínas Serina-Treonina Quinasas/clasificación , Proteínas Serina-Treonina Quinasas/ultraestructura , Programas Informáticos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico/genética , Bases de Datos de Proteínas , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Análisis de Secuencia de Proteína , Especificidad por Sustrato/genéticaRESUMEN
BACKGROUND: Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed. METHODOLOGY/PRINCIPAL FINDINGS: We tested the effect of nine potential heterogenous nucleating agents on crystallization of ten test proteins in a sparse matrix screen. Several nucleating agents induced crystal formation under conditions where no crystallization occurred in the absence of the nucleating agent. Four nucleating agents: dried seaweed; horse hair; cellulose and hydroxyapatite, had a considerable overall positive effect on crystallization success. This effect was further enhanced when these nucleating agents were used in combination with each other. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the addition of heterogeneous nucleating agents increases the chances of crystal formation when using sparse matrix screens.
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Cristalografía por Rayos X/métodos , Proteínas/química , Bioquímica/métodos , Celulosa/química , Cristalización , Dextranos/química , Difusión , Durapatita/química , Modelos Biológicos , Estructura Molecular , Desnaturalización Proteica , Dióxido de Silicio/química , Solubilidad , Titanio/químicaRESUMEN
LC-MS/MS was used to identify secreted proteins in the Antarctic archaeon Methanococcoides burtonii. Seven proteins possessing a classical class 1 signal peptide were identified in the supernatant from cultures grown at 4 and 23 degrees C. The proteins included a putative S-layer cell surface protein, cell surface protein involved with cell adhesion, and trypsin-like serine protease. Protease activity was detected in the secreted fraction, and the signal peptide cleavage site of the protease was confirmed using Edman sequencing. The expression profile of putative cell surface proteins suggests a requirement for cell interactions during growth at low temperature. Sequences of the secreted proteins were used to compile a dataset containing a further 32 predicted secreted proteins from the Methanosarcinaceae. Many of these proteins were also S-layer cell surface proteins with a variety of predicted roles, particularly in cell-cell interaction. Computational analysis of signal peptides revealed a preference for lysine in the n-region, leucine in the h-region, and a eucaryal-type cleavage site, highlighting the mosaic nature of signal peptides in Archaea. This is the first study to experimentally characterize secreted proteins from a cold-adapted archaeon and provides new insight and a functional dataset for studying secretion in Archaea.
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Proteínas Arqueales/análisis , Biología Computacional/métodos , Methanosarcinaceae/química , Señales de Clasificación de Proteína/genética , Proteómica/métodos , Secuencia de Aminoácidos , Regiones Antárticas , Proteínas Arqueales/genética , Cromatografía Liquida , Espectrometría de Masas , Datos de Secuencia Molecular , Compuestos Organofosforados , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Using isotope coded affinity tag (ICAT) chromatography and liquid chromatography-mass spectrometry, 163 proteins were identified from the cold-adapted archaeon, Methanococcoides burtonii. 14 proteins were differentially expressed during growth at 4 degrees C and 23 degrees C. Knowledge of protein abundance, protein identity and gene arrangement was used to determine mechanisms of cold adaptation. Growth temperature was found to affect proteins involved in energy generation and biosynthesis linked to methanogenesis, membrane transport, transcription and protein folding, as well as affecting the expression of two hypothetical proteins. Pooling the data from this ICAT study with data from a previous two-dimensional gel electrophoresis study highlighted consistencies and differences between the two methods, and led us to conclude that the two approaches were generally complementary. This is the first report of ICAT applied to Archaea, or for the study of cold adaptation in any organism.
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Adaptación Fisiológica , Frío , Methanosarcinaceae/fisiología , Proteómica , Cromatografía de Afinidad , Espectrometría de MasasRESUMEN
Using liquid chromatography-mass spectrometry, 528 proteins were identified that are expressed during growth at 4 degrees C in the cold adapted archaeon, Methanococcoides burtonii. Of those, 135 were annotated previously as unique or conserved hypothetical proteins. We have performed a comprehensive, integrated analysis of the latter proteins using threading, InterProScan, predicted subcellular localization and visualization of conserved gene context across multiple prokaryotic genomes. Functional information was obtained for 55 proteins, providing new insight into the physiology of M. burtonii. Many of the proteins were predicted to be involved in DNA/RNA binding or modification and cell signaling, suggesting a complex, uncharacterized regulatory network controlling cellular processes during growth at low-temperature. Novel enzymatic functions were predicted for several proteins, including a putative candidate gene for the posttranslational modification of the key methanogenesis enzyme coenzyme M methyl reductase. A bacterial-like CRISPR locus was identified as a strong candidate for archaeal-bacterial lateral gene transfer. Gene context analysis proved a valuable augmentation to the other predictive methods in several cases, by revealing conserved gene associations and annotations in other microbial genomes. Our results underscore the importance of addressing the "hypothetical protein problem" for a complete understanding of cell physiology.
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Frío , Methanosarcinaceae/metabolismo , Proteoma , Adaptación Fisiológica , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Methanosarcinaceae/genética , Methanosarcinaceae/fisiología , Oxidación-ReducciónRESUMEN
UNLABELLED: We have developed a rapid, automated screening system and online database to detect foreign sequences of archaeal origin in human expressed sequence tags. The aim of the screening is to detect transcripts that may be derived from novel, putative archaeal pathogens or symbionts. AVAILABILITY: http://psychro.bioinformatics.unsw.edu.au/pathogen/index.php.
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Algoritmos , Archaea/genética , Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Almacenamiento y Recuperación de la Información/métodos , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Secuencia Conservada/genética , Evolución Molecular , Pruebas Genéticas/métodos , Genoma Arqueal , Genoma Humano , Humanos , Internet , Sistemas en Línea , Homología de Secuencia de Ácido NucleicoRESUMEN
The Pseudomonas aeruginosa dipZ gene has been cloned and sequenced. Whereas disruption of Escherichia coli dipZ (dsbD), the hydrophilic C-terminal domain of which has been deduced to be periplasmic and to function as a protein-disulfide reductase, leads to the absence of c-type cytochromes, disruption of P. aeruginosa dipZ attenuated, but did not abolish, holo-c-type cytochrome biosynthesis. Comparison of the P. aeruginosa DipZ sequence with three other DipZ sequences indicated that there are not only two conserved cysteine residues in the C-terminal hydrophilic domain, but also two more in the central highly hydrophobic domain. The latter would be located toward the centre of two of the eight membrane-spanning alpha-helices predicted to compose the hydrophobic central domain of DipZ. Both these cysteine residues, plus other transmembrane helix residues, notably prolines and glycines, are also conserved in a group of membrane proteins, related to Bacillus subtilis CcdA, which lack the N- and C-terminal hydrophilic domains of the DipZ proteins. It is proposed that DipZ of P. aeruginosa and other organisms transfers reducing power from the cytoplasm to the periplasm through reduction and reoxidation of an intramembrane disulfide bond, or other mechanism involving these cysteine residues, and that this function can also be performed by B. subtilis CcdA and other CcdA-like proteins. The failure of dipZ disruption to abolish c-type cytochrome synthesis in P. aeruginosa suggests that, in contrast to the situation in E. coli, the absence of DipZ can be compensated for by one or more other proteins, for example a CcdA-like protein acting in tandem with one or more thioredoxin-like proteins.
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Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Genes Bacterianos , Proteínas de la Membrana/genética , Proteína Disulfuro Reductasa (Glutatión)/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Grupo Citocromo c/biosíntesis , Escherichia coli/genética , Marcación de Gen , Haemophilus influenzae/genética , Datos de Secuencia Molecular , Neisseria gonorrhoeae/genética , Sistemas de Lectura Abierta , Oxidorreductasas , Fenotipo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Tiorredoxinas/genéticaRESUMEN
The gene for cytochrome cd1 nitrite reductase of Paracoccus pantotrophus, a protein of known crystal structure, is nirS. This gene is shown to be flanked by genes previously recognized in other organisms to encode proteins involved in the control of its transcription (nirI) and the biosynthesis of the d1 cofactor (nirE). Northern blot analysis has established under anaerobic conditions that a monocistronic transcript is produced from nirS, in contrast to observations with other denitrifying bacteria in which arrangement of flanking genes is different and the messages produced are polycistronic. The lack of a transcript under aerobic conditions argues against a role for cytochrome cd1 in the previously proposed aerobic denitrification pathway in Pa. pantotrophus. A putative rho-independent transcription termination sequence immediately following nirS, and preceding nirE, can be identified. The independent transcription of nirS and nirE indicates that it should be possible to produce site-directed mutants of nirS borne on a plasmid in a nirS deletion mutant. The transcript start point for nirS has been determined by two complementary techniques, 5'-RACE (Rapid amplification of cDNA 5' ends) and primer extension. It is 29 bp upstream of the AUG of nirS. An anaerobox, which presumably binds Nnr, is centred a further 41.5 bp upstream of the transcript start. No standard sigma70 DNA sequence motifs can be identified, but a conserved sequence (T-T-GIC-C-G/C-G/C) can be found in approximately the same position (-16) upstream of the transcript starts of nirS and nirI, whose products are both involved in the conversion of nitrite to nitric oxide.
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Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Nitrito Reductasas/genética , Nitrito Reductasas/metabolismo , Paracoccus/enzimología , Paracoccus/genética , Transcripción Genética , Secuencia de Bases , Northern Blotting , Citocromos , Cartilla de ADN , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Regiones Promotoras Genéticas , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
Genome sequence data of the cold-adapted archaeon, Methanococcoides burtonii, was linked to liquid chromatography-mass spectrometry analysis of the expressed-proteome to define the key biological processes functioning at 4 degrees C. 528 proteins ranging in pI from 3.5 to 13.2, and 3.5-230 kDa, were identified. 133 identities were for hypothetical proteins, and the analysis of these is described separately (Goodchild et al. manuscript in preparation). DNA replication and cell division involves eucaryotic-like histone and MC1-family DNA binding proteins, and 2 bacterial-like FtsZ proteins. Eucaryotic-like, core RNA polymerase machinery, a bacterial-like antiterminator, and numerous bacterial-like regulators enable transcription. Motility involves flagella synthesis regulated by a bacterial-like chemotaxis system. Lsmalpha and Lsmgamma were coexpressed raising the possibility of homo- and hetero-oligomeric complexes functioning in RNA processing. Expression of FKBP-type and cyclophilin-type peptidyl-prolyl cis-trans isomerases highlights the importance of protein folding, and novel characteristics of folding in the cold. Thirteen proteins from a superoperon system encoding proteasome and exosome subunits were expressed, supporting the functional interaction of transcription and translation pathways in archaea. Proteins involved in every step of methylotropic methanogenesis were identified. CO(2) appears to be fixed by a modified Calvin cycle, and by carbon monoxide dehydrogenase. Biosynthesis involves acetyl-CoA conversion to pyruvate by a non-oxidative pentose phosphate pathway, and gluconeogenesis for the conversion of pyruvate to carbohydrates. An incomplete TCA cycle may supply biosynthetic intermediates for amino acid biosynthesis. A novel finding was the expression of Tn11- and Tn12-family transposases, which has implications for genetic diversity and fitness of natural populations. Characteristics of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.
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Adaptación Fisiológica/genética , Proteínas Bacterianas/análisis , Frío , Methanococcus/química , Proteómica/métodos , Archaea/química , Archaea/genética , Archaea/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Cromatografía Líquida de Alta Presión , Genoma Bacteriano , Espectrometría de Masas , Methanococcus/genética , Methanococcus/fisiología , Operón , Proteómica/instrumentaciónRESUMEN
A global view of the biology of the cold-adapted archaeon Methanococcoides burtonii was achieved using proteomics. Proteins specific to growth at 4 degrees C versus T(opt) (23 degrees C) were identified by mass spectrometry using the draft genome sequence of M. burtonii. mRNA levels were determined for all genes identified by proteomics, and specific enzyme assays confirmed the protein expression results. Key aspects of cold adaptation related to transcription, protein folding and metabolism, including specific roles for RNA polymerase subunit E, a response regulator and peptidyl prolyl cis/trans isomerase. Heat shock protein DnaK was expressed during growth at T(opt), indicating that growth at 'optimal' temperatures was stressful for this cold-adapted organism. Expression of trimethylamine methyltransferase involves contiguous translation of two open reading frames, which is likely to result from incorporation of pyrrolysine at an amber stop codon. Thermal regulation in M. burtonii is achieved through complex gene expression events involving gene clusters and operons, through to protein modifications.
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Adaptación Fisiológica/genética , Proteínas Arqueales/biosíntesis , Frío , Regulación de la Expresión Génica Arqueal , Methanosarcinaceae/metabolismo , Proteómica/métodos , Regiones Antárticas , Proteínas Arqueales/genética , Genes Arqueales/genética , Genoma Arqueal , Methanosarcinaceae/enzimología , Methanosarcinaceae/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Most serpins irreversibly inactivate specific serine proteinases of the chymotrypsin family. Inhibitory serpins are unusual proteins in that their native structure is metastable, and rapid conversion to a relaxed state is required to trap target enzymes in a covalent complex. The evolutionary origin of the serpin fold is unresolved, and while serpins in animals are known to be involved in the regulation of a remarkable diversity of metabolic processes, the physiological functions of homologues from other phyla are unknown. Addressing these questions, here we analyze serpin genes identified in unicellular eukaryotes: the green alga Chlamydomonas reinhardtii, the dinoflagellate Alexandrium tamarense, and the human pathogens Entamoeba spp., Eimera tenella, Toxoplasma gondii, and Giardia lamblia. We compare these sequences to others, particularly those in the complete genome sequences of Archaea, where serpins were found in only 4 of 13 genera, and Bacteria, in only 9 of 56 genera. The serpins from unicellular organisms appear to be phylogenetically distinct from all of the clades of higher eukaryotic serpins. Most of the sequences from unicellular organisms have the characteristics of inhibitory serpins, and where multiple serpin genes are found in one genome, variability is displayed in the region of the reactive-center loop important for specificity. All the unicellular eukaryotic serpins have large hydrophobic or positively charged residues at the putative PI position. In contrast, none of the prokaryotic serpins has a residue of these types at the predicted P1 position, but many have smaller, neutral residues. Serpin evolution is discussed.
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Archaea/genética , Bacterias/genética , Evolución Molecular , Serpinas/química , Serpinas/genética , Secuencia de Aminoácidos , Animales , Archaea/enzimología , Bacterias/enzimología , Secuencia Conservada , Entamoeba histolytica/enzimología , Entamoeba histolytica/genética , Células Eucariotas/química , Humanos , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de Proteína , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genéticaRESUMEN
We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gln and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15 degrees -98 degrees C) were used to generate 1111 modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gln, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60 degrees C, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes from psychrophiles to hyperthermophiles.