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Search Results: 1 - 10 of 333781 matches for " Patrick S. Schnable "
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The roles of aldehyde dehydrogenases (ALDHs) in the PDH bypass of Arabidopsis
Yanling Wei, Ming Lin, David J Oliver, Patrick S Schnable
BMC Biochemistry , 2009, DOI: 10.1186/1471-2091-10-7
Abstract: To test for the presence of the PDH bypass in the sporophytic tissue of plants, Arabidopsis plants homozygous for mutant alleles of all three Family 2 ALDH genes were fed with 14C-ethanol along with wild type controls. Comparisons of the incorporation rates of 14C-ethanol into fatty acids in mutants and wild type controls provided direct evidence for the presence of the PDH bypass in sporophytic tissue. Among the three Family 2 ALDHs, one of the two mitochondrial ALDHs (ALDH2B4) appears to be the primary contributor to this pathway. Surprisingly, single, double and triple ALDH mutants of Arabidopsis did not exhibit detectable phenotypes, even though a Family 2 ALDH gene is required for normal anther development in maize.The PDH bypass is active in sporophytic tissue of plants. Blocking this pathway via triple ALDH mutants does not uncover obvious visible phenotypes.Aldehydes vary in length and in characteristics of their alkyl chains but all are usually deleterious to biological systems due to their chemical reactivity. Aldehyde dehydrogenases (ALDHs, EC 1.2.1) oxidize aldehydes into carboxylic acids, using NAD+ or NADP+ as a co-factor. As such ALDHs play an important role in detoxifying aldehydes that are generated endogenously or introduced from the environment. ALDHs are very diverse in that some only use either NAD+ or NADP+ as the co-factor, while others can use both, some oxidize only a limited number of aldehydes, while others have broader substrate spectra, and ALDHs exist in various subcellular compartments, including the cytosol, mitochondria, plastids and microsomes.Over 550 ALDH genes have been identified across virtually all species, and those from eukaryotes have been classified into more than 20 families [1]. Family 2 ALDHs are mitochondrial or cytosolic homotetrameric enzymes. The well studied human mitochondrial Family 2 ALDH, ALDH2, detoxifies acetaldehyde generated via alcohol intake [2]. Family 2 ALDHs in plants have gained attention since the cl
Loss of RNA–Dependent RNA Polymerase 2 (RDR2) Function Causes Widespread and Unexpected Changes in the Expression of Transposons, Genes, and 24-nt Small RNAs
Yi Jia,Damon R. Lisch,Kazuhiro Ohtsu,Michael J. Scanlon,Dan Nettleton,Patrick S. Schnable
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000737
Abstract: Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA–dependent RNA polymerase 2 (RDR2) is a component of the RNA–directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA–seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2–sensitive and RDR2–resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.
Gene Mapping via Bulked Segregant RNA-Seq (BSR-Seq)
Sanzhen Liu, Cheng-Ting Yeh, Ho Man Tang, Dan Nettleton, Patrick S. Schnable
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036406
Abstract: Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. BSA requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples by analyzing the same RNA-Seq data using an empirical Bayesian approach. In addition, analysis of the RNA-Seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost. In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. By subjecting the reference allele of gl3 to BSR-Seq, we were able to map the gl3 locus to an ~2 Mb interval. The single gene located in the ~2 Mb mapping interval whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independent transposon induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the biosynthesis of very-long-chain fatty acids.
Direct calibration of PICKY-designed microarrays
Hui-Hsien Chou, Arunee Trisiriroj, Sunyoung Park, Yue-Ie C Hsing, Pamela C Ronald, Patrick S Schnable
BMC Bioinformatics , 2009, DOI: 10.1186/1471-2105-10-347
Abstract: Using synthesized samples with known concentrations of specific oligonucleotides, a series of microarray experiments was conducted to evaluate microarrays designed by PICKY, an oligo microarray design software tool, and to test a direct microarray calibration method based on the PICKY-predicted, thermodynamically closest nontarget information. The complete set of microarray experiment results is archived in the GEO database with series accession number GSE14717. Additional data files and Perl programs described in this paper can be obtained from the website http://www.complex.iastate.edu webcite under the PICKY Download area.PICKY-designed microarray probes are highly reliable over a wide range of hybridization temperatures and sample concentrations. The microarray calibration method reported here allows researchers to experimentally optimize their hybridization conditions. Because this method is straightforward, uses existing microarrays and relatively inexpensive synthesized samples, it can be used by any lab that uses microarrays designed by PICKY. In addition, other microarrays can be reanalyzed by PICKY to obtain the thermodynamically closest nontarget information for calibration.PICKY is an optimal oligo microarray design software developed for large and complex genomes [1]. PICKY-estimated DNA annealing temperatures for probes can deviate from actual annealing temperatures because some potentially important parameters are unavailable to its design algorithms, such as variations in the salt composition of hybridization buffers, effects of partially immobilized probes on the microarray surface, nonlinear and multistage nontarget annealing with a probe, effects of incorporated dye molecules on transcript annealing efficiency with a probe, and effects of additional chemicals (e.g., SDS, formamide or DMSO) in the hybridization buffers. These parameters vary with lab environments, and their influence on hybridization kinetics can only be experimentally measured. Be
Regulation of Small RNA Accumulation in the Maize Shoot Apex
Fabio T. S. Nogueira equal contributor,Daniel H. Chitwood equal contributor,Shahinez Madi,Kazuhiro Ohtsu,Patrick S. Schnable,Michael J. Scanlon,Marja C. P. Timmermans
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000320
Abstract: MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial–abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.
Mu Transposon Insertion Sites and Meiotic Recombination Events Co-Localize with Epigenetic Marks for Open Chromatin across the Maize Genome
Sanzhen Liu,Cheng-Ting Yeh,Tieming Ji,Kai Ying,Haiyan Wu,Ho Man Tang,Yan Fu,Dan Nettleton,Patrick S. Schnable
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000733
Abstract: The Mu transposon system of maize is highly active, with each of the ~50–100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar ~215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ~965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination.
Laser Microdissection of Narrow Sheath Mutant Maize Uncovers Novel Gene Expression in the Shoot Apical Meristem
Xiaolan Zhang,Shahinez Madi,Lisa Borsuk,Dan Nettleton,Robert J Elshire,Brent Buckner,Diane Janick-Buckner,Jon Beck,Marja Timmermans,Patrick S Schnable,Michael J Scanlon
PLOS Genetics , 2007, DOI: 10.1371/journal.pgen.0030101
Abstract: Microarrays enable comparative analyses of gene expression on a genomic scale, however these experiments frequently identify an abundance of differentially expressed genes such that it may be difficult to identify discrete functional networks that are hidden within large microarray datasets. Microarray analyses in which mutant organisms are compared to nonmutant siblings can be especially problematic when the gene of interest is expressed in relatively few cells. Here, we describe the use of laser microdissection microarray to perform transcriptional profiling of the maize shoot apical meristem (SAM), a ~100-μm pillar of organogenic cells that is required for leaf initiation. Microarray analyses compared differential gene expression within the SAM and incipient leaf primordium of nonmutant and narrow sheath mutant plants, which harbored mutations in the duplicate genes narrow sheath1 (ns1) and narrow sheath2 (ns2). Expressed in eight to ten cells within the SAM, ns1 and ns2 encode paralogous WUSCHEL1-like homeobox (WOX) transcription factors required for recruitment of leaf initials that give rise to a large lateral domain within maize leaves. The data illustrate the utility of laser microdissection-microarray analyses to identify a relatively small number of genes that are differentially expressed within the SAM. Moreover, these analyses reveal potentially conserved WOX gene functions and implicate specific hormonal and signaling pathways during early events in maize leaf development.
High-Resolution Genotyping via Whole Genome Hybridizations to Microarrays Containing Long Oligonucleotide Probes
Yan Fu,Nathan M. Springer,Kai Ying,Cheng-Ting Yeh,A. Leonardo Iniguez,Todd Richmond,Wei Wu,Brad Barbazuk,Dan Nettleton,Jeff Jeddeloh,Patrick S. Schnable
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0014178
Abstract: To date, microarray-based genotyping of large, complex plant genomes has been complicated by the need to perform genome complexity reduction to obtain sufficiently strong hybridization signals. Genome complexity reduction techniques are, however, tedious and can introduce unwanted variables into genotyping assays. Here, we report a microarray-based genotyping technology for complex genomes (such as the 2.3 GB maize genome) that does not require genome complexity reduction prior to hybridization. Approximately 200,000 long oligonucleotide probes were identified as being polymorphic between the inbred parents of a mapping population and used to genotype two recombinant inbred lines. While multiple hybridization replicates provided ~97% accuracy, even a single replicate provided ~95% accuracy. Genotyping accuracy was further increased to >99% by utilizing information from adjacent probes. This microarray-based method provides a simple, high-density genotyping approach for large, complex genomes.
The Maize glossy13 Gene, Cloned via BSR-Seq and Seq-Walking Encodes a Putative ABC Transporter Required for the Normal Accumulation of Epicuticular Waxes
Li Li, Delin Li, Sanzhen Liu, Xiaoli Ma, Charles R. Dietrich, Heng-Cheng Hu, Gaisheng Zhang, Zhiyong Liu, Jun Zheng, Guoying Wang, Patrick S. Schnable
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0082333
Abstract: Aerial plant surfaces are covered by epicuticular waxes that among other purposes serve to control water loss. Maize glossy mutants originally identified by their “glossy” phenotypes exhibit alterations in the accumulation of epicuticular waxes. By combining data from a BSR-Seq experiment and the newly developed Seq-Walking technology, GRMZM2G118243 was identified as a strong candidate for being the glossy13 gene. The finding that multiple EMS-induced alleles contain premature stop codons in GRMZM2G118243, and the one knockout allele of gl13, validates the hypothesis that gene GRMZM2G118243 is gl13. Consistent with this, GRMZM2G118243 is an ortholog of AtABCG32 (Arabidopsis thaliana), HvABCG31 (barley) and OsABCG31 (rice), which encode ABCG subfamily transporters involved in the trans-membrane transport of various secondary metabolites. We therefore hypothesize that gl13 is involved in the transport of epicuticular waxes onto the surfaces of seedling leaves.
Heritable Epigenetic Variation among Maize Inbreds
Steve R. Eichten equal contributor,Ruth A. Swanson-Wagner equal contributor,James C. Schnable,Amanda J. Waters,Peter J. Hermanson,Sanzhen Liu,Cheng-Ting Yeh,Yi Jia,Karla Gendler,Michael Freeling,Patrick S. Schnable,Matthew W. Vaughn ,Nathan M. Springer
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002372
Abstract: Epigenetic variation describes heritable differences that are not attributable to changes in DNA sequence. There is the potential for pure epigenetic variation that occurs in the absence of any genetic change or for more complex situations that involve both genetic and epigenetic differences. Methylation of cytosine residues provides one mechanism for the inheritance of epigenetic information. A genome-wide profiling of DNA methylation in two different genotypes of Zea mays (ssp. mays), an organism with a complex genome of interspersed genes and repetitive elements, allowed the identification and characterization of examples of natural epigenetic variation. The distribution of DNA methylation was profiled using immunoprecipitation of methylated DNA followed by hybridization to a high-density tiling microarray. The comparison of the DNA methylation levels in the two genotypes, B73 and Mo17, allowed for the identification of approximately 700 differentially methylated regions (DMRs). Several of these DMRs occur in genomic regions that are apparently identical by descent in B73 and Mo17 suggesting that they may be examples of pure epigenetic variation. The methylation levels of the DMRs were further studied in a panel of near-isogenic lines to evaluate the stable inheritance of the methylation levels and to assess the contribution of cis- and trans- acting information to natural epigenetic variation. The majority of DMRs that occur in genomic regions without genetic variation are controlled by cis-acting differences and exhibit relatively stable inheritance. This study provides evidence for naturally occurring epigenetic variation in maize, including examples of pure epigenetic variation that is not conditioned by genetic differences. The epigenetic differences are variable within maize populations and exhibit relatively stable trans-generational inheritance. The detected examples of epigenetic variation, including some without tightly linked genetic variation, may contribute to complex trait variation.
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