bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genomic rearrangements and transcriptional analysis of the spliced leader‐associated retrotransposon in RNA interference‐deficient Trypanosoma brucei
Authors: Patrick Kristin L. , Luz Paula M. , Ruan Jia‐peng , Shi Huafang , Ullu Elisabetta , Tschudi Christian 
Published: August 2010

SummaryThe Trypanosoma brucei genome is colonized by the site‐specific non‐LTR retrotransposon SLACS, or spliced leader‐associated conserved sequence, which integrates exclusively into the spliced leader (SL) RNA genes. Although there is evidence that the RNA interference (RNAi) machinery regulates SLACS transcript levels, we do not know whether RNAi deficiency affects the genomic stability of SLACS, nor do we understand the mechanism of SLACS transcription. Here, we report that prolonged culturing of RNAi‐deficient T. brucei cells, but not wild‐type cells, results in genomic rearrangements of SLACS. Furthermore, two populations of SLACS transcripts persist in RNAi‐deficient cells: a full‐length transcript of approximately 7 kb and a heterogeneous population of small SLACS transcripts ranging in size from 450 to 550 nt. We provide evidence that SLACS transcription initiates at the +1 of the interrupted SL RNA gene and proceeds into the 5′ UTR and open reading frame 1 (ORF1). This transcription is carried out by an RNA polymerase with α‐amanitin sensitivity reminiscent of SL RNA synthesis and is dependent on the SL RNA promoter. Additionally, we show that both sense and antisense small SLACS transcripts originate from ORF1 and that they are associated with proteins in vivo. We speculate that the small SLACS transcripts serve as substrates for the production of siRNAs to regulate SLACS expression.
Acetylation of histone H4K4 is cell cycle regulated and mediated by HAT3 in Trypanosoma brucei
Authors: Siegel T. Nicolai , Kawahara Taemi , DeGrasse Jeffrey A. , Janzen Christian J. , Horn David , Cross George A. M. 
Published: August 2010

SummaryPost‐translational histone modifications have been studied intensively in several eukaryotes. It has been proposed that these modifications constitute a ‘histone code’ that specifies epigenetic information for transcription regulation. With a limited number of histone‐modifying enzymes, implying less redundancy, Trypanosoma brucei represents an excellent system in which to investigate the function of individual histone modifications and histone‐modifying enzymes. In this study, we characterized the acetylation of lysine 4 of histone H4 (H4K4), the most abundant acetylation site in T. brucei histones. Because of the large sequence divergence of T. brucei histones, we generated highly specific antibodies to acetylated and unmodified H4K4. Immunofluorescence microscopy and Western blots with sorted cells revealed a strong enrichment of unmodified H4K4 in S phase and suggested a G1/G0‐specific masking of the site, owing to non‐covalently binding factors. Finally, we showed that histone acetyltransferase 3 (HAT3) is responsible for H4K4 acetylation and that treatment of cells with the protein synthesis inhibitor cycloheximide led to an almost instantaneous loss of unmodified H4K4 sites. As HAT3 is located inside the nucleus, our findings suggest that newly synthesized histone H4 with an unmodified K4 is imported rapidly into the nucleus, where it is acetylated, possibly irreversibly.
A small protein from the bop–brp intergenic region of Halobacterium salinarum contains a zinc finger motif and regulates bop and crtB1 transcription
Authors: Tarasov Valery Y. , Besir Hüseyin , Schwaiger Rita , Klee Kathrin , Furtwängler Katarina , Pfeiffer Friedhelm , Oesterhelt Dieter 
Published: August 2010

SummaryBacteriorhodopsin, the photosynthetic protein of Halobacterium salinarum, is optimally expressed under anaerobic growth conditions. We identified Brz (OE3104F, bacteriorhodopsin‐regulating zinc finger protein), a new regulator of the bop gene. It is a small protein with a zinc finger motif, encoded directly upstream of the bop gene in the same orientation. Deletion of the brz gene caused a large decrease of bop mRNA levels as shown by Northern blot and microarray analysis. A similar effect was obtained by site‐directed mutagenesis of Cys and His residues in the zinc finger motif, indicating the importance of this motif for the function of the protein. In silico analysis of the genomes from H. salinarum and other archaea revealed a large family of similar small zinc finger motif proteins, some of which may also be involved in transcription regulation of their adjacent genes.
Molecular basis of gene regulation by the THI‐box riboswitch
Authors: Ontiveros‐Palacios Nancy , Smith Angela M. , Grundy Frank J. , Soberon Mario , Henkin Tina M. , Miranda‐Ríos Juan 
Published: August 2010

SummaryRiboswitches are genetic control elements located mainly within the 5′ untranslated regions of messenger RNAs. These RNA elements undergo conformational changes that modulate gene expression upon binding of regulatory signals including vitamins, amino acids, nucleobases and uncharged tRNA. The thiamin pyrophosphate (TPP)‐binding riboswitch (THI‐box) is found in all three kingdoms of life and can regulate gene expression at the levels of premature termination of transcription, initiation of translation and mRNA splicing. The THI‐box is composed of two parallel stacked helices bound by another helix in a three‐way junction. We performed an in vivo expression analysis of mutants with substitutions in conserved bases located at the interior and terminal loops of the Escherichia coli thiM THI‐box, which is translationally regulated, and observed two different phenotypic classes. One class exhibited high expression during growth in the presence or absence of thiamin, while the second class exhibited low expression regardless of the presence of thiamin. Accessibility of the Shine‐Dalgarno region of the RNA following the addition of TPP was monitored by means of an oligonucleotide‐dependent RNase H cleavage assay, and binding of 30S ribosomal subunits. These studies showed that high‐ and low‐expression mutant RNAs are locked in the non‐repressive and repressive conformations respectively.
Post‐transcriptional cross‐talk between pro‐ and anti‐colonization pili biosynthesis systems in Vibrio cholerae
Authors: Hsiao Ansel , Toscano Kristin , Zhu Jun 
Published: August 2010

SummaryThe pathogen Vibrio cholerae modulates the expression of many genes in order to transition from its environmental reservoir to its niche in the human host. Among these are genes encoding two related Type IV pili, the mannose‐sensitive haemagglutinin (MSHA) pilus, which aids V. cholerae persistence in aquatic environments but causes clearance of bacteria by host immune defences, and the toxin co‐regulated pilus (TCP) required for colonization. These antagonistic effects are resolved transcriptionally by the regulator ToxT, which represses msh genes while activating tcp genes during infection. We show that these two pili systems are also intertwined post‐transcriptionally through the ToxT‐regulated pre‐pilin peptidase TcpJ. We found that the major MSHA pilin, MshA, was degraded in V. cholerae in a TcpJ‐dependent fashion. In a heterologous Escherichia coli system, TcpJ can recognize both MshA and its cognate substrate, the TCP subunit TcpA, but that processing by TcpJ causes the degradation of MshA. Through site‐directed mutagenesis and chimeric pilin analysis, we show that this process targets a combination of MshA N‐terminal motifs and depends on the proteolytic activity of TcpJ. Moreover, overexpression of tcpJ partially restored the ability of bacteria unable to transcriptionally downregulate msh genes to colonize infant mice. These findings describe co‐ordinated proteolysis as a regulatory mechanism in V. cholerae and illustrate this organism's adaptability in the face of dramatic environmental changes.
The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomycetes
Authors: Tiffert Yvonne , Supra Petra , Wurm Reinhild , Wohlleben Wolfgang , Wagner Rolf , Reuther Jens 
Published: August 2010

Summary Streptomyces coelicolor GlnR is a global regulator that controls genes involved in nitrogen metabolism. By genomic screening 10 new GlnR targets were identified, including enzymes for ammonium assimilation (glnII, gdhA), nitrite reduction (nirB), urea cleavage (ureA) and a number of biochemically uncharacterized proteins (SCO0255, SCO0888, SCO2195, SCO2400, SCO2404, SCO7155). For the GlnR regulon, a GlnR binding site which comprises the sequence gTnAc‐n6‐GaAAc‐n6‐GtnAC‐n6‐GAAAc‐n6 has been found. Reverse transcription analysis of S. coelicolor and the S. coelicolor glnR mutant revealed that GlnR activates or represses the expression of its target genes. Furthermore, glnR expression itself was shown to be nitrogen‐dependent. Physiological studies of S. coelicolor and the S. coelicolor glnR mutant with ammonium and nitrate as the sole nitrogen source revealed that GlnR is not only involved in ammonium assimilation but also in ammonium supply. blast analysis demonstrated that GlnR‐homologous proteins are present in different actinomycetes containing the glnA gene with the conserved GlnR binding site. By DNA binding studies, it was furthermore demonstrated that S. coelicolor GlnR is able to interact with these glnA upstream regions. We therefore suggest that GlnR‐mediated regulation is not restricted to Streptomyces but constitutes a regulon conserved in many actinomycetes.
Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans
Authors: Araujo‐Bazán Lidia , Peñalva Miguel A. , Espeso Eduardo A. 
Published: August 2010

SummaryAbpA, SlaB and AmpA, three demonstrated components of the endocytic internalization machinery, are strongly polarized in Aspergillus nidulans hyphae, forming a ring that embraces the hyphal tip, leaving an area of exclusion at the apex. AbpA, a prototypic endocytic internalization marker, localizes to highly motile and transient (average half life, 24 ± 5 s) peripheral punctate structures overlapping with actin patches, which also predominate in the tip. SlaB also localizes to peripheral patches, but these are markedly more abundant and cortical than those of AbpA. In contrast to its polarized distribution in hyphae, endocytic patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarize as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along the hyphae, the apical predominance and the spatial organization of actin patches and of the above endocytic machinery proteins as a slightly subapical ring strongly suggests that tight spatial coupling of apical secretion and subapical compensatory endocytosis underlies hyphal growth. In agreement, the phenotype of a null slaB allele indicates that endocytosis is essential.
The gapped xylem mutant identifies a common regulatory step in secondary cell wall deposition
Authors: Turner Simon R. , Hall Madeleine 
Published: August 2010

SummaryThe phenotype of the novel gapped xylem (gpx) mutant is described. gpx plants exhibit gaps in the xylem in positions where xylem elements would normally be located. These gaps are not part of the transpiration stream and result in gpx plants having fewer functional xylem elements. The gaps are due to the absence of a secondary cell wall in developing xylem elements, resulting in complete degradation of these elements during cell death, and illustrate the importance of the secondary cell wall in retaining a functional xylem element following programmed cell death. Consequently the gpx phenotype suggests that the processes of secondary cell wall formation and cell death are independently regulated in developing xylem. gpx plants also exhibit a highly irregular pattern of secondary cell wall thickening in interfascicular cells, with some cells apparently undergoing little or no secondary cell wall deposition. Secondary cell wall deposition in plants involves the co‐ordinate regulation of several complex metabolic pathways. The gpx mutant identifies a key step involved in regulating the deposition of secondary cell wall material in both xylem and interfascicular cells, and suggests that a common regulatory step controls secondary cell wall formation in these diverse cell types. The gpx mutant offers a unique opportunity to elucidate the mechanism by which the complex processes involved in secondary cell wall formation are co‐ordinately regulated.
A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron‐splicing mutant
Authors: Schock Irene , Gregan Juraj , Steinhauser Siegfried , Schweyen Rudolf , Brennicke Axel , Knoop Volker 
Published: August 2010

SummaryAutocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo. In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2‐1 and atmrs2‐2. Atmrs2‐1, but not atmrs2‐2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2‐1 and yeast MRS2 but not in AtMRS2‐2. This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2‐1. A differing, non‐mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts. Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome. Similarity searches with the psi‐blast algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters. In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild‐type levels in the yeast mutant on complementation with atmrs2‐1.
Application of high‐performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids
Authors: Fraser Paul D. , Pinto M. Elisabete S. , Holloway Daniel E. , Bramley Peter M. 
Published: August 2010

SummaryThe application of high‐performance liquid chromatography (HPLC) using a C30 reverse‐phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identification and quantification of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure.
Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus
Authors: Kropat Janette , Oster Ulrike , Rüdiger Wolfhart , Beck Christoph F. 
Published: August 2010

SummaryChlorophyll precursors Mg‐protoporphyrin IX and its monomethylester are candidates for plastid‐derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+‐containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs‐1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light‐induced increase in the levels of Mg2+‐containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light‐induced rise in pool levels and induction of the HSP70 genes. Similarly, pre‐gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor‐treated cells and in pre‐gametes could be circumvented by the exogenous addition of Mg‐protoporphyrin IX in the dark. This suggests an essential role for light‐induced Mg‐protoporphyrin IX accumulation in this chloroplast‐to‐nucleus signalling pathway. However, accumulation of this porphyrin in the dark – presumably in the chloroplast – did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated.
Plant sucrose‐H+ symporters mediate the transport of vitamin H
Authors: Ludwig Andreas , Stolz Jürgen , Sauer Norbert 
Published: August 2010

SummaryA cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake‐deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH‐group inhibitor p‐chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP), suggesting an energy‐dependent biotin‐H+ symport mechanism. The transport activity could contribute to the so‐far uncharacterized plant sucrose‐H+ symporter AtSUC5 which mediates the energy‐dependent transport of biotin and sucrose, and restores growth of the biotin transport‐deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin‐H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.
Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance
Authors: Sunkar Ramanjulu , Kaplan Boaz , Bouché Nicolas , Arazi Tzahi , Dolev Dvora , Talke Ina N. , Maathuis Frans J.M. , Sanders Dale , Bouchez David , Fromm Hillel 
Published: August 2010

SummaryRecently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4ΔC) from which its C‐terminal, with the calmodulin‐binding domain and part of the putative cyclic nucleotide‐binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full‐length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T‐DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.
The maize LAG1‐O mutant suggests that reproductive cell lineages show unique gene expression patterns early in vegetative development
Authors: Giedt Christopher D. , Weil Clifford F. 
Published: August 2010

SummaryPatterns of transposable element activity often provide useful information about how and when organisms regulate gene expression. The maize lowered Ac/Ds germinal reversion 1 (LAG1)‐O mutation causes unusually low rates of germinal reversion by Ac/Ds‐induced alleles even though these same alleles revert frequently and early in somatic development. LAG1‐O suppresses Ds transposition at multiple, unlinked loci, and does not affect Spm elements, indicating that the mutation acts in trans and may be specific to Ac/Ds elements. Our data suggest that LAG1‐O suppression gradually reduces Ac/Ds activity in the meristem and newly formed leaves until, by the floral transition, transposition is undetectable even with PCR‐based assays. This suppression persists during tassel development and does not appear to be released until some point after meiosis. Competitive RT–PCR results show no difference in Ac transposase mRNA levels between LAG1‐O and lag1+ tassels, suggesting that suppression is post‐transcriptional. The pattern of LAG1‐O expression is consistent with a model in which at least some gene expression specific to those meristem cells that will ultimately give rise to floral tissue and therefore gametes begins very early in plant development, and then persists throughout development.
Identification of genes encoding receptor‐like protein kinases as possible targets of pathogen‐ and salicylic acid‐induced WRKY DNA‐binding proteins in Arabidopsis
Authors: Du Liqun , Chen Zhixiang 
Published: August 2010

SummaryTo understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen‐ and salicylic acid (SA)‐induced DNA‐binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W‐box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor‐like protein kinases (RLK), each consisting of an N‐terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C‐terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W‐box elements in its promoter region were recognized by both purified WRKY proteins and SA‐induced W‐box binding activities from SA‐treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein‐binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen‐ and SA‐induced W‐box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti‐microbial activities, but also genes encoding proteins with regulatory functions.