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Dan Nettleton

Publications and source records attributed to Dan Nettleton.

At least 19 recordsLinked to original sources
PubMed2006

Gene expression profiling in Salmonella Choleraesuis-infected porcine lung using a long oligonucleotide microarray.

Understanding the transcriptional response to pathogenic bacterial infection within food animals is of fundamental and applied interest. To determine the transcriptional response to Salmonella enterica serovar Choleraesuis (SC) infection, a 13,297-oligonucleotide swine array was used to analyze RNA from control, 24-h postinoculation (hpi), and 48-hpi porcine lung tissue from pigs infected with SC. In total, 57 genes showed differential expression (p < 0.001; false discovery rate = 12%). Quantitative real-time PCR (qRT-PCR) of 61 genes was used to confirm the microarray results and to identify pathways responding to infection. Of the 33 genes identified by microarray analysis as differentially expressed, 23 were confirmed by qRT-PCR results. A novel finding was that two transglutaminase family genes (TGM1 and TGM3) showed dramatic increases in expression postinoculation; combined with several other apoptotic genes, they indicated the induction of apoptotic pathways during SC infection. A predominant T helper 1-type immune response occurred during infection, with interferon gamma (IFNG) significantly increased at 48 hpi. Genes induced by IFNs (GBP1, GBP2, C1S, C1R, MHC2TA, PSMB8, TAP1, TAP2) showed increased expression during porcine lung infection. These data represent the first thorough investigation of gene regulation pathways that control an important porcine respiratory and foodborne bacterial infection.

Animals↗
PubMed2006

Scanning microarrays at multiple intensities enhances discovery of differentially expressed genes.

MOTIVATION: Scanning parameters are often overlooked when optimizing microarray experiments. A scanning approach that extends the dynamic data range by acquiring multiple scans of different intensities has been developed. RESULTS: Data from each of three scan intensities (low, medium, high) were analyzed separately using multiple scan and linear regression approaches to identify and compare the sets of genes that exhibit statistically significant differential expression. In the multiple scan approach only one-third of the differentially expressed genes were shared among the three intensities, and each scan intensity identified unique sets of differentially expressed genes. The set of differentially expressed genes from any one scan amounted to < 70% of the total number of genes identified in at least one scan. The average signal intensity of genes that exhibited statistically significant changes in expression was highest for the low-intensity scan and lowest for the high-intensity scan, suggesting that low-intensity scans may be best for detecting expression differences in high-signal genes, while high-intensity scans may be best for detecting expression differences in low-signal genes. Comparison of the differentially expressed genes identified in the multiple scan and linear regression approaches revealed that the multiple scan approach effectively identifies a subset of statistically significant genes that linear regression approach is unable to identify. Quantitative RT-PCR (qRT-PCR) tests demonstrated that statistically significant differences identified at all three scan intensities can be verified. AVAILABILITY: The data presented can be viewed at http://www.ncbi.nlm.nih.gov/geo/ under GEO accession no. GSE3017.

Gene Expression Profiling↗
PubMed2006

A novel approach for characterizing expression levels of genes duplicated by polyploidy.

Studying gene expression in polyploids is complicated by genomewide gene duplication and the problem of distinguishing transcript pools derived from each of the two homeologous genomes such as the A- and D-genomes of allotetraploid Gossypium. Short oligonucleotide probes designed to specifically target several hundred homeologous gene pairs of Gossypium were printed on custom NimbleGen microarrays. These results demonstrate that relative expression levels of homeologous genes may be measured by microarrays and that deviation from equal expression levels of homeologous loci may be common in the allotetraploid nucleus of Gossypium.

Base Sequence↗
PubMed2006

Gene expression programs during shoot, root, and callus development in Arabidopsis tissue culture.

Shoots can be regenerated from Arabidopsis (Arabidopsis thaliana) root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium. Regenerating tissues can be directed along different developmental pathways leading to the formation of shoots, new roots, or callus by transferring to the appropriate organ induction medium. Using gene-profiling methods, we identified groups of genes that serve as molecular signatures of the different developmental processes, i.e. genes that were specifically up- or down-regulated on one developmental pathway, but not on others. One transcription factor gene that was up-regulated during early shoot development was RAP2.6L (At5g13330), a member of the ERF (ethylene response factor) subfamily B-4 of the ERF/APETALA2 transcription factor gene family. RAP2.6L functions in shoot regeneration because T-DNA knockdown mutations in the gene reduced the efficiency of shoot formation in tissue culture, but not normal embryo or seedling development. RAP2.6L promoter:beta-glucuronidase fusions demonstrated that the up-regulation of the gene during shoot regeneration was, at least in part, transcriptionally controlled. The promoter:beta-glucuronidase fusions also demonstrated that RAP2.6L expression was localized to the shoot and emerging leaves, but expression declined in the leaf lamina as leaves expanded. T-DNA knockdown mutations in RAP2.6L reduced the expression of many genes that are normally up-regulated during shoot development including CUP-SHAPED COTYLEDON2 that is involved in shoot meristem specification. Thus, RAP2.6L appears to be part of a network involved in regulating the expression of many other genes in shoot regeneration.

Arabidopsis↗
PubMed2006

All possible modes of gene action are observed in a global comparison of gene expression in a maize F1 hybrid and its inbred parents.

Heterosis is the phenomenon whereby the progeny of particular inbred lines have enhanced agronomic performance relative to both parents. Although several hypotheses have been proposed to explain this fundamental biological phenomenon, the responsible molecular mechanisms have not been determined. The maize inbred lines B73 and Mo17 produce a heterotic F1 hybrid. Global patterns of gene expression were compared in seedlings of these three genotypes by using a microarray that contains 13,999 cDNAs. Using an estimated 15% false discovery rate as a cutoff, 1,367 ESTs (9.8%) were identified as being significantly differentially expressed among genotypes. All possible modes of gene action were observed, including additivity, high- and low-parent dominance, underdominance, and overdominance. The largest proportion of the ESTs (78%; 1,062 of 1,367) exhibited expression patterns that are not statistically distinguishable from additivity. Even so, 22% of the differentially regulated ESTs exhibited nonadditive modes of gene expression. Classified on the basis of significant pairwise comparisons of genotype means, 181 of these 305 nonadditive ESTs exhibited high-parent dominance and 23 exhibited low-parent dominance. In addition, 44 ESTs exhibited underdominance or overdominance. These findings are consistent with the hypothesis that multiple molecular mechanisms, including overdominance, contribute to heterosis.

Expressed Sequence Tags↗
PubMed2006

Transcriptional profiling of myostatin-knockout mice implicates Wnt signaling in postnatal skeletal muscle growth and hypertrophy.

Myostatin is an inhibitor of skeletal muscle growth. Disruption of the Mstn gene in mice results in muscles that weigh two to three times those of controls, but precisely how myostatin signals to exert its effects on muscle is unclear. We used the Affymetrix GeneChip system to identify differences in gene expression between myostatin null and wild-type mice. The results indicated a switch in muscle fiber type, from slow to fast, in the absence of myostatin. They also indicated that myostatin may act upstream of Wnt pathway components. Notably, it repressed expression of Wnt4. Wnt4 was capable of stimulating satellite cell proliferation, while inhibition of Wnt signaling down-regulated satellite cell proliferation. This evidence points to a role for Wnt/calcium signaling in the growth and maintenance of postnatal skeletal muscle. This study offers new insight into potential downstream targets of myostatin, which will be beneficial for elucidation of the mechanism through which myostatin acts to inhibit muscle growth.

Animals↗
PubMed2006

Stage-specific suppression of basal defense discriminates barley plants containing fast- and delayed-acting Mla powdery mildew resistance alleles.

Nonspecific recognition of pathogen-derived general elicitors triggers the first line of plant basal defense, which in turn, preconditions the host towards resistance or susceptibility. To elucidate how basal defense responses influence the onset of Mla (mildew resistance locus a)-specified resistance, we performed a meta-analysis of GeneChip mRNA expression for 155 basal defense-related genes of barley (Hordeum vulgare) challenged with Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. In plants containing the fast-acting Mla1, Mla6, or Mla13 alleles, transcripts hyper-accumulated from 0 to 16 h after inoculation (hai) in both compatible and incompatible interactions. Suppression of basal defense-related transcripts was observed after 16 hai only in compatible interactions, whereas these transcripts were sustained or increased in incompatible interactions. By contrast, in plants containing wild-type and mutants of the delayed-acting Mla12 allele, an early hyper-induction of transcripts from 0 to 8 hai was observed, but the expression of many of these genes is markedly suppressed from 8 to 16 hai. These results suggest that the inhibition of basal defense facilitates the development of haustoria by the pathogen, consequently delaying the onset of host resistance responses. Thus, we hypothesize that the regulation of basal defense influences host-cell accessibility to the fungal pathogen and drives allelic diversification of gene-specific resistance phenotypes.

Alleles↗
PubMed2006

Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays.

Mycoplasma hyopneumoniae, the causative agent of swine enzootic pneumonia and a major component of the porcine respiratory disease complex, continues to confound swine producers despite control programmes worldwide. The disease is chronic and self-limiting, but the host is subject to immunopathological changes that potentiate respiratory disease associated with other pathogens. The response of M. hyopneumoniae to environmental stress is of interest because of its relevance to virulence mechanisms in other bacterial pathogens. One of these stressors, iron deprivation, is a prominent feature of the host innate immune response, and most certainly impacts growth of mycoplasmas in vivo. To study this, microarray technology was applied to the transcriptome analysis of M. hyopneumoniae during iron deprivation. An array consisting of 632 of the 698 ORFs in the genome was used to compare the mRNA isolated from organisms grown under normal laboratory conditions with that from organisms subjected to iron deprivation with the chelator 2,2'-dipyridyl. This analysis identified 27 genes that were either up- or down-regulated in response to low-iron growth conditions (P<0.01), with an estimated false discovery rate below 10 %. These included genes encoding transport proteins, enzymes involved in energy metabolism, and components of the translation process. Ten of the 27 identified genes had no assigned function. These studies indicate that M. hyopneumoniae can respond to changes in environmental conditions, but the mechanism employed remains unknown.

2,2'-Dipyridyl↗
PubMed2006

Identifying genes associated with a quantitative trait or quantitative trait locus via selective transcriptional profiling.

Genetical genomics is an approach that blends the mapping of quantitative trait loci (QTL) with microarray analysis. The approach can be used to identify associations between the allelic state of a genomic region and a gene's transcript abundance. However, the large number of microarrays required for adequate power results in high material and labor costs that prevent wide adoption of the genetical genomics strategy outside of some well-funded laboratories. We present a method called selective transcriptional profiling that involves selecting an optimal subset of individuals to microarray from a larger set of individuals for which relatively inexpensive quantitative trait and molecular marker data are available. We show how to use microarray data from the selected individuals, along with the trait and marker data from all individuals, to identify genes whose transcript abundance is associated with a quantitative trait of interest through linkage to a trait QTL or correlation with the trait. Our methods for selection and analysis are derived within a missing data framework.

Alleles↗
PubMed2006

Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays.

Bacterial pathogens undergo stress during host colonization and disease processes. These stresses result in changes in gene expression to compensate for potentially lethal environments developed in the host during disease. Mycoplasma hyopneumoniae colonizes the swine epithelium and causes a pneumonia that predisposes the host to enhanced disease from other pathogens. How M. hyopneumoniae responds to changing environments in the respiratory tract during disease progression is not known. In fact, little is known concerning the capabilities of mycoplasmas to respond to changing growth environments. With limited genes, mycoplasmas are thought to possess only a few mechanisms for gene regulation. A microarray consisting of 632 of the 698 open reading frames of M. hyopneumoniae was constructed and used to study gene expression differences during a temperature shift from 37 degrees C to 42 degrees C, a temperature swing that might be encountered during disease. To enhance sensitivity, a unique hexamer primer set was employed for generating cDNA from only mRNA species. Our analysis identified 91 genes that had significant transcriptional differences in response to heat shock conditions (P < 0.01) with an estimated false-discovery rate of 4 percent. Thirty-three genes had a change threshold of 1.5-fold or greater. Many of the heat shock proteins previously characterized in other bacteria were identified as significant in this study as well. A proportion of the identified genes (54 of 91) currently have no assigned function.

Gene Expression Profiling↗
PubMed2005

Isolation, characterization, and pericycle-specific transcriptome analyses of the novel maize lateral and seminal root initiation mutant rum1.

The monogenic recessive maize (Zea mays) mutant rootless with undetectable meristems 1 (rum1) is deficient in the initiation of the embryonic seminal roots and the postembryonic lateral roots at the primary root. Lateral root initiation at the shoot-borne roots and development of the aerial parts of the mutant rum1 are not affected. The mutant rum1 displays severely reduced auxin transport in the primary root and a delayed gravitropic response. Exogenously applied auxin does not induce lateral roots in the primary root of rum1. Lateral roots are initiated in a specific cell type, the pericycle. Cell-type-specific transcriptome profiling of the primary root pericycle 64 h after germination, thus before lateral root initiation, via a combination of laser capture microdissection and subsequent microarray analyses of 12k maize microarray chips revealed 90 genes preferentially expressed in the wild-type pericycle and 73 genes preferentially expressed in the rum1 pericycle (fold change >2; P-value <0.01; estimated false discovery rate of 13.8%). Among the 51 annotated genes predominately expressed in the wild-type pericycle, 19 genes are involved in signal transduction, transcription, and the cell cycle. This analysis defines an array of genes that is active before lateral root initiation and will contribute to the identification of checkpoints involved in lateral root formation downstream of rum1.

Biological Transport↗
PubMed2005

Genetic regulation of gene expression during shoot development in Arabidopsis.

The genetic control of gene expression during shoot development in Arabidopsis thaliana was analyzed by combining quantitative trait loci (QTL) and microarray analysis. Using oligonucleotide array data from 30 recombinant inbred lines derived from a cross of Columbia and Landsberg erecta ecotypes, the Arabidopsis genome was scanned for marker-by-gene linkages or so-called expression QTL (eQTL). Single-feature polymorphisms (SFPs) associated with sequence disparities between ecotypes were purged from the data. SFPs may alter the hybridization efficiency between cDNAs from one ecotype with probes of another ecotype. In genome scans, five eQTL hot spots were found with significant marker-by-gene linkages. Two of the hot spots coincided with classical QTL conditioning shoot regeneration, suggesting that some of the heritable gene expression changes observed in this study are related to differences in shoot regeneration efficiency between ecotypes. Some of the most significant eQTL, particularly those at the shoot regeneration QTL sites, tended to show cis-chromosomal linkages in that the target genes were located at or near markers to which their expression was linked. However, many linkages of lesser significance showed expected "trans-effects," whereby a marker affects the expression of a target gene located elsewhere on the genome. Some of these eQTL were significantly linked to numerous genes throughout the genome, suggesting the occurrence of large groups of coregulated genes controlled by single markers.

Arabidopsis↗
PubMed2005

BarleyBase--an expression profiling database for plant genomics.

BarleyBase (BB) (www.barleybase.org) is an online database for plant microarrays with integrated tools for data visualization and statistical analysis. BB houses raw and normalized expression data from the two publicly available Affymetrix genome arrays, Barley1 and Arabidopsis ATH1 with plans to include the new Affymetrix 61K wheat, maize, soybean and rice arrays, as they become available. BB contains a broad set of query and display options at all data levels, ranging from experiments to individual hybridizations to probe sets down to individual probes. Users can perform cross-experiment queries on probe sets based on observed expression profiles and/or based on known biological information. Probe set queries are integrated with visualization and analysis tools such as the R statistical toolbox, data filters and a large variety of plot types. Controlled vocabularies for gene and plant ontologies, as well as interconnecting links to physical or genetic map and other genomic data in PlantGDB, Gramene and GrainGenes, allow users to perform EST alignments and gene function prediction using Barley1 exemplar sequences, thus, enhancing cross-species comparison.

Arabidopsis↗
PubMed2005

Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation.

A novel porcine cDNA array, containing 1,015 PCR products selected for embryonic expression, was used for transcriptional profiling of conceptuses at four stages of peri-implantation development. Total conceptus RNA from small spherical, large spherical, tubular, and filamentous stages was amplified, converted to cDNA, and hybridized to membranes. Initially, normalized signal intensities obtained using cDNA from total RNA or from amplified RNA were compared. Uniform distribution of P-values associated with t-tests conducted for each gene indicated no evidence that amplification introduced bias. Analysis of data obtained by using amplified targets and the novel array identified genes differentially expressed across stages. Such genes were identified by testing for significant stage effects in gene-specific mixed models. A total of nine genes were declared differentially expressed. Six of the nine genes had P-values less than 0.001, and a false discovery rate of approximately 17% was associated with this significance threshold. Two out of six genes were significant when using the Bonferroni method to control the probability of one or more false positives. The other three genes had P-values between 0.001 and 0.01 and exhibited differences greater than twofold between stages. All four genes selected for confirmation (steroidogenic acute regulatory protein, interleukin 1 beta, transforming growth factor beta 3, and thymosin beta 10) were shown to be differentially expressed by using quantitative real time RT-PCR. Our study shows that RNA amplification is useful for transcriptional profiling with limiting porcine embryonic RNA, and that this novel targeted array can detect differential gene expression during trophoblastic elongation. Finally, our results contribute to an increased understanding of the temporal patterns of expression of known genes controlling conceptus development, as well as identify novel genes also differentially regulated during implantation.

Animals↗
PubMed2005

Validation of a first-generation long-oligonucleotide microarray for transcriptional profiling in the pig.

A first-generation porcine oligonucleotide set, representing 13,297 cDNAs and ESTs, has been designed by Qiagen-Operon for transcriptional profiling. To validate this set, microarrays containing each 70-mer oligonucleotide, referred to as the Qiagen-NRSP8 array, were hybridized with targets from porcine adult liver, lung, muscle, or small intestine. Transcriptome analyses showed that 11,328 of the oligonucleotides demonstrated expression in at least one tissue. Statistical analyses revealed that 1810 genes showed differential expression among tissues (Bonferroni adjusted p < 0.05). Biological pathways identified by DAVID/EASE analysis using a list of 423 tissue-selective genes matched archetypal pathways in the corresponding human or mouse tissue. Real-time quantitative PCR confirmed expression patterns for 9 of 11 genes tested. Our results demonstrate that this first-generation porcine oligonucleotide array is informative and the specificity is high. This is essential validation for investigators using the Qiagen-NRSP8 array for porcine functional genomics and for using the pig in modeling important physiological problems.

Animals↗
PubMed2005

Transformation of maize with the p1 transcription factor directs production of silk maysin, a corn earworm resistance factor, in concordance with a hierarchy of floral organ pigmentation.

The maize p1 gene encodes an R2R3-MYB transcription factor that controls the biosynthesis of red flavonoid pigments in floral tissues of the maize plant. Genetic and quantitative trait locus analyses have also associated the p1 gene with the synthesis of maysin, a flavone glycoside from maize silks that confers natural resistance to corn earworm. Here, we show directly that the p1 gene induces maysin accumulation in silk tissues. Transformation of maize plants that had low or no silk maysin with p1 transgenes elevated silk maysin concentrations to levels sufficient for corn earworm abiosis. The p1 transgenes also conferred red pigment to pericarp, cob, husk and tassel tissues, as expected; however, different subsets of these tissues were pigmented within individual transgenic plants. Statistical analysis shows that the pigmentation patterns observed amongst the p1 transgenic plants conform to a hierarchy that is similar to the temporal ordering of floral organ initiation. We propose that the observed hierarchy of pigmentation patterns is conferred by variation due to epigenetic control of the p1 transgenes. The production of plants with improved traits through genetic engineering can depend in large part on the achievement of tight organ-specific expression of the introduced transgenes. Our results demonstrate that the production of transgenic plants using a promoter with well-defined tissue specificity, such as the p1 promoter, can result in unexpected variation in tissue specificity amongst the resulting transgenic plants.

Journal Article↗
PubMed2004

Genomic neighborhoods for Arabidopsis retrotransposons: a role for targeted integration in the distribution of the Metaviridae.

BACKGROUND: Retrotransposons are an abundant component of eukaryotic genomes. The high quality of the Arabidopsis thaliana genome sequence makes it possible to comprehensively characterize retroelement populations and explore factors that contribute to their genomic distribution. RESULTS: We identified the full complement of A. thaliana long terminal repeat (LTR) retroelements using RetroMap, a software tool that iteratively searches genome sequences for reverse transcriptases and then defines retroelement insertions. Relative ages of full-length elements were estimated by assessing sequence divergence between LTRs: the Pseudoviridae were significantly younger than the Metaviridae. All retroelement insertions were mapped onto the genome sequence and their distribution was distinctly non-uniform. Although both Pseudoviridae and Metaviridae tend to cluster within pericentromeric heterochromatin, this association is significantly more pronounced for all three Metaviridae sublineages (Metavirus, Tat and Athila). Among these, Tat and Athila are strictly associated with pericentromeric heterochromatin. CONCLUSIONS: The non-uniform genomic distribution of the Pseudoviridae and the Metaviridae can be explained by a variety of factors including target-site bias, selection against integration into euchromatin and pericentromeric accumulation of elements as a result of suppression of recombination. However, comparisons based on the age of elements and their chromosomal location indicate that integration-site specificity is likely to be the primary factor determining distribution of the Athila and Tat sublineages of the Metaviridae. We predict that, like retroelements in yeast, the Athila and Tat elements target integration to pericentromeric regions by recognizing a specific feature of pericentromeric heterochromatin.

Arabidopsis↗