Biomedical subjects

Iain W Manfield

Publications and source records attributed to Iain W Manfield.

11 recordsLinked to original sources

PubMed2007

Conservation, convergence, and divergence of light-responsive, circadian-regulated, and tissue-specific expression patterns during evolution of the Arabidopsis GATA gene family.

In vitro analyses of plant GATA transcription factors have implicated some proteins in light-mediated and circadian-regulated gene expression, and, more recently, the analysis of mutants has uncovered further diverse roles for plant GATA factors. To facilitate function discovery for the 29 GATA genes in Arabidopsis (Arabidopsis thaliana), we have experimentally verified gene structures and determined expression patterns of all family members across adult tissues and suspension cell cultures, as well as in response to light and signals from the circadian clock. These analyses have identified two genes that are strongly developmentally light regulated, expressed predominantly in photosynthetic tissue, and with transcript abundance peaking before dawn. In contrast, several GATA factor genes are light down-regulated. The products of these light-regulated genes are candidates for those proteins previously implicated in light-regulated transcription. Coexpression of these genes with well-characterized light-responsive transcripts across a large microarray data set supports these predictions. Other genes show additional tissue-specific expression patterns suggesting novel and unpredicted roles. Genome-wide analysis using coexpression scatter plots for paralogous gene pairs reveals unexpected differences in cocorrelated gene expression profiles. Clustering the Arabidopsis GATA factor gene family by similarity of expression patterns reveals that genes of recent descent do not uniformly show conserved current expression profiles, yet some genes showing more distant evolutionary origins have acquired common expression patterns. In addition to defining developmental and environmental dynamics of GATA transcript abundance, these analyses offer new insights into the evolution of gene expression profiles following gene duplication events.

Amino Acid Motifs↗

PubMed2006

Arabidopsis Co-expression Tool (ACT): web server tools for microarray-based gene expression analysis.

The Arabidopsis Co-expression Tool, ACT, ranks the genes across a large microarray dataset according to how closely their expression follows the expression of a query gene. A database stores pre-calculated co-expression results for approximately 21,800 genes based on data from over 300 arrays. These results can be corroborated by calculation of co-expression results for user-defined sub-sets of arrays or experiments from the NASC/GARNet array dataset. Clique Finder (CF) identifies groups of genes which are consistently co-expressed with each other across a user-defined co-expression list. The parameters can be altered easily to adjust cluster size and the output examined for optimal inclusion of genes with known biological roles. Alternatively, a Scatter Plot tool displays the correlation coefficients for all genes against two user-selected queries on a scatter plot which can be useful for visual identification of clusters of genes with similar r-values. User-input groups of genes can be highlighted on the scatter plots. Inclusion of genes with known biology in sets of genes identified using CF and Scatter Plot tools allows inferences to be made about the roles of the other genes in the set and both tools can therefore be used to generate short lists of genes for further characterization. ACT is freely available at www.Arabidopsis.leeds.ac.uk/ACT.

Algorithms↗

PubMed2006

Transcript analysis reveals an extended regulon and the importance of protein-protein co-operativity for the Escherichia coli methionine repressor.

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.

Base Sequence↗

PubMed2006

The Arabidopsis co-expression tool (ACT): a WWW-based tool and database for microarray-based gene expression analysis.

We present a new WWW-based tool for plant gene analysis, the Arabidopsis Co-Expression Tool (ACT), based on a large Arabidopsis thaliana microarray data set obtained from the Nottingham Arabidopsis Stock Centre. The co-expression analysis tool allows users to identify genes whose expression patterns are correlated across selected experiments or the complete data set. Results are accompanied by estimates of the statistical significance of the correlation relationships, expressed as probability (P) and expectation (E) values. Additionally, highly ranked genes on a correlation list can be examined using the novel clique finder tool to determine the sets of genes most likely to be regulated in a similar manner. In combination, these tools offer three levels of analysis: creation of correlation lists of co-expressed genes, refinement of these lists using two-dimensional scatter plots, and dissection into cliques of co-regulated genes. We illustrate the applications of the software by analysing genes encoding functionally related proteins, as well as pathways involved in plant responses to environmental stimuli. These analyses demonstrate novel biological relationships underlying the observed gene co-expression patterns. To demonstrate the ability of the software to develop testable hypotheses on gene function within a defined biological process we have used the example of cell wall biosynthesis genes. The resource is freely available at http://www.arabidopsis.leeds.ac.uk/ACT/

Arabidopsis↗

PubMed2005

Molecular characterization of DNA sequences from the Primula vulgaris S-locus.

Primula species provide possibly the best known examples of heteromorphic flower development and this breeding system has attracted considerable attention, including that of Charles Darwin. However, despite considerable recent advances in molecular genetics, nothing is known about the molecular basis of floral heteromorphy. The first molecular marker for the Primula S-locus is reported here. This DNA sequence was identified by random amplification of polymorphic DNA (RAPD)-PCR, further defined as a sequence characterized amplified region (SCAR) marker, and subsequently shown to correspond to a restriction fragment length polymorphism (RFLP) that is linked to the thrum allele of the Primula S-locus. The sequence of 8.8 kb of genomic DNA encompassing this thrum-specific RFLP is presented. Analysis of this DNA reveals a highly repetitive sequence structure similar to that found at the S-locus in other species; it also contains sequences similar to elements of a Gypsy-like retrotransposon. The identification of a specific DNA sequence associated with the thrum allele of the Primula S-locus provides the first molecular probe with which to investigate the molecular basis of heteromorphic flower development in Primula.

Base Sequence↗

PubMed2004

Novel cell wall architecture of isoxaben-habituated Arabidopsis suspension-cultured cells: global transcript profiling and cellular analysis.

The herbicide isoxaben is a highly specific and potent inhibitor of cellulose synthesis in plants. Nevertheless, suspension-cultured cells can be habituated to grow in high concentrations of isoxaben, and apparently compensate for the disruption of cellulose synthesis by the modulation of other cell wall components. We have habituated Arabidopsis cells to isoxaben and characterized the cellular and genetic consequences. Near whole-genome transcript profiling implicated novel genes in cell wall assembly and extended our understanding of the activity of known cell wall-related genes including glycosyltransferases involved in cellulose and pectin biosynthesis. Habituation does not appear to be mediated by stress response processes, nor by functional redundancy within the cellulose synthase (AtCesA) family. Uniquely, amongst the cellulose synthase superfamily, AtCslD5 was highly upregulated and may play a role in the biosynthesis of the novel walls of habituated cells. In silico analysis of differentially expressed genes with unknown functions identified a putative glycosyltransferase and collagen-like putative cell wall protein.

Adaptation, Biological↗

PubMed2003

Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA.

The 876-aa protein AreA regulates the expression of numerous genes involved in nitrogen metabolism in Aspergillus nidulans, and interacts with GATA sequences upstream of the relevant genes. We have carried out limited proteolysis of the C-terminal domain of the AreA protein in order to identify possible structural domains within the protein. A stable 156-amino-acid fragment was identified that contained the zinc finger region, and this sequence was cloned and expressed in E. coli. Fluorescence spectroscopy of the purified protein showed that the proteolytic domain was folded and could be denatured by high concentrations of urea (approximately 4 M), exhibiting a sharp transition. Fluorescence spectroscopy was also used to monitor binding to a DNA duplex containing the AreA recognition site, demonstrating tight binding of the domain to its DNA recognition sequence. The DNA binding affinity of the domain is comparable with that of the native AreA protein and much higher than that of the minimal zinc finger region of AreA.

Aspergillus nidulans↗

PubMed2003

A biaryl peptide crosslink in a MetJ peptide model confers cooperative, nonspecific binding to DNA that ablates both repressor binding and in vitro transcription.

The MetJ repressor is the archetypal example of the beta-ribbon-helix-helix DNA binding motif. A model of the MetJ beta-ribbon (residues 22-28) was prepared by forming a dityrosine crosslinked dimer from the heptapeptide KKYTVSI. Using SPR, the peptide dimer 2 was shown to bind to dsDNA under physiologically relevant conditions, whereas the monomeric peptide did not. The peptide dimer appeared to inhibit binding of the MetJ repressor to natural met operators. Based on the stoichiometry of binding, the binding of peptide dimer 2 seems both highly co-operative and to lack sequence specificity. Peptide binding also appears to prevent transcription in vitro.

Bacterial Proteins↗

PubMed2002

Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ.

We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.

Bacterial Proteins↗

PubMed2002

Arabidopsis thaliana GATA factors: organisation, expression and DNA-binding characteristics.

Many light-responsive promoters contain GATA motifs and a number of nuclear proteins have been defined that interact with these elements. Type-IV zinc-finger proteins have been extensively characterised in animals and fungi and are referred to as GATA factors by virtue of their affinity for promoter elements containing this sequence. We previously identified cDNA sequences representing four Arabidopsis thaliana type-TV zinc-finger proteins. Here we define the organisation and expression of GATA-1, GATA-2, GATA-3 and GATA-4 as well as DNA-binding characteristics of their encoded proteins. Transcripts from all four genes can be detected in all tissues examined suggesting that they are not developmentally regulated at the level of transcription. In vitro binding experiments with Escherichia coli-derived recombinant proteins were performed using motifs previously defined as targets for nuclear GATA-binding proteins. These studies reveal differences in DNA binding specificity of GATA-1 as compared to the other three proteins. In vivo protein-DNA interactions monitored by yeast one-hybrid assays reveal different binding characteristics as compared to those defined with E. coli-derived recombinant protein. Trans-activation of gene expression by the four Arabidopsis proteins via some, but not all, DNA elements tested indicates that the Arabidopsis proteins can form functional interactions with previously defined promoter elements containing GATA motifs. We conclude that the Arabidopsis type-IV zinc-finger proteins may represent the previously defined family of nuclear GATA-binding proteins implicated in light-responsive transcription.

Amino Acid Sequence↗

PubMedDate not supplied

Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10.

Peroxisomes are organelles found in all eukaryotic cells. Peroxisomes import integral membrane proteins post-translationally, and PEX19 is a predominantly cytosolic, farnesylated protein of mammalian and yeast cells that binds multiple peroxisome membrane proteins and is required for their correct targeting/insertion to the peroxisome membrane. We report the characterisation of the Arabidopsisthaliana homologue of PEX19 which is a predominantly cytosolic protein. AtPEX19 is encoded by two genes (designated AtPEX19-1 and AtPEX19-2) that are expressed in all tissues and at all developmental stages of the plant. Quantitative real time PCR shows that AtPEX19-1 and AtPEX19-2 have distinct expression profiles. Using in vitro translation and co-immunoprecipitation AtPEX19-1 was shown to bind to the Arabidopsis peroxisomal membrane protein PEX10. Additionally, bacterially expressed recombinant AtPEX19-1 was able to bind a fusion protein consisting of the C-terminus of PEX10 and glutathione S-transferase in pull-down assays, thereby demonstrating that non-farnesylated AtPEX19 can interact with the C-terminus of AtPEX10. Purified recombinant AtPEX19-1 was analysed by gel filtration chromatography and was found to have a molecular weight consistent with it forming a dimer and a dimer was detected in Arabidopsis cell extracts that was slightly destabilised in the presence of DTT. Moreover, cross-linking studies of native AtPEX19 suggest that in vivo it is the dimeric species of the protein that preferentially forms complexes with other proteins.

Arabidopsis↗