RNA extraction, probe preparation, and competitive hybridization for transcriptional profiling using Neurospora crassa long-oligomer DNA microarrays

Maternal transcripts are accumulated during oocyte growth and drive early embryonic development; therefore, their characterisation is a relevant factor for predicting fertility. DNA microarrays have been the method of choice for transcriptional profiling, but this method has some limitations when applied to domestic species because it relies upon existing knowledge about genome sequence and offers a limited quantitative evaluation. These limits are overcome by next-generation sequencing technology. The aim of the work was to define a reference standard for bovine fertility determining the list and the level of transcripts stored in fully grown oocytes collected from heifers (H) and to compare this pattern with that of adult repeat breeders (RB). Oocytes were collected by ovum pick-up (OPU) from 5 Italian Dappled Red heifers of 11 to 15 months of age that became pregnant at the following oestrus and from 4 adult cows of the same breed with an age of 4 to 7 years, classified as repeat breeders after they failed to become pregnant for a minimum of 3 consecutive AI. In both groups, oocytes were aspirated from follicles of 4 to 6 mm in diameter. Each oocyte was carefully denuded and immediately snap frozen in liquid nitrogen. Oocytes from each animal were pooled together (range 4 to 11) and analysed as a single sample. Total RNA extraction was performed by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, from both mRNA and non-polyadenylated transcripts, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (NuGEN Inc., San Carlos, CA, USA). Purified cDNA was ligated directly into an Illumina sequencing library using TruSeq DNA Sample Prep kit (Illumina Inc., San Diego, CA, USA). Sequencing was performed on Illumina HiSEqn 2000 in the 50-bp long single-read set-up, at a 4-plex of multiplexing level, producing 30 to 40 million reads per sample. Data were annotated using the cDNA ENSEMBL UMD 3.1.67 database. On average, the number of transcripts present in each sample was 15 438 ± 766 in H and 15 624 ± 768 in RB oocytes. Nineteen thousand one hundred sixty-one transcripts were detected at least in one sample, and 12 174 were detected in all samples. The comparison between H and RB showed that 598 transcripts out of 19 161 (3.12%) and 437 out of 12 174 (3.59%) are expressed at a significantly different level (P < 0.05) in the 2 groups. Taking into consideration only the transcripts detected in all the samples, with an expression rate of at least 10-fold different and a P < 0.05 we identified 39 genes. Seventeen transcripts were more abundant in RB oocytes, whereas 22 were downregulated. This is the first analysis of the oocyte transcriptome performed with deep sequencing technology. The method enabled us to compile a full list of transcripts that are found in highly competent oocytes. A direct comparison with low-quality oocytes indicated that quantitative differences of transcripts level are limited to a small subpopulation of key transcripts. Supported by PRIN 2008.

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Biofilm Formation in Pseudomonas aeruginosa: Fimbrial cup Gene Clusters Are Controlled by the Transcriptional Regulator MvaT

Journal of Bacteriology ◽

10.1128/jb.186.9.2880-2890.2004 ◽

2004 ◽

Vol 186 (9) ◽

pp. 2880-2890 ◽

Cited By ~ 102

Author(s):

Isabelle Vallet ◽

Stephen P. Diggle ◽

Rachael E. Stacey ◽

Miguel Cámara ◽

Isabelle Ventre ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Dna Microarrays ◽

Transcriptional Profiling ◽

Bacterial Pathogen ◽

Gene Clusters ◽

Northern Blotting ◽

Negative Regulator ◽

Regulatory Control ◽

Homologous Gene

ABSTRACT Pseudomonas aeruginosa is an opportunistic bacterial pathogen which poses a major threat to long-term-hospitalized patients and individuals with cystic fibrosis. The capacity of P. aeruginosa to form biofilms is an important requirement for chronic colonization of human tissues and for persistence in implanted medical devices. Various stages of biofilm formation by this organism are mediated by extracellular appendages, such as type IV pili and flagella. Recently, we identified three P. aeruginosa gene clusters that were termed cup (chaperone-usher pathway) based on their sequence relatedness to the chaperone-usher fimbrial assembly pathway in other bacteria. The cupA gene cluster, but not the cupB or cupC cluster, is required for biofilm formation on abiotic surfaces. In this study, we identified a gene (mvaT) encoding a negative regulator of cupA expression. Such regulatory control was confirmed by several approaches, including lacZ transcriptional fusions, Northern blotting, and transcriptional profiling using DNA microarrays. MvaT also represses the expression of the cupB and cupC genes, although the extent of the regulatory effect is not as pronounced as with cupA. Consistent with this finding, mvaT mutants exhibit enhanced biofilm formation. Although the P. aeruginosa genome contains a highly homologous gene, mvaU, the repression of cupA genes is MvaT specific. Thus, MvaT appears to be an important regulatory component within a complex network that controls biofilm formation and maturation in P. aeruginosa.

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In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase

Microbiology ◽

10.1099/mic.0.064675-0 ◽

2014 ◽

Vol 160 (2) ◽

pp. 243-260 ◽

Cited By ~ 10

Author(s):

Öykü İrigül-Sönmez ◽

Türkan E. Köroğlu ◽

Büşra Öztürk ◽

Ákos T. Kovács ◽

Oscar P. Kuipers ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stationary Phase ◽

Exponential Growth ◽

Dna Microarrays ◽

Antibiotic Production ◽

Transcriptional Profiling ◽

Mobility Shift ◽

Carbohydrate Utilization ◽

Altered Expression ◽

Gel Mobility Shift

The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 mono-cistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR + wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase.

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Dissecting Colony Development of Neurospora crassa Using mRNA Profiling and Comparative Genomics Approaches

Eukaryotic Cell ◽

10.1128/ec.00195-08 ◽

2008 ◽

Vol 7 (9) ◽

pp. 1549-1564 ◽

Cited By ~ 51

Author(s):

Takao Kasuga ◽

N. Louise Glass

Keyword(s):

Neurospora Crassa ◽

Transcriptional Profiling ◽

Fundamental Aspect ◽

Colony Development ◽

Published Data ◽

Middle Section ◽

Data Set ◽

Expression Of Genes ◽

Spatiotemporal Regulation ◽

Genetic Mechanisms

ABSTRACT Colony development, which includes hyphal extension, branching, anastomosis, and asexual sporulation, is a fundamental aspect of the life cycle of filamentous fungi; genetic mechanisms underlying these phenomena are poorly understood. We conducted transcriptional profiling during colony development of the model filamentous fungus Neurospora crassa, using 70-mer oligonucleotide microarrays. Relative mRNA expression levels were determined for six sections of defined age excised from a 27-h-old N. crassa colony. Functional category analysis showed that the expression of genes involved in cell membrane biosynthesis, polar growth, and cellular signaling was enriched at the periphery of the colony. The relative expression of genes involved in protein synthesis and energy production was enriched in the middle section of the colony, while sections of the colony undergoing asexual development (conidiogenesis) were enriched in expression of genes involved in protein/peptide degradation and unclassified proteins. A cross-examination of the N. crassa data set with a published data set of Aspergillus niger revealed shared patterns in the spatiotemporal regulation of gene orthologs during colony development. At present, less than 50% of genes in N. crassa have functional annotation, which imposes the chief limitation on data analysis. Using an evolutionary approach, we observed that the expression of phylogenetically conserved groups of genes was enriched in the middle section of an N. crassa colony whereas expression of genes unique to euascomycete species and of N. crassa orphan genes was enriched at the colony periphery and in the older, conidiating sections of a fungal colony.

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Production of the signalling molecule, autoinducer-2, by Neisseria meningitidis: lack of evidence for a concerted transcriptional response

Microbiology ◽

10.1099/mic.0.26185-0 ◽

2003 ◽

Vol 149 (7) ◽

pp. 1859-1869 ◽

Cited By ~ 19

Author(s):

Joanne E. Dove ◽

Kazutoyo Yasukawa ◽

Colin R. Tinsley ◽

Xavier Nassif

Keyword(s):

Quorum Sensing ◽

Neisseria Meningitidis ◽

Dna Microarrays ◽

Transcriptional Profiling ◽

Cell Signalling ◽

Transcriptional Response ◽

Autoinducer 2 ◽

Signalling Molecule ◽

A Cell ◽

Quorum Sensing Molecule

Neisseria meningitidis is a Gram-negative bacterium which is an important causative agent of septicaemia and meningitis. LuxS has been shown to be involved in the biosynthesis of a quorum sensing molecule, autoinducer-2 (AI-2), known to play a role in virulence in Escherichia coli, as well as other bacteria. Evidence that serogroup B of N. meningitidis produces AI-2, along with the observation that a luxS mutant of this strain had attenuated virulence in an infant rat model of bacteraemia, led to further investigation of the role of this quorum sensing molecule in N. meningitidis. In this study, it is demonstrated that AI-2 is not involved in regulating growth of meningococci, either in culture or in contact with epithelial cells. Furthermore, transcriptional profiling using DNA microarrays shows an absence of the concerted regulation seen in other bacteria. Taken together, these data suggest that in N. meningitidis, AI-2 may be a metabolic by-product and not a cell-to-cell signalling molecule.

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Escherichia coli Spotted Double-Strand DNA Microarrays: RNA Extraction, Labeling, Hybridization, Quality Control, and Data Management

Functional Genomics ◽

10.1385/1-59259-364-x:61 ◽

2003 ◽

pp. 61-78 ◽

Cited By ~ 12

Author(s):

Arkady B. Khodursky ◽

Jonathan A. Bernstein ◽

Brian J. Peter ◽

Virgil Rhodius ◽

Volker F. Wendisch ◽

...

Keyword(s):

Escherichia Coli ◽

Quality Control ◽

Data Management ◽

Dna Microarrays ◽

Rna Extraction ◽

Double Strand Dna

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Transcriptional profiling and localization of GUL-1, a COT-1 pathway component, in Neurospora crassa

Fungal Genetics and Biology ◽

10.1016/j.fgb.2019.01.010 ◽

2019 ◽

Vol 126 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Inbal Herold ◽

David Kowbel ◽

Diego L. Delgado-Álvarez ◽

Marisela Garduño-Rosales ◽

Rosa R. Mouriño-Pérez ◽

...

Keyword(s):

Neurospora Crassa ◽

Transcriptional Profiling

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Transcriptional Profiling of Epidermal Keratinocytes: Comparison of Genes Expressed in Skin, Cultured Keratinocytes, and Reconstituted Epidermis, Using Large DNA Microarrays

Journal of Investigative Dermatology ◽

10.1111/j.1523-1747.2003.12611.x ◽

2003 ◽

Vol 121 (6) ◽

pp. 1459-1468 ◽

Cited By ~ 49

Author(s):

Alix Gazel ◽

Patricia Ramphal ◽

Martin Rosdy ◽

Bart De wever ◽

Carine Tornier ◽

...

Keyword(s):

Dna Microarrays ◽

Transcriptional Profiling ◽

Epidermal Keratinocytes ◽

Cultured Keratinocytes

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Transcriptional Profiling of Cross Pathway Control in Neurospora crassa and Comparative Analysis of the Gcn4 and CPC1 Regulons

Eukaryotic Cell ◽

10.1128/ec.00078-07 ◽

2007 ◽

Vol 6 (6) ◽

pp. 1018-1029 ◽

Cited By ~ 59

Author(s):

Chaoguang Tian ◽

Takao Kasuga ◽

Matthew S. Sachs ◽

N. Louise Glass

Keyword(s):

Amino Acid ◽

Neurospora Crassa ◽

Regulatory Networks ◽

Transcriptional Profiling ◽

Trna Synthetase ◽

Network Evolution ◽

Amino Acid Starvation ◽

Bzip Transcription Factor ◽

Biosynthetic Genes ◽

Data Set

ABSTRACT Identifying and characterizing transcriptional regulatory networks is important for guiding experimental tests on gene function. The characterization of regulatory networks allows comparisons among both closely and distantly related species, providing insight into network evolution, which is predicted to correlate with the adaptation of different species to particular environmental niches. One of the most intensely studied regulatory factors in the yeast Saccharomyces cerevisiae is the bZIP transcription factor Gcn4p. Gcn4p is essential for a global transcriptional response when S. cerevisiae experiences amino acid starvation. In the filamentous ascomycete Neurospora crassa, the ortholog of GCN4 is called the cross pathway control-1 (cpc-1) gene; it is required for the ability of N. crassa to induce a number of amino acid biosynthetic genes in response to amino acid starvation. Here, we deciphered the CPC1 regulon by profiling transcription in wild-type and cpc-1 mutant strains with full-genome N. crassa 70-mer oligonucleotide microarrays. We observed that at least 443 genes were direct or indirect CPC1 targets; these included 67 amino acid biosynthetic genes, 16 tRNA synthetase genes, and 13 vitamin-related genes. Comparison among the N. crassa CPC1 transcriptional profiling data set and the Gcn4/CaGcn4 data sets from S. cerevisiae and Candida albicans revealed a conserved regulon of 32 genes, 10 of which are predicted to be directly regulated by Gcn4p/CPC1. The 32-gene conserved regulon comprises mostly amino acid biosynthetic genes. The comparison of regulatory networks in species with clear orthology among genes sheds light on how gene interaction networks evolve.

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Characterization of Biofilm Formation by the Bioleaching Acidophilic Bacterium Acidithiobacillus Ferrooxidans by a Microarray Transcriptome Analysis

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.71-73.175 ◽

2009 ◽

Vol 71-73 ◽

pp. 175-178 ◽

Cited By ~ 14

Author(s):

Mario A. Vera ◽

Thore Rohwerder ◽

Soeren Bellenberg ◽

Wolfgang Sand ◽

Y. Denis ◽

...

Keyword(s):

Biofilm Formation ◽

Dna Microarrays ◽

Cell Envelope ◽

Expression Patterns ◽

Rna Extraction ◽

Metal Sulfide ◽

Bacterial Attachment ◽

Leaching Bacteria ◽

Metabolic Activities ◽

Sessile Cells

Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.

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