Propidium monoazide (PMA) and ethidium bromide monoazide(EMA) improve DNA array and high-throughput sequencing ofporcine reproductive and respiratory syndrome virus identification

Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses.

DNA-Array-Technologie

Es wurde ein genomischer DNA-Array der Modellpflanze Arabidopsis thaliana mit einer 13.800 EST-Klone umfassenden cDNA-Bibliothek entwickelt und in der Genexpressionsanalyse der pflanzlichen Pathogenabwehr eingesetzt. Mittels PCR-Amplifikation sind 13.000 PCR-Produkte der cDNA-Fragmente hergestellt worden, mit denen 66 genomische Arabidopsis-Arrays auf Nylon und Polypropylen als Trägermaterial hergestellt werden konnten. Die Validierung mit Fluoreszenz- und Radiaktivhybridisierung sowie der Vergleich von drei Normalisierungsmethoden führte zu reproduzierbaren Ergebnissen bei hohem Korrelationskoeffizienten. Die etablierte DNA-Array-Technologie wurde zur Genexpressionsanalyse der pathogeninduzierten Abwehrmechanismen der Pflanze Arabidopsis thaliana in den ersten 24 Stunden nach Infektion mit dem avirulenten Bakterium Pseudomonas syringae pv. tomato eingesetzt. In einer Auswahl von 75 Genen der Stoffwechselwege Glycolyse, Citrat-Cyclus, Pentosephosphat-Cyclus und Glyoxylatmetabolismus konnte für 25 % der Gene, im Shikimat-, Tryptophan- und Phenylpropanoidsyntheseweg für 60 % der Gene eine erhöhte Transkriptionsrate nachgewiesen werden. Die Ergebnisse dieser Arbeit stimmen mit experimentellen Daten verschiedener unabhängiger Studien zur pflanzlichen Pathogenantwort überein. Darüberhinaus sind erstmals Transkriptionsprofile von bisher auf Transkriptionsebene nicht untersuchten Genen erstellt worden. Diese Ergebnisse bestätigen die transkriptionelle Aktivierung ganzer Stoffwechselwege und gewähren erstmals einen Einblick in die koordinierte differentielle Transkription ganzer Stoffwechselwege während der Pathogenabwehr.

Can eukaryotic cells monitor the presence of unreplicated DNA?

Completion of DNA replication before mitosis is essential for genome stability and cell viability. Cellular controls called checkpoints act as surveillance mechanisms capable of detecting errors and blocking cell cycle progression to allow time for those errors to be corrected. An important question in the cell cycle field is whether eukaryotic cells possess mechanisms that monitor ongoing DNA replication and make sure that all chromosomes are fully replicated before entering mitosis, that is whether a replication-completion checkpoint exists. From recent studies with smc5–smc6 mutants it appears that yeast cells can enter anaphase without noticing that replication in the ribosomal DNA array was unfinished. smc5–smc6 mutants are proficient in all known cellular checkpoints, namely the S phase checkpoint, DNA-damage checkpoint, and spindle checkpoint, thus suggesting that none of these checkpoints can monitor the presence of unreplicated segments or the unhindered progression of forks in rDNA. Therefore, these results strongly suggest that normal yeast cells do not contain a DNA replication-completion checkpoint.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Lineage relationship of prostate cancer cell types based on gene expression

Background: Prostate tumor heterogeneity is a major factor in disease management. Heterogeneity could be due to multiple cancer cell types with distinct gene expression. Of clinical importance is the so-called cancer stem cell type. Cell type-specific transcriptomes are used to examine lineage relationship among cancer cell types and their expression similarity to normal cell types including stem/progenitor cells. Methods: Transcriptomes were determined by Affymetrix DNA array analysis for the following cell types. Putative prostate progenitor cell populations were characterized and isolated by expression of the membrane transporter ABCG2. Stem cells were represented by embryonic stem and embryonal carcinoma cells. The cancer cell types were Gleason pattern 3 (glandular histomorphology) and pattern 4 (aglandular) sorted from primary tumors, cultured prostate cancer cell lines originally established from metastatic lesions, xenografts LuCaP 35 (adenocarcinoma phenotype) and LuCaP 49 (neuroendocrine/small cell carcinoma) grown in mice. No detectable gene expression differences were detected among serial passages of the LuCaP xenografts. Results: Based on transcriptomes, the different cancer cell types could be clustered into a luminal-like grouping and a non-luminal-like (also not basal-like) grouping. The non-luminal-like types showed expression more similar to that of stem/progenitor cells than the luminal-like types. However, none showed expression of stem cell genes known to maintain stemness. Conclusions: Non-luminal-like types are all representatives of aggressive disease, and this could be attributed to the similarity in overall gene expression to stem and progenitor cell types.

Gene expression relationship between prostate cancer cells of Gleason 3, 4 and normal epithelial cells as revealed by cell type-specific transcriptomes

Background: Prostate cancer cells in primary tumors have been typed CD10(-)/CD13(-)/CD24(hi)/CD26(+)/CD38(lo)/CD44(-)/CD104(-). This CD phenotype suggests a lineage relationship between cancer cells and luminal cells. The Gleason grade of tumors is a descriptive of tumor glandular differentiation. Higher Gleason scores are associated with treatment failure. Methods: CD26(+) cancer cells were isolated from Gleason 3+3 (G3) and Gleason 4+4 (G4) tumors by cell sorting, and their gene expression or transcriptome was determined by Affymetrix DNA array analysis. Dataset analysis was used to determine gene expression similarities and differences between G3 and G4 as well as to prostate cancer cell lines and histologically normal prostate luminal cells. Results: The G3 and G4 transcriptomes were compared to those of prostatic cell types of non-cancer, which included luminal, basal, stromal fibromuscular, and endothelial. A principal components analysis of the various transcriptome datasets indicated a closer relationship between luminal and G3 than luminal and G4. Dataset comparison also showed that the cancer transcriptomes differed substantially from those of prostate cancer cell lines. Conclusions: Genes differentially expressed in cancer are potential biomarkers for cancer detection, and those differentially expressed between G3 and G4 are potential biomarkers for disease stratification given that G4 cancer is associated with poor outcomes. Differentially expressed genes likely contribute to the prostate cancer phenotype and constitute the signatures of these particular cancer cell types.

Pipeline for macro- and microarray analyses

The pipeline for macro- and microarray analyses (PMmA) is a set of scripts with a web interface developed to analyze DNA array data generated by array image quantification software. PMmA is designed for use with single- or double-color array data and to work as a pipeline in five classes (data format, normalization, data analysis, clustering, and array maps). It can also be used as a plugin in the BioArray Software Environment, an open-source database for array analysis, or used in a local version of the web service. All scripts in PMmA were developed in the PERL programming language and statistical analysis functions were implemented in the R statistical language. Consequently, our package is a platform-independent software. Our algorithms can correctly select almost 90% of the differentially expressed genes, showing a superior performance compared to other methods of analysis. The pipeline software has been applied to 1536 expressed sequence tags macroarray public data of sugarcane exposed to cold for 3 to 48 h. PMmA identified thirty cold-responsive genes previously unidentified in this public dataset. Fourteen genes were up-regulated, two had a variable expression and the other fourteen were down-regulated in the treatments. These new findings certainly were a consequence of using a superior statistical analysis approach, since the original study did not take into account the dependence of data variability on the average signal intensity of each gene. The web interface, supplementary information, and the package source code are available, free, to non-commercial users at http://ipe.cbmeg.unicamp.br/pub/PMmA.

Cellular gene expression altered by human cytomegalovirus: Global monitoring with oligonucleotide arrays

Mechanistic insights to viral replication and pathogenesis generally have come from the analysis of viral gene products, either by studying their biochemical activities and interactions individually or by creating mutant viruses and analyzing their phenotype. Now it is possible to identify and catalog the host cell genes whose mRNA levels change in response to a pathogen. We have used DNA array technology to monitor the level of ≈6,600 human mRNAs in uninfected as compared with human cytomegalovirus-infected cells. The level of 258 mRNAs changed by a factor of 4 or more before the onset of viral DNA replication. Several of these mRNAs encode gene products that might play key roles in virus-induced pathogenesis, identifying them as intriguing targets for further study.

A genomic approach to gene fusion technology

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

Changes in global gene expression patterns during development and maturation of the rat kidney

We set out to define patterns of gene expression during kidney organogenesis by using high-density DNA array technology. Expression analysis of 8,740 rat genes revealed five discrete patterns or groups of gene expression during nephrogenesis. Group 1 consisted of genes with very high expression in the early embryonic kidney, many with roles in protein translation and DNA replication. Group 2 consisted of genes that peaked in midembryogenesis and contained many transcripts specifying proteins of the extracellular matrix. Many additional transcripts allied with groups 1 and 2 had known or proposed roles in kidney development and included LIM1, POD1, GFRA1, WT1, BCL2, Homeobox protein A11, timeless, pleiotrophin, HGF, HNF3, BMP4, TGF-α, TGF-β2, IGF-II, met, FGF7, BMP4, and ganglioside-GD3. Group 3 consisted of transcripts that peaked in the neonatal period and contained a number of retrotransposon RNAs. Group 4 contained genes that steadily increased in relative expression levels throughout development, including many genes involved in energy metabolism and transport. Group 5 consisted of genes with relatively low levels of expression throughout embryogenesis but with markedly higher levels in the adult kidney; this group included a heterogeneous mix of transporters, detoxification enzymes, and oxidative stress genes. The data suggest that the embryonic kidney is committed to cellular proliferation and morphogenesis early on, followed sequentially by extracellular matrix deposition and acquisition of markers of terminal differentiation. The neonatal burst of retrotransposon mRNA was unexpected and may play a role in a stress response associated with birth. Custom analytical tools were developed including “The Equalizer” and “eBlot,” which contain improved methods for data normalization, significance testing, and data mining.

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.

Inheritance of the CENP-A chromatin domain is spatially and temporally constrained at human centromeres.

BACKGROUND: Chromatin containing the histone variant CENP-A (CEN chromatin) exists as an essential domain at every centromere and heritably marks the location of kinetochore assembly. The size of the CEN chromatin domain on alpha satellite DNA in humans has been shown to vary according to underlying array size. However, the average amount of CENP-A reported at human centromeres is largely consistent, implying the genomic extent of CENP-A chromatin domains more likely reflects variations in the number of CENP-A subdomains and/or the density of CENP-A nucleosomes within individual subdomains. Defining the organizational and spatial properties of CEN chromatin would provide insight into centromere inheritance via CENP-A loading in G1 and the dynamics of its distribution between mother and daughter strands during replication. RESULTS: Using a multi-color protein strategy to detect distinct pools of CENP-A over several cell cycles, we show that nascent CENP-A is equally distributed to sister centromeres. CENP-A distribution is independent of previous or subsequent cell cycles in that centromeres showing disproportionately distributed CENP-A in one cycle can equally divide CENP-A nucleosomes in the next cycle. Furthermore, we show using extended chromatin fibers that maintenance of the CENP-A chromatin domain is achieved by a cycle-specific oscillating pattern of new CENP-A nucleosomes next to existing CENP-A nucleosomes over multiple cell cycles. Finally, we demonstrate that the size of the CENP-A domain does not change throughout the cell cycle and is spatially fixed to a similar location within a given alpha satellite DNA array. CONCLUSIONS: We demonstrate that most human chromosomes share similar patterns of CENP-A loading and distribution and that centromere inheritance is achieved through specific placement of new CENP-A near existing CENP-A as assembly occurs each cell cycle. The loading pattern fixes the location and size of the CENP-A domain on individual chromosomes. These results suggest that spatial and temporal dynamics of CENP-A are important for maintaining centromere identity and genome stability.

Array de DNA per identificar espècies de Fusarium: ampliació d'un array existent per incloure espècies del Sud d'Europa

Projecte de recerca elaborat a partir d’una estada al Department for Feed and Food Hygiene del National Veterinary Institute, Noruega, entre novembre i desembre del 2006. Els grans de cereal poden estar contaminats amb diferents espècies de Fusarium capaces de produir metabolits secundaris altament tòxics com trichotecenes, fumonisines o moniliformines. La correcta identificació d’aquestes espècies és de gran importància per l’assegurament del risc en l’àmbit de la salut humana i animal. La identificació de Fusarium en base a la seva morfologia requereix coneixements taxonòmics i temps; la majoria dels mètodes moleculars permeten la identificació d’una única espècie diana. Per contra, la tecnologia de microarray ofereix l’anàlisi paral•lel d’un alt nombre de DNA dianes. En aquest treball, s’ha desenvolupat un array per a la identificació de les principals espècies de Fusarium toxigèniques del Nord i Sud d’Europa. S’ha ampliat un array ja existent, per a la detecció de les espècies de Fusarium productores de trichothecene i moniliformina (predominants al Nord d’Europa), amb l’addició de 18 sondes de DNA que permeten identificar les espècies toxigèniques més abundants al Sud d’Europa, les qual produeixen majoritàriament fumonisines. Les sondes de captura han estat dissenyades en base al factor d’elongació translació- 1 alpha (TEF-1alpha). L’anàlisi de les mostres es realitza mitjançant una única PCR que permet amplificar part del TEF-1alpha seguida de la hibridació al xip de Fusarium. Els resultats es visualitzen mitjançant un mètode de detecció colorimètric. El xip de Fusarium desenvolupat pot esdevenir una eina útil i de gran interès per a l’anàlisi de cereals presents en la cadena alimentària.