RESUMO
Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.
Assuntos
Bactérias , Coenzimas , Euryarchaeota , Mesna , Anaerobiose , Archaea/metabolismo , Bactérias/metabolismo , Coenzimas/biossíntese , Euryarchaeota/metabolismo , Mesna/metabolismo , Metano/metabolismo , Oxirredução , Fosfatos/metabolismoRESUMO
A particular type of miniature ceramic vessel locally known as "veneneras" is occasionally found during archaeological excavations in the Maya Area. To date, only one study of a collection of such containers successfully identified organic residues through coupled chromatography-mass spectrometry methods. That study identified traces of nicotine likely associated with tobacco. Here we present a more complete picture by analyzing a suite of possible complementary ingredients in tobacco mixtures across a collection of 14 miniature vessels. The collection includes four different vessel forms and allows for the comparison of specimens which had previously formed part of museum exhibitions with recently excavated, untreated containers. Archaeological samples were compared with fresh as well as cured reference materials from two different species of tobacco (Nicotiana tabacum and N. rustica). In addition, we sampled six more plants which are linked to mind-altering practices through Mesoamerican ethnohistoric or ethnographic records. Analyses were conducted using UPLC-MS metabolomics-based analytical techniques, which significantly expand the possible detection of chemical compounds compared to previous biomarker-focused studies. Results include the detection of more than 9000 residual chemical features. We trace, for the first time, the presence of Mexican marigold (Tagetes lucida) in presumptive polydrug mixtures.
Assuntos
Cerâmica/análise , Metabolômica/métodos , Nicotiana/química , Uso de Tabaco/história , Arqueologia , Cromatografia Líquida de Alta Pressão , História Antiga , Humanos , México , Análise de Componente Principal , Espectrometria de Massas em TandemRESUMO
Residues from ancient artifacts can help identify which plant species were used for their psychoactive properties, providing important information regarding the deep-time co-evolutionary relationship between plants and humans. However, relying on the presence or absence of one or several biomarkers has limited the ability to confidently connect residues to particular plants. We describe a comprehensive metabolomics-based approach that can distinguish closely related species and provide greater confidence in species use determinations. An ~1430-year-old pipe from central Washington State not only contained nicotine, but also had strong evidence for the smoking of Nicotiana quadrivalvis and Rhus glabra, as opposed to several other species in this pre-contact pipe. Analysis of a post-contact pipe suggested use of different plants, including the introduced trade tobacco, Nicotiana rustica. Ancient residue metabolomics provides a new frontier in archaeo-chemistry, with greater precision to investigate the evolution of drug use and similar plant-human co-evolutionary dynamics.
RESUMO
ATP is not only an essential metabolite of cellular biochemistry but also acts as a signal in the extracellular milieu. In plants, extracellular ATP is monitored by the purinergic receptor P2K1. Recent studies have revealed that extracellular ATP acts as a damage-associated molecular pattern in plants, and its signaling through P2K1 is important for mounting an effective defense response against various pathogenic microorganisms. Biotrophic and necrotrophic pathogens attack plants using different strategies, to which plants respond accordingly with salicylate-based or jasmonate/ethylene-based defensive signaling, respectively. Interestingly, defense mediated by P2K1 is effective against pathogens of both lifestyles, raising the question of the level of interplay between extracellular ATP signaling and that of jasmonate, ethylene, and salicylate. To address this issue, we analyzed ATP-induced transcriptomes in wild-type Arabidopsis (Arabidopsis thaliana) seedlings and mutant seedlings defective in essential components in the signaling pathways of jasmonate, ethylene, and salicylate (classic defense hormones) as well as a mutant and an overexpression line of the P2K1 receptor. We found that P2K1 function is crucial for faithful ATP-induced transcriptional changes and that a subset of genes is more responsive in the P2K1 overexpression line. We also found that more than half of the ATP-responsive genes required signaling by one or more of the pathways for the classical defense hormones, with the jasmonate-based signaling being more critical than others. By contrast, the other ATP-responsive genes were unaffected by deficiencies in signaling for any of the classical defense hormones. These ATP-responsive genes were highly enriched for defense-related Gene Ontology terms. We further tested the ATP-induced genes in knockout mutants of transcription factors, demonstrating that MYCs acting downstream of the jasmonate receptor complex and calmodulin-binding transcription activators are nuclear transducers of P2K1-mediated extracellular ATP signaling.
Assuntos
Trifosfato de Adenosina/metabolismo , Arabidopsis/metabolismo , Transcriptoma , Arabidopsis/genética , Ciclopentanos/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Salicilatos/metabolismo , Plântula/genética , Plântula/metabolismo , Transdução de Sinais/genéticaRESUMO
Chemical analysis of residues contained in the matrix of stone smoking pipes reveal a substantial direct biomolecular record of ancient tobacco (Nicotiana) smoking practices in the North American interior northwest (Plateau), in an area where tobacco was often portrayed as a Euro-American-introduced postcontact trade commodity. Nicotine, a stimulant alkaloid and biomarker for tobacco, was identified via ultra-performance liquid chromatography-mass spectrometry in 8 of 12 analyzed pipes and pipe fragments from five sites in the Columbia River Basin, southeastern Washington State. The specimens date from 1200 cal BP to historic times, confirming the deep time continuity of intoxicant use and indigenous smoking practices in northwestern North America. The results indicate that hunting and gathering communities in the region, including ancestral Nez Perce peoples, established a tobacco smoking complex of wild (indigenous) tobacco well before the main domesticated tobacco (Nicotiana tabacum) was introduced by contact-era fur traders and settlers after the 1790s. This is the longest continuous biomolecular record of ancient tobacco smoking from a single region anywhere in the world-initially during an era of pithouse development, through the late precontact equestrian era, and into the historic period. This contradicts some ethnohistorical data indicating that kinnikinnick, or bearberry (Arctostaphylos uva-ursi) was the primary precontact smoke plant in the study area. Early use likely involved the management and cultivation of indigenous tobaccos (Nicotiana quadrivalvis or Nicotiana attenuata), species that are today exceedingly rare in the region and seem to have been abandoned as smoke plants after the entry of trade tobacco.
Assuntos
Arqueologia/métodos , Fumar Tabaco/história , Indígena Americano ou Nativo do Alasca/genética , Cromatografia Gasosa-Espectrometria de Massas/métodos , História Antiga , Humanos , Indígenas Norte-Americanos/história , Nicotina/análise , América do Norte , Nicotiana/genética , Nicotiana/metabolismo , Estados UnidosRESUMO
Porcine mammary fatty tissues represent an abundant source of natural biomaterial for generation of breast-specific extracellular matrix (ECM). Here we report the extraction of total ECM proteins from pig breast fatty tissues, the fabrication of hydrogel and porous scaffolds from the extracted ECM proteins, the structural properties of the scaffolds (tissue matrix scaffold, TMS), and the applications of the hydrogel in human mammary epithelial cell spatial cultures for cell surface receptor expression, metabolomics characterization, acini formation, proliferation, migration between different scaffolding compartments, and in vivo tumor formation. This model system provides an additional option for studying human breast diseases such as breast cancer.
Assuntos
Mama/citologia , Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Proteínas da Matriz Extracelular/química , Hidrogéis/química , Alicerces Teciduais/química , Tecido Adiposo/química , Animais , Materiais Biocompatíveis/química , Mama/química , Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Cocultura/métodos , Células Epiteliais/química , Células Epiteliais/metabolismo , Feminino , Humanos , Metaboloma , Porosidade , SuínosRESUMO
Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones.
Assuntos
Chalconas/biossíntese , Engenharia Metabólica , Plantas/genética , Plantas/metabolismo , Vias Biossintéticas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.
Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Detecção Precoce de Câncer/métodos , Fezes/química , Proteínas HMGA/genética , Metaboloma , Polipose Adenomatosa do Colo/genética , Animais , Ácidos e Sais Biliares/metabolismo , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismoRESUMO
Colorectal cancer (CRC) remains a leading cause of cancer death worldwide, despite the fact that it is a curable disease when diagnosed early. The development of new screening methods to aid in early diagnosis or identify precursor lesions at risk for progressing to CRC will be vital to improving the survival rate of individuals predisposed to CRC. Metabolomics is an advancing area that has recently seen numerous applications to the field of cancer research. Altered metabolism has been studied for many years as a means to understand and characterize cancer. However, further work is required to establish standard procedures and improve our ability to identify distinct metabolomic profiles that can be used to diagnose CRC or predict disease progression. The present study demonstrates the use of direct infusion traveling wave ion mobility mass spectrometry to distinguish metabolic profiles from CRC samples and matched non-neoplastic epithelium as well as metastatic and primary tumors at different stages of disease (T1-T4). By directly infusing our samples, the analysis time was reduced significantly, thus increasing the speed and efficiency of this method compared to traditional metabolomics platforms. Partial least squares discriminant analysis was used to visualize differences between the metabolic profiles of sample types and to identify the specific m/z features that led to this differentiation. Identification of the distinct m/z features was made using the human metabolome database. We discovered alterations in fatty acid biosynthesis and oxidative, glycolytic, and polyamine pathways that distinguish tumors from non-malignant colonic epithelium as well as various stages of CRC. Although further studies are needed, our results indicate that colonic epithelial cells undergo metabolic reprogramming during their evolution to CRC, and the distinct metabolites could serve as diagnostic tools or potential targets in therapy or primary prevention. Graphical Abstract Colon tissue biopsy samples were collected from patients after which metabolites were extracted via sonication. Two-dimensional data were collected via IMS in tandem with MS (IMMS). Data were then interpreted statistically via PLS-DA. Scores plots provided a visualization of statistical separation and groupings of sample types. Loading plots allowed identification of influential ion features. Lists of these features were exported and analyzed for specific differences. Direct comparisons of the ion features led to the identification and comparative analyses of candidate biomarkers. These differences were then expressed visually in charts and tables.
Assuntos
Neoplasias Colorretais/metabolismo , Cromatografia Líquida/métodos , Neoplasias Colorretais/patologia , Humanos , Espectrometria de Massas/métodos , Metástase NeoplásicaRESUMO
Although significant progress has been made in the diagnosis and treatment of colorectal cancer (CRC), it remains a leading cause of cancer death worldwide. Early identification and removal of polyps that may progress to overt CRC is the cornerstone of CRC prevention. Expression of the High Mobility Group A1 (HMGA1) gene is significantly elevated in CRCs as compared with adjacent, nonmalignant tissues. We investigated metabolic aberrations induced by HMGA1 overexpression in small intestinal and colonic epithelium using traveling wave ion mobility mass spectrometry (TWIMMS) in a transgenic model in which murine Hmga1 was misexpressed in colonic epithelium. To determine if these Hmga1-induced metabolic alterations in mice were relevant to human colorectal carcinogenesis, we also investigated tumors from patients with CRC and matched, adjacent, nonmalignant tissues. Multivariate statistical methods and manual comparisons were used to identify metabolites specific to Hmga1 and CRC. Statistical modeling of data revealed distinct metabolic patterns in Hmga1 transgenics and human CRC samples as compared with the control tissues. We discovered that 13 metabolites were specific for Hmga1 in murine intestinal epithelium and also found in human CRC. Several of these metabolites function in fatty acid metabolism and membrane composition. Although further validation is needed, our results suggest that high levels of HMGA1 protein drive metabolic alterations that contribute to CRC pathogenesis through fatty acid synthesis. These metabolites could serve as potential biomarkers or therapeutic targets.
Assuntos
Polipose Adenomatosa do Colo/fisiopatologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/patologia , Proteína HMGA1a/fisiologia , Mucosa Intestinal/patologia , Neoplasias Colorretais/metabolismo , Proteína HMGA1a/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Espectrometria de Massas em TandemRESUMO
The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3µM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,ß-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds.
Assuntos
Chalconas/metabolismo , Malus , NADP/metabolismo , Acil Coenzima A/metabolismo , Arabidopsis/metabolismo , Clonagem de Organismos , Cumarínicos/química , Cumarínicos/metabolismo , Flores/genética , Flores/metabolismo , Malus/química , Malus/enzimologia , Malus/genética , Malus/metabolismo , Estrutura Molecular , Oxirredutases/metabolismo , Polifenóis/químicaRESUMO
Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum 'Folva') and their proteome investigated. Proteins were resolved by one-dimensional gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different search programs, a total of 1,060 nonredundant proteins were identified in a quantitative manner using normalized spectral counts including as many as 5-fold more "extreme" proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression spanned 1,800-fold and included nearly all components of the electron transport chain, tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of prominent posttranslational modifications revealed that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms.
Assuntos
Mitocôndrias/metabolismo , Tubérculos/metabolismo , Proteoma/metabolismo , Solanum tuberosum/metabolismo , Arabidopsis/metabolismo , Cromatografia Líquida , Bases de Dados de Proteínas , Fluorescência , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Peptídeos/metabolismo , Epiderme Vegetal/citologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteoma/química , Frações Subcelulares/metabolismo , Espectrometria de Massas em Tandem , Nicotiana/citologiaRESUMO
The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-ß-bisabolene synthase from ginger rhizome, and α-humulene synthase and ß-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (-)-caryolan-1-ol synthase and α-zingiberene/ß-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-ß-turmerone, are produced from (-)-α-zingiberene and (-)-ß-sesquiphellandrene, respectively, via α-zingiberene/ß-sesquiphellandrene oxidase and a still unidentified dehydrogenase.
Assuntos
Alquil e Aril Transferases/fisiologia , Curcuma/metabolismo , Extratos Vegetais/farmacologia , Terpenos/química , Zingiber officinale/metabolismo , Alquil e Aril Transferases/química , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular , Códon , Primers do DNA/genética , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Etiquetas de Sequências Expressas , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Modelos Químicos , Conformação Molecular , Filogenia , Extratos Vegetais/química , Rizoma/química , Temperatura , Leveduras/metabolismoRESUMO
Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5µM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC-MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.
Assuntos
Ácido Benzoico/farmacologia , Bryopsida/enzimologia , Metiltransferases/química , Proteínas de Plantas/química , Compostos de Sulfidrila/química , Sequência de Aminoácidos , Bryopsida/classificação , Bryopsida/genética , Ativação Enzimática , Escherichia coli/química , Escherichia coli/genética , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Metilação , Metiltransferases/genética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Nicotiana/efeitos dos fármacos , Nicotiana/enzimologia , Nicotiana/genéticaRESUMO
BACKGROUND: The template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo. RESULTS: In this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background. CONCLUSIONS: Our results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection.
Assuntos
DNA Complementar/biossíntese , Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA/metabolismo , Análise de Sequência de DNA/métodos , Sequência de Bases , DNA Complementar/genética , DNA Complementar/metabolismo , Biblioteca Gênica , Isomerismo , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/enzimologia , Oligonucleotídeos/química , Oligonucleotídeos/genética , Células Vegetais , Plantas/genética , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.