RESUMO
The gold standard for determining the tumorigenic potential of human cancer cells is a xenotransplantation into immunodeficient mice. Higher tumorigenicity of cells is associated with earlier tumor onset. Here, we used xenotransplantation to assess the tumorigenic potential of human breast cancer cells following RNA interference-mediated inhibition of over 5000 genes. We identify 16 candidate tumor suppressors, one of which is the zinc-finger transcription factor SALL1. Analyzing this particular molecule in more detail, we show that inhibition of SALL1 correlates with reduced levels of CDH1, an important contributor to epithelial-to-mesenchymal transition. Furthermore, SALL1 expression led to an increased migration and more than twice as many cells expressing a cancer stem cell signature. Also, SALL1 expression correlates with the survival of breast cancer patients. These findings cast new light on a gene that has previously been described to be relevant during embryogenesis, but not carcinogenesis.
Assuntos
Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Antígenos CD , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Caderinas/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Intervalo Livre de Doença , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Modelos de Riscos Proporcionais , Interferência de RNA , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here, we determine the contribution of epigenetically silenced genes to the development of PDAC. We analyzed enriched, highly methylated DNAs from PDACs, chronic pancreatitis (CP) and normal tissues using CpG island microarrays and identified WNK2 as a prominent candidate tumor suppressor gene being downregulated early in PDAC development. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanIN), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in tumor than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expressions were lower in PDAC and CP compared with normal tissues both in patients and mouse models. Overexpression of WNK2 led to reduced cell growth, and WNK2 expression in tissues correlated negatively with pERK1/2 expression, a downstream target of WNK2 responsible for cell proliferation. Downregulation of WNK2 by promoter hypermethylation occurs early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway.
Assuntos
Carcinoma Ductal Pancreático/genética , Metilação de DNA/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG/genética , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/patologia , Pancreatite Crônica/genética , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/biossíntese , RNA Mensageiro/biossínteseRESUMO
BACKGROUND: Large-scale international collaboration is essential to decipher relevant information in the context of omics-scale interrogations in cancer research. This is even more important for rare and fatal diseases like pancreas cancer (PC). METHODS: The COST Action BM1204 is a unique platform to facilitate the collaboration of a broad range of European and international PC multidisciplinary research groups in order to: (1) integrate knowledge and experience in a multidisciplinary way 'from cell to society', (2) promote the application of uniform study tools and protocols, (3) foster their optimal use by early-stage researchers, (4) enhance the mobility and training of researchers, and (5) disseminate the results produced to the broader society. RESULTS: This Action will develop novel interdisciplinary tools for collaborative research to improve our understanding of PC and its prevention, diagnosis and treatment. It also aims to answer questions related to the etiology, early detection, evidence-based and personalized treatment, and health management for PC. Furthermore, the Action will contribute to new insights into PC personalized medicine and beyond as well as to the understanding of complex and rare diseases taking PC as a best practice example. The Action aims at attracting young scholars across a range of disciplines in collaboration with more experienced researchers and enhancing active European participation in the international scenario of PC research. CONCLUSION: The ultimate aim is to foster PC research in Europe and to coordinate this effort with other international initiatives to reduce disease mortality.
Assuntos
Pesquisa Biomédica , Disseminação de Informação , Cooperação Internacional , Neoplasias Pancreáticas , Saúde Pública , Pesquisa Translacional Biomédica , Europa (Continente) , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , Doenças Raras/diagnóstico , Doenças Raras/terapiaRESUMO
AIM: Breast cancer (BC) is one of the most common forms of cancer amongst females. Early diagnosis, prognosis and therapy plays crucial role in the survival of patients with breast cancer. The study was aimed on identification of potential markers for early BC diagnostics by means of genome-wide comparative analysis of gene expression in cancer and normal tissue of breast. METHODS: The analysis of gene expression in 15 invasive adenocarcinoma specimens and 15 normal breast tissue was conducted using the full-genome microarrays Sentrix HumanWD-6V3 BeadChip (Illumina). Methylation of TP53INK1 and TUSС5 promoters was interrogated by the combined bisulfite restriction analysis (COBRA). RESULTS: Analysis of gene expression in the samples of breast adenocarcinoma revealed abnormal expression of more than 2,300 genes. While genes TFF1, S100P, ERBB2, TOP2A, CDF15, HOOK1, DNAJC12, CORO2A were up-regulated in cancer, decreased expression was found for genes TUSC5, SFRP1, PPPQR1B, NTRK4, TIMP4, BARD1, AKR1C2, TP53INK1 and others. Analysis of DNA methylation of TUSC5 by COBRA revealed higher levels of exon 1 methylation (11/12) in samples of breast cancer, whereas the gene was essentially unmethylated in matched normal appearing tissue of breast (2/12). TP53INK1 gene was methylated neither in cancer nor in normalcy. CONCLUSION: A total of 149 genes exhibited the highest difference in expression in cancer versus normal appearing tissue of breast. Most prominent down-regulated candidates, TUSC5 and TP53INK1, were reported for the first time in breast cancer and may be considered as potential markers of the disease. Aberrant DNA hypermethylation of TUSC5 suggests epigenetic mechanism of cancer associated down-regulation.
Assuntos
Adenocarcinoma/genética , Neoplasias da Mama/genética , Metilação de DNA , Proteínas de Membrana/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Proteínas de Transporte/biossíntese , Regulação para Baixo , Epigênese Genética/genética , Feminino , Estudo de Associação Genômica Ampla , Proteínas de Choque Térmico/biossíntese , Humanos , Proteínas de Membrana/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma , Proteínas Supressoras de Tumor/metabolismoRESUMO
RNA interference (RNAi) screens have recently emerged as an exciting new tool for studying gene function in mammalian cells. In order to facilitate those studies, short hairpin RNA (shRNA) expression libraries covering the entire human transcriptome have become commercially available. To make use of the full potential of such large-scale shRNA libraries, microarray-based methods have been developed to analyze complex pooled RNAi screens. In terms of microarray analysis, different strategies have been pursued by different research groups, largely influenced by the employed shRNA library. In this review, we compare the three major shRNA expression libraries with a focus on their suitability for a microarray-based analysis of pooled screens. We analyze and compare approaches previously used to perform pooled RNAi screens and point out their advantages as well as limitations.
RESUMO
BACKGROUND: Although aggressive resection of pulmonary metastases prolongs the survival of patients with metastatic colorectal cancer, there is a need for predictive pathologic parameters to understand the key molecular events of metastatic progression. The aim of this study was to verify immunohistochemical markers in addition to established clinical parameters after surgery. METHODS: From our subset of patients undergoing resection of pulmonary metastases from metastatic colorectal carcinoma, we analyzed 39 patients (23 men and 16 women) between 2003 and 2007. Only patients who met the criteria for a potentially curative operation were included. All patients were analyzed with regard to age and sex, primary tumor location, stage of the primary tumor, history of hepatic metastases, number of pulmonary metastases, pre-thoracotomy carcinoembryonic (CEA) serum antigen level, and the presence of thoracic lymph node metastasis. Furthermore, we immunohistochemically investigated the expression of vascular endothelial growth factor (VEGF)-D, FBJ murine osteosarcoma viral oncogene homolog B (FOS-B), and melanoma antigen (MAGE)-A in the surgical specimens of pulmonary metastatic lesions. RESULTS: The overall 3-year survival was 50.6 %. A significantly longer survival was observed with multivariate analysis in patients with a pre-thoracotomy serum carcinoembryonic antigen level of no more than 4.2 ng/mL ( P = 0.001), and Dukes stage A or B primary tumor ( P = 0.001). A significantly longer recurrence-free survival was observed with multivariate analysis in patients without thoracic lymph node involvement compared to patients with pulmonary and/or mediastinal lymph node metastases ( P = 0.006). The stage of the primary tumor remained significant ( P = 0.029), and FOS-B expression in tumor cells showed a trend towards favorable recurrence-free survival after pulmonary metastasectomy ( P = 0.059). No statistically significant difference was found in the overall survival rate or recurrence-free survival rate of patients with expression of VEGF-D or MAGE-A antigen in pulmonary metastatic tumor cells. CONCLUSIONS: Our results suggest that in addition to clinically prognostic factors, FOS-B expression has a debatable impact on patient survival. We conclude that the evaluation of molecular and clinical prognostic parameters at the time of pulmonary metastasectomy offers a greater understanding of the metastatic process and provides important information for patient selection.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma/química , Neoplasias Colorretais/patologia , Imuno-Histoquímica , Neoplasias Pulmonares/química , Pneumonectomia/mortalidade , Proteínas Proto-Oncogênicas c-fos/análise , Idoso , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/sangue , Antígeno Carcinoembrionário/sangue , Carcinoma/mortalidade , Carcinoma/secundário , Carcinoma/cirurgia , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/cirurgia , Metástase Linfática , Masculino , Antígenos Específicos de Melanoma , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Medição de Risco , Toracotomia/mortalidade , Fatores de Tempo , Resultado do Tratamento , Fator D de Crescimento do Endotélio Vascular/análiseRESUMO
Expression profiling analysis offers great opportunities for the identification of novel molecular targets, drug discovery, development, and validation. The beauty of microarray analysis of gene expression is that it can be used to screen the expression of tens of thousands of genes in parallel and to identify appropriate molecular targets for therapeutic intervention. Toward identifying novel therapeutic options, natural products, notably from medicinal plants used in traditional Chinese medicine (TCM), have been thoroughly investigated. Increased knowledge of the molecular mechanisms of TCM-derived drugs could be achieved through application of modern molecular technologies including transcript profiling. In the present review, we introduce a brief introduction to the field of microarray technology and disclose its role in target identification and validation. Moreover, we provide examples for applications regarding molecular target discovery in medicinal plants derived TCM. This could be an attractive strategy for the development of novel and improved therapeutics.
RESUMO
UNLABELLED: Uterine leiomyoma is a most common benign neoplasm in women of reproductive age. It arises from the myometrial compartment of the uterus and may transform in some cases to a malignant phenotype. AIM: To identify the genes involved in pathogenesis of uterine leiomyoma. METHODS: We have studied differential gene expression in matched tissue samples of leiomyoma and normal myometrium from the very same people utilizing a cDNA microarray screening method. We also compared our results with previously published microarray data to identify the overlapping gene alterations. RESULTS: Based on this comparison we can divide genes deregulated in our study into two groups. The first group comprises genes that to our knowledge have not been previously reported as deregulated in fibroids: CLDN1, FGF7 (KGF), HNRPM, ISOC1, MAGEC1 (CT7), MAPK12, RFC, TIE1, TNFRSF21 (DR6). The second group consists of genes identified also in previous studies: CCND1 (BCL1), CDKN1A (P21), CRABP2, FN1 and SOX4 (EVI16). In our study FN1 was the most up-regulated gene, occupying the place between the myometrium and fibroids ranging from 2.07 to 3.64, depending of the probe molecule used for detection. CONCLUSIONS: Newly identified genes may be regarded as potential diagnostic or prognostic markers of uterine leiomyoma and thus may be very useful as new therapeutic candidates.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leiomioma/metabolismo , Miométrio/metabolismo , Neoplasias Uterinas/metabolismo , DNA Complementar/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Leiomioma/genética , Leiomioma/patologia , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologiaRESUMO
Genomic DNA methylation pattern (methylome) represents epigenetic program of a cell. It controls expression of genetic information. In tumor cells, significant alterations in DNA methylation take place, which can be identified as one of the earliest and most consistent features of tumorigenesis. Detailed survey of methylcytosines' distribution in genome is extremely important for understanding of real tumor etiology and early diagnostics. Progress in the field has been hampered by the unavailability of methods for large-scale determination of methylation patterns. Nowadays, variety of techniques is in development that allow for highly parallel regime of samples analysis (high-throughput analysis) or large loci DNA profiling (large-scale analysis). Aim of the work is to consider the main trends in the field of new methods development. The principles of the most frequently used approaches to DNA methylation studies are reviewed as well as their application and results. Most attention is paid to DNA microarrays as a technology of choice for epigenetic tumor analysis (oligonucleotide microarrays, BAC-arrays etc.). Alternative DNA sequencing based techniques are discussed, which can soon take on the leadership. Results of a large-scale analysis can be used for identification of new epigenetic markers and epigenetic classification of neoplasia.
Assuntos
Metilação de DNA , Genoma Humano/genética , Neoplasias/genética , Análise de Sequência de DNA/métodos , Animais , Humanos , Neoplasias/diagnóstico , Neoplasias/patologiaRESUMO
BACKGROUND/OBJECTIVES: Proinflammatory cytokines such as interleukin-1beta are known to be synthesized in oral gingivitis and periodontitis and lead to the activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Although numerous effects of interleukin-1beta on mesenchymal cells are known, e.g. up-regulation of intercellular adhesion molecule-1 in endothelial cells, little is known of the effects of interleukin-1beta on oral keratinocytes. The purpose of the present study was to seek interleukin-1beta-mediated alterations in mRNA gene transcription and a putative activation of NF-kappaB in oral gingival keratinocytes. METHODS: As an in vitro model for gingivitis and periodontitis, immortalized human gingival keratinocytes (IHGK) were stimulated with the proinflammatory cytokine interleukin-1beta. An epithelia-specific cDNA microarray was used to analyze mRNA expression profiles from IHGK cells treated with 200 units interleukin-1beta/ml for 3, 6, 9, 12, and 24 h. Indirect immunofluorescence was carried out to detect NF-kappaB in IHGK following interleukin-1beta treatment. RESULTS: Detailed analysis revealed distinct patterns of time-dependent changes, including genes induced or repressed early (3-6 h) or late (12-24 h) after interleukin-1beta treatment. Differentially expressed genes were involved in (i) cell stress, (ii) DNA repair, (iii) cell cycle and proliferation, (iv) anti-pathogen response, (v) extracellular matrix turnover, and (vi) angiogenesis. A large number of genes were responsive to NF-kappaB and induction was concomitant with nuclear translocation of the p65 RelA subunit of NF-kappaB. Interestingly, many of these genes contain multiple NF-kappaB binding sites in their promoters. CONCLUSION: Analysis of altered gene expression allows identification of gene networks associated with inflammatory responses. In addition to a number of well-known genes involved in gingivitis and periodontitis, we identified novel candidates that might be associated with the onset and maintenance of an inflammatory disease.
Assuntos
Gengiva/metabolismo , Gengivite/genética , Interleucina-1beta/fisiologia , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Ativação Transcricional/fisiologia , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Transformada , Citocinas/biossíntese , Enzimas Reparadoras do DNA/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Gengiva/citologia , Gengivite/metabolismo , Proteínas de Choque Térmico/biossíntese , Humanos , Metaloproteinases da Matriz/biossíntese , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regiões Promotoras Genéticas , Transporte Proteico , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
BACKGROUND: While the genome sequences for a variety of organisms are now available, the precise number of the genes encoded is still a matter of debate. For the human genome several stringent annotation approaches have resulted in the same number of potential genes, but a careful comparison revealed only limited overlap. This indicates that only the combination of different computational prediction methods and experimental evaluation of such in silico data will provide more complete genome annotations. In order to get a more complete gene content of the Drosophila melanogaster genome, we based our new D. melanogaster whole-transcriptome microarray, the Heidelberg FlyArray, on the combination of the Berkeley Drosophila Genome Project (BDGP) annotation and a novel ab initio gene prediction of lower stringency using the Fgenesh software. RESULTS: Here we provide evidence for the transcription of approximately 2,600 additional genes predicted by Fgenesh. Validation of the developmental profiling data by RT-PCR and in situ hybridization indicates a lower limit of 2,000 novel annotations, thus substantially raising the number of genes that make a fly. CONCLUSIONS: The successful design and application of this novel Drosophila microarray on the basis of our integrated in silico/wet biology approach confirms our expectation that in silico approaches alone will always tend to be incomplete. The identification of at least 2,000 novel genes highlights the importance of gathering experimental evidence to discover all genes within a genome. Moreover, as such an approach is independent of homology criteria, it will allow the discovery of novel genes unrelated to known protein families or those that have not been strictly conserved between species.
Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Genes de Insetos/fisiologia , Genoma , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Análise por Conglomerados , Biologia Computacional/métodos , Biologia Computacional/estatística & dados numéricos , Perfilação da Expressão Gênica/estatística & dados numéricos , Hibridização In Situ/métodos , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Valor Preditivo dos Testes , Pseudogenes/genética , Interferência de RNA/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.
Assuntos
Metabolismo Energético , Genoma Bacteriano , Fases de Leitura Aberta/genética , Pseudomonas putida/genética , Proteínas de Bactérias/genética , Sequência de Bases , Genes Bacterianos/genética , Dados de Sequência Molecular , Filogenia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas putida/metabolismoRESUMO
A system was establishedfor the parallel synthesis of peptide library arrays in afully automated manner Synthesis takes place in blocks made of polyoxymethylene that hold during all synthesis steps a polypropylene membrane of 8 x 12 cm. Yields are in the nanomole range, obtained at a low consumption of reagents. The current setup is based on a commercially available pipetting robot and supports the generation of 1536 different oligomers/run. Much higher array densities are possible because the membranes are amicable to spot diameters of down to 200 microm, naturally at a cost of the absolute amount produced of each oligomer The method was put to use for the creation of arrayed libraries of peptide nucleic acids (PNAs). These can be employed both as a source of PNA molecules applied individually in experimentation subsequent to their release or as intact oligomer arrays in hybridization analyses.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biblioteca de Peptídeos , Ácidos Nucleicos Peptídicos/síntese química , Sequência de Bases , Biotecnologia , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligonucleotídeos/síntese químicaRESUMO
Correspondence analysis is an explorative computational method for the study of associations between variables. Much like principal component analysis, it displays a low-dimensional projection of the data, e.g., into a plane. It does this, though, for two variables simultaneously, thus revealing associations between them. Here, we demonstrate the applicability of correspondence analysis to and high value for the analysis of microarray data, displaying associations between genes and experiments. To introduce the method, we show its application to the well-known Saccharomyces cerevisiae cell-cycle synchronization data by Spellman et al. [Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Botstein, D. & Futcher, B. (1998) Mol. Biol. Cell 9, 3273-3297], allowing for comparison with their visualization of this data set. Furthermore, we apply correspondence analysis to a non-time-series data set of our own, thus supporting its general applicability to microarray data of different complexity, underlying structure, and experimental strategy (both two-channel fluorescence-tag and radioactive labeling).
Assuntos
Interpretação Estatística de Dados , Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiae , Transcrição Gênica , Ciclo Celular , Proteínas de Ciclo Celular/genética , Saccharomyces cerevisiae/genéticaRESUMO
Gene arrays containing all currently known open reading frames of Bacillus subtilis were used to examine the general stress response of Bacillus. By proteomics, transcriptional analysis, transposon mutagenesis, and consensus promoter-based screening, 75 genes had previously been described as sigma(B)-dependent general stress genes. The present gene array-based analysis confirmed 62 of these already known general stress genes and detected 63 additional genes subject to control by the stress sigma factor sigma(B). At least 24 of these 125 sigma(B)-dependent genes seemed to be subject to a second, sigma(B)-independent stress induction mechanism. Therefore, this transcriptional profiling revealed almost four times as many regulon members as the proteomic approach, but failure of confirmation of all known members of the sigma(B) regulon indicates that even this approach has not yet elucidated the entire regulon. Most of the sigma(B)-dependent general stress proteins are probably located in the cytoplasm, but 25 contain at least one membrane-spanning domain, and at least 6 proteins appear to be secreted. The functions of most of the newly described genes are still unknown. However, their classification as sigma(B)-dependent stress genes argues that their products most likely perform functions in stress management and help to provide the nongrowing cell with multiple stress resistance. A comprehensive screening program analyzing the multiple stress resistance of mutants with mutations in single stress genes is in progress. The first results of this program, showing the diminished salt resistance of yjbC and yjbD mutants compared to that of the wild type, are presented. Only a few new sigma(B)-dependent proteins with already known functions were found, among them SodA, encoding a superoxide dismutase. In addition to analysis of the sigma(B)-dependent general stress regulon, a comprehensive list of genes induced by heat, salt, or ethanol stress in a sigma(B)-independent manner is presented. Perhaps the most interesting of the sigma(B)-independent stress phenomena was the induction of the extracytoplasmic function sigma factor sigma(W) and its entire regulon by salt shock.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Análise de Sequência com Séries de Oligonucleotídeos , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Sequência de Bases , Genoma Bacteriano , Dados de Sequência Molecular , Fator sigma/genética , Fator sigma/metabolismoRESUMO
A problem in many sequencing projects is the final closure of gaps left in the clone libraries, which serve as templates for sequencing, because of uncloned or unclonable genomic areas. By use of the Xylella fastidiosa genome as a test system, we present here an approach to generate, in a directed manner, sequence information from those gaps. We suggest using the complete clone library as a competitor against the genomic DNA of interest in a subtractive hybridization procedure similar to representational difference analysis (RDA). The resulting sequence information can be used to screen selectively other clone resources or serve directly for gap closure.
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Análise de Sequência de DNA/métodos , DNA Bacteriano/genética , Genoma Bacteriano , Biblioteca Genômica , Hibridização de Ácido Nucleico/métodos , Xanthomonas/genéticaRESUMO
Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N:-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de DNA , DNA Complementar/genética , DNA Complementar/metabolismo , Sensibilidade e EspecificidadeRESUMO
As part of the German Neurospora crassa genome project, physical clone maps of linkage groups II and V of N. crassa were generated by hybridization-based mapping. To this end, two different types of clone library were used: (1) a bacterial artificial clone library of 15-fold genome coverage and an average insert size of 69 kb, and (2) three cosmid libraries--each cloned in a different vector--with 17-fold coverage and 34 kb average insert size. For analysis, the libraries were arrayed on filters. At the first stage, chromosome-specific sublibraries were selected by hybridization of the respective chromosomal DNA fragments isolated from pulsed-field electrophoresis gels. Subsequently, the sublibraries were exhaustively ordered by single clone hybridizations. Eventually, the global libraries were used again for gap filling. By this means, physical maps were generated that consist of 13 and 21 contigs, respectively, and form the basis of the current sequencing effort on the two chromosomes.