bluuebunny/crossref_metadata_2025 huggingface dataset:
Inversion within the haloalkaliphilic virus φCh1 DNA results in differential expression of structural proteins
Authors: Rössler N. , Klein R. , Scholz H. , Witte A.
Published: April 2004

The sequence of φCh1 contains an open reading frame (int1) in the central part of its genome that belongs to the λ integrase family of site‐specific recombinases. Sequence similarities to known integrases include the highly conserved tetrad R‐H‐R‐Y. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR‐L and IR‐R), which are orientated in an inverted direction. Here, we show that a recombination active region exists in the genome of φCh1: the number of those repeats, non‐homologous regions within the repeat clusters IR‐L and IR‐R and the orientation of the int1 gene vary in a given virus population. Within this study, we identified circular intermediates, composed of the int1 gene and the inwards orientated repeat regions IR‐L and IR‐R, which could be involved in the recombination process itself. IR‐L and IR‐R are embedded within ORF34 and ORF36 respectively. As a consequence of the inversion within this region of φCh1, the C‐terminal parts of the proteins encoded by ORF34 and 36 are exchanged. Both proteins, expressed in Escherichia coli, interact with specific antisera against whole virus particles, indicating that they could be parts of φCh1 virions. Expression of the protein(s) in Natrialba magadii could be detected 98 h after inoculation, which is similar to other structural proteins of φCh1. Taken together, the data show that the genome of φCh1 contains an invertible region that codes for a recombinase and structural proteins. Inversion of this segment results in a variation of these structural proteins.
Unequal access of chromosomal regions to each other in Salmonella: probing chromosome structure with phage λ integrase‐mediated long‐range rearrangements
Authors: Garcia‐Russell Nathalie , Harmon Timothy G. , Le Tien Q. , Amaladas Nelusha H. , Mathewson Richard D. , Segall Anca M.
Published: April 2004

We have investigated the fluidity of the Salmonella chromosome architecture using the phage lambda site‐specific recombination system as a probe. We determined how chromosome position affects the extent of integrase‐mediated recombination between pairs of inversely oriented att sites at various loci. We also investigated the accessibility of each chromosomal att site to an extrachromosomal partner carried on a low‐copy plasmid. Recombination events were assayed by semi‐quantitative polymerase chain reaction of the attP product. The extent of recombination between the chromosome and the plasmid was generally higher than intrachromosomal recombination except for two loci, araA::attL and galT::attL, which gave no detectable recombination with any other locus. Based on 20 intervals, we found that chromosomal locations are not equally accessible to each other. Although multiple factors probably affect accessibility, the most important is the specific combination of the end‐points used. Neither the size of the intervals nor the accessibility of individual end‐points to extrachromosomal sequences is as important. These results suggest that the chromosome is not completely fluid but rather organized in some way, with barriers that limit the movement of DNA within the cell. The nature of the barriers involved in chromosomal organization remains to be determined.
Dispensable PDZ domain of Escherichia coli YaeL essential protease
Authors: Bohn Chantal , Collier Justine , Bouloc Philippe
Published: April 2004

In Escherichia coli, adaptation to extra‐cytoplasmic stress relies on σE activation to induce a rescue pathway. Under non‐stressed conditions, σE is sequestered by the inner membrane protein RseA. Extra‐cytoplasmic stress activates DegS‐dependent cleavage of RseA, rendering RseA sensitive to further degradation by the YaeL protease. YaeL contains two motifs characteristic of a family of metallo‐proteases, as well as a periplasmic PDZ domain. We report results of mutational analyses of the YaeL domains. Surprisingly, expression in a strain depleted for wild‐type YaeL or YaeL variants having a 40 amino acid deletion of the PDZ domain or amino acid substitutions of conserved amino acids of the YaeL PDZ domain did not affect cell viability. The proteolytic activity against RseA of these YaeL variants became independent of DegS. These observations suggest that the YaeL PDZ domain exerts a negative control on YaeL activity. Rather than being involved in substrate recognition, the PDZ domain of YaeL is likely to act as an inhibitor of proteolytic activity.
Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP
Authors: Plumbridge Jacqueline , Pellegrini Olivier
Published: April 2004

The chitobiose operon, chbBCARFG, encodes genes for the transport and degradation of the N‐acetylglucosamine disaccharide, chitobiose. Chitobiose is transported by the phosphotransferase system (PTS) producing chitobiose‐6P which is hydrolysed to GlcNAc‐6P by the chbF gene product and then further degraded by the nagBA gene products. Expression of the chb operon is repressed by NagC, which regulates genes involved in amino sugar metabolism. The inducer for NagC is GlcNAc‐6P. NagC binds to two sites separated by 115 bp and the transcription start point of the chb operon lies within the downstream NagC operator. In addition the chb operon encodes its own specific regulator, ChbR, an AraC‐type dual repressor–activator, which binds to two direct repeats of 19 bp located between the two NagC sites. ChbR is necessary for transcription activation in the presence of chitobiose in vivo. Induction of the operon also requires CAP, which binds to a site upstream of the ChbR repeats. In the absence of chitobiose both NagC and ChbR act as repressors. Together these three factors cooperate in switching chb expression from the repressed to the activated state. The need for two specific inducing signals, one for ChbR to activate the expression of the operon and a second for NagC to relieve its repression, ensure that the chb operon is only induced when there is sufficient flux through the combined chb‐nag metabolic pathway to activate expression of both the chb and nag operons.
Lipopolysaccharide O antigen status of Yersinia enterocolitica O:8 is essential for virulence and absence of O antigen affects the expression of other Yersinia virulence factors
Authors: Bengoechea José Antonio , Najdenski Hristo , Skurnik Mikael
Published: April 2004

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram‐negative bacteria. Although much attention has been given to the biological effects of its lipid A portion, a great body of evidence indicates that its O chain polysaccharide (O antigen) portion plays an important role in the bacterium–host interplay. In this work we have studied in‐depth the role of the O antigen in Yersinia enterocolitica serotype O:8 pathogenesis. We made a detailed virulence analysis of three mutants having different O antigen phenotypes: (i) LPS with no O antigen (rough mutant); (ii) LPS with one O unit (semirough mutant) and (iii) LPS with random distribution of O antigen chain lengths. We demonstrated that these LPS O antigen mutants were attenuated in virulence regardless of the infection route used. Co‐infection experiments revealed that the rough and semirough mutants were severely impaired in their ability to colonize the Peyer's patches and in contrast to the wild‐type strain they did not colonize spleen and liver. The mutant with random distribution of O antigen chain lengths, however, survived better but started to be cleared from mouse organs after 8 days. As an explanation to this attenuation we present here evidence that other Yersinia virulence factors depend on the presence of O antigen for their proper function and/or expression. We demonstrated that in the rough mutant: (i) the YadA function but not its expression was altered; (ii) Ail was not expressed and (iii) inv expression was downregulated. On the other hand, expression of flhDC, the flagellar master regulatory operon, was upregulated in this mutant with a concomitant increase in the production of flagellins. Finally, expression of yplA, encoding for the Yersinia phospholipase A, was also upregulated accompanied by an increased flagellar type III secretion system mediated secretion of YplA to culture medium. Together these findings suggest that the absence of O antigen in the outer membrane of Yersinia either directly or indirectly, for example through a cellular or membrane stress, could act as a regulatory signal.
Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract
Authors: Hendrixson David R. , DiRita Victor J.
Published: April 2004

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature‐tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81–176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl‐accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP‐domain containing proteins of C. jejuni revealed one other required for wild‐type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.
Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata
Authors: D’Haeze Wim , Glushka John , De Rycke Riet , Holsters Marcelle , Carlson Russell W.
Published: April 2004

During lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its host plant, Sesbania rostrata, via the formation of outer cortical infection pockets, a process that is characterized by a massive production of H2O2. Infection threads guide bacteria from infection pockets towards nodule primordia. Previously, two mutants were constructed that produce lipopolysaccharides (LPSs) similar to one another but different from the wild‐type LPS, and that are affected in extracellular polysaccharide (EPS) production. Mutant ORS571‐X15 was blocked at the infection pocket stage and unable to produce EPS. The other mutant, ORS571‐oac2, was impaired in the release from infection threads and was surrounded by a thin layer of EPS in comparison to the wild‐type strain that produced massive amounts of EPS. Structural characterization revealed that EPS purified from cultured and nodule bacteria was a linear homopolysaccharide of α‐1,3‐linked 4,6‐O‐(1‐carboxyethylidene)‐d‐galactosyl residues. In situ H2O2 localization demonstrated that increased EPS production during early stages of invasion prevented the incorporation of H2O2 inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H2O2. In addition, ex planta assays confirmed a positive correlation between increased EPS production and enhanced protection against H2O2.
Origins and evolution of isoprenoid lipid biosynthesis in archaea
Authors: Boucher Yan , Kamekura Masahiro , Doolittle W. Ford
Published: April 2004

A characteristic feature of the domain archaea are the lipids forming the hydrophobic core of their cell membrane. These unique lipids are composed of iso‐prenoid side‐chains stereospecifically ether linked to sn‐glycerol‐1‐phosphate. Recently, considerable progress has been made in characterizing the enzymes responsible for the synthesis of archaeal lipids. However, little is known about their evolution. To better understand how this unique biosynthetic apparatus came to be, large‐scale database surveys and phylogenetic analyses were performed. All characterized enzymes involved in the biosynthesis of isoprenoid side‐chains and the glycerol phosphate backbone along with their assembly in ether lipids were included in these analyses. The sequence data available in public databases was complemented by an in‐depth sampling of isoprenoid lipid biosynthesis genes from multiple genera of the archaeal order Halobacteriales, allowing us to look at the evolution of these enzymes on a smaller phylogenetic scale. This investigation of the isoprenoid biosynthesis apparatus of archaea on small and large phylogenetic scales reveals that it evolved through a combination of evolutionary processes, including the co‐option of ancestral enzymes, modification of enzymatic specificity, orthologous and non‐orthologous gene displacement, integration of components from eukaryotes and bacteria and lateral gene transfer within and between archaeal orders.
Requirement for RecFOR‐mediated recombination in priA mutant
Authors: Grompone Gianfranco , Sanchez Nicolas , Dusko Ehrlich S. , Michel Bénédicte
Published: April 2004

Restart of arrested replication forks is an important process and PriA, the main Escherichia coli replication restart protein, is essential for viability under any condition that increases the frequency of fork arrest. In priA mutant, replication forks are arrested by spontaneously occurring roadblocks and blocked replication forks persist as a result of the defect in replication restart. In the present work, we analysed how recombination proteins contribute to the viability of the priA mutant. RecFOR‐mediated homologous recombination occurs in a large fraction of priA mutant cells, indicating a frequent formation of DNA single strand gaps and their recombinational repair. This high level of homologous recombination renders the proteins that resolve Holliday junctions recombination intermediates essential for viability. When homologous recombination is blocked at early steps by recFOR or recA inactivation, exonuclease V‐mediated DNA degradation is required for full viability of priA mutants, indicating that unrepaired gaps are broken and that DNA degradation of the broken DNA allows the formation of viable cells. Models for the formation of single strand DNA gaps consequently to a replication restart defect and for gap processing are proposed.
A lysR‐type regulator is involved in the negative regulation of genes encoding selenium‐free hydrogenases in the archaeon Methanococcus voltae
Authors: Sun Junsong , Klein Albrecht
Published: April 2004

The archaeon Methanococcus voltae encodes two pairs of NiFe‐hydrogenase isoenzymes. One hydrogenase of each pair contains selenium in the active site, whereas the other one is selenium‐free. The gene groups for the selenium‐free hydrogenases, called vhc and frc, are linked by a common intergenic region. They are only transcribed under selenium limitation. A protein binding to a negative regulatory element involved in the regulation of the two operons was purified by DNA‐affinity chromatography. Through the identification of the corresponding gene the protein was found to be a LysR‐type regulator. It was named HrsM (hydrogenase gene regulator, selenium dependent in M. voltae). hrsM knockout mutants constitutively transcribed the vhc and frc operons in the presence of selenium. A putative HrsM binding site was also detected in the intergenic region in front of the hrsM gene. Northern blot analysis indicated that the hrsM gene might be autoregulated.
Bacterial mitosis: partitioning protein ParA oscillates in spiral‐shaped structures and positions plasmids at mid‐cell
Authors: Ebersbach Gitte , Gerdes Kenn
Published: April 2004

The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis‐acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells with a single plasmid focus, the focus located preferentially at mid‐cell. In cells with two foci, these located at quarter‐cell positions. In the absence of ParB and parC1/parC2, ParA‐GFP formed stationary helices extending from one end of the nucleoid to the other. In the presence of ParB and parC1/parC2, ParA‐GFP oscillated in spiral‐shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid‐cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid and subsequently separate them into daughter cells.
Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor
Authors: Ermolaeva Svetlana , Novella Susana , Vega Yolanda , Ripio Maria‐Teresa , Scortti Mariela , Vázquez‐Boland José A.
Published: April 2004

Virulence genes from the facultative intracellular pathogen Listeria monocytogenes are controlled by the transcriptional regulator PrfA. Although PrfA synthesis is activated at 37°C, PrfA‐dependent expression remains low in rich medium. However, a strong induction of the PrfA regulon is observed when L. monocytogenes is cultured in the presence of activated charcoal. Here, we show that the ‘charcoal effect’ results from the adsorption of a diffusible autorepressor substance released by L. monocytogenes during exponential growth. Analyses using an L. monocytogenes strain in which the prfA gene is expressed constitutively at 37°C from a plasmid indicate that the autoregulatory substance represses PrfA‐dependent expression by inhibiting PrfA activity. PrfA presumably functions via an allosteric activation mechanism. The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA. Our data indicate that the listerial autorepressor is a low‐molecular‐weight hydrophobic substance. We suggest that this diffusible substance mediates a quorum‐sensing mechanism by which L. monocytogenes restricts the expression of its PrfA virulence regulon. This autoregulatory pathway could serve L. monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37°C. It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA‐dependent virulence factors in the PrfA‐activating cytosolic compartment.
Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism
Authors: Ito Kaoru , Tanaka Takayoshi , Hatta Rieko , Yamamoto Mikihiro , Akimitsu Kazuya , Tsuge Takashi
Published: April 2004

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce different host‐specific toxins and cause diseases on different plants. The strawberry pathotype produces host‐specific AF‐toxin and causes Alternaria black spot of strawberry. This pathotype is also pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype that produces AK‐toxin. The strawberry pathotype produces two related molecular species, AF‐toxins I and II: toxin I is toxic to both strawberry and pear, and toxin II is toxic only to pear. Previously, we isolated a cosmid clone pcAFT‐1 from the strawberry pathotype that contains three genes involved in AF‐toxin biosynthesis. Here, we have identified a new gene, designated AFTS1, from pcAFT‐1. AFTS1 encodes a protein with similarity to enzymes of the aldo‐ketoreductase superfamily. Targeted mutation of AFTS1 diminished the host range of the strawberry pathotype: ΔaftS1 mutants were pathogenic to pear, but not to strawberry, as is the Japanese pear pathotype. These mutants were found to produce AF‐toxin II, but not AF‐toxin I. These data represent a novel example of how the host range of a plant pathogenic fungus can be restricted by modification of secondary metabolism.
A single‐amino acid substitution in the sixth leucine‐rich repeat of barley MLA6 and MLA13 alleviates dependence on RAR1 for disease resistance signaling
Authors: Halterman Dennis A. , Wise Roger P.
Published: April 2004

Interactions between barley and the powdery mildew pathogen, Blumeria graminis f. sp. hordei, (Bgh) are determined by unique combinations of host resistance genes, designated Mildew‐resistance locus (Ml), and cognate pathogen avirulence genes. These interactions occur both dependent and independent of Rar1 (required for Mla12 resistance) and Sgt1 (Suppressor of G‐two allele of skp1), which are differentially required for diverse plant disease‐resistance pathways. We have isolated two new functional Mla alleles, Rar1‐independent Mla7 and Rar1‐dependent Mla10, as well as the Mla paralogs, Mla6‐2 and Mla13‐2. Utilizing the inherent diversity amongst Mla‐encoded proteins, we identified the only two amino acids exclusively conserved in RAR1‐dependent MLA6, MLA10, MLA12, and MLA13 that differ at the corresponding position in RAR1‐independent MLA1 and MLA7. Two‐ and three‐dimensional modeling places these residues on a predicted surface of the sixth leucine‐rich repeat (LRR) domain at positions distinct from those within the β‐sheets hypothesized to determine resistance specificity. Site‐directed mutagenesis of these residues indicates that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence. These results demonstrate that a single‐amino acid substitution in the sixth MLA LRR can alter host signaling but not resistance specificity to B. graminis.
Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development
Authors: Ye Rui , Yao Quan‐Hong , Xu Zhi‐Hong , Xue Hong‐Wei
Published: April 2004

An efficient yeast‐based system was developed for the isolation of plant cDNAs encoding transcription factors (TFs) and proteins with transcription activation functions (co‐activators). The system consists of two vectors: (i) a reporter vector (pG221) harboring the iso‐1‐cytochrome c (CYC1) core promoter and the β‐galactosidase (lacZ) gene; and (ii) a cDNA library construction vector (pYF503), which yields a library of plant peptides fused to the GAL4‐binding domain (GAL4‐BD). Expression of a peptide harboring the characteristics of a transcriptional activator leads to expression of lacZ, allowing for selection of relevant colonies. TFs during rice embryo development were isolated through this system. Approximately 200 confirmed positive colonies were obtained from screening 106 yeast colonies, and sequence analysis of conserved domains identified 75 independent cDNAs, 20 of which encoded plant TFs or co‐activators, including members of the APETALA2 (AP2)/ethylene‐responsive element‐binding protein (EREBP), MYB and growth‐regulating factor (GRF) families. Peptides encoded by 13 of the isolated cDNAs were classified as potential TFs or co‐activators because of the presence of conserved TF‐like domains. Additionally, 2, 11, and 13 clones encoded kinases, chromosome‐related proteins, and unknown proteins, respectively, while the remaining 16 cDNAs were associated with specific functions seemingly unrelated to TFs. Expression pattern analysis of selected TF‐encoding genes via RT‐PCR revealed that these genes were expressed during seed development, with differential transcription observed during various stages. This work provides informative hints for further study of the regulatory mechanism of rice seed development and illustrates an identification strategy that will be of practical value for the isolation of TFs and co‐activators associated with specific plant developmental processes.
Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses
Authors: Lohar Dasharath Prasad , Schaff Jennifer E. , Laskey James G. , Kieber Joseph J. , Bilyeu Kristin D. , Bird David McK.
Published: April 2004

We used the cytokinin‐responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a β‐glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root‐knot nematode (RKN) second‐stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode–plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6‐(Δ2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode‐induced root galls per plant, than control hairy roots.
A link between sterol biosynthesis, the cell wall, and cellulose in Arabidopsis
Authors: Schrick Kathrin , Fujioka Shozo , Takatsuto Suguru , Stierhof York‐Dieter , Stransky Harald , Yoshida Shigeo , Jürgens Gerd
Published: April 2004

A crucial role for sterols in plant growth and development is underscored by the identification of three Arabidopsis sterol biosynthesis mutants that exhibit embryonic defects: fackel (fk), hydra1 (hyd1), and sterol methyltransferase 1/cephalopod (smt1/cph). We have taken a dual approach of sterol profiling and ultrastructural analysis to investigate the primary defects underlying the mutant phenotypes. Comprehensive gas chromatography (GC)–MS analysis of hyd1 in comparison to fk reveals an abnormal accumulation of unique sterol intermediates in each case. Sterol profiling of the fk hyd1 double mutant provides genetic evidence that FK C‐14 reductase acts upstream of HYD1 C‐8,7 isomerase. Despite distinct differences in sterol profiles, fk and hyd1 as well as smt1/cph share ultrastructural features such as incomplete cell walls and aberrant cell wall thickenings in embryonic and post‐embryonic tissues. The common defects are coupled with ectopic callose and lignin deposits. We show that all three mutants exhibit a deficiency in cellulose, but are not reduced in pectin and sugars of the cell wall and cytosol. The sterol biosynthesis inhibitors 15‐azasterol and fenpropimorph also cause cell wall gaps in dividing root cells and a reduction in bulk cellulose, corroborating that the cell wall abnormalities are due to altered sterol composition. Our results demonstrate that sterols are crucial for cellulose synthesis in the building of the plant cell wall.
The victorin‐induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity
Authors: Curtis Marc J. , Wolpert Thomas J.
Published: April 2004

In this study, we determined the timing of events associated with cell death induced by the host‐selective toxin, victorin. We show that the victorin‐induced collapse in mitochondrial transmembrane potential (Δψm), indicative of a mitochondrial permeability transition (MPT), on a per cell basis, did not occur simultaneously in the entire mitochondrial population. The loss of Δψm in a predominant population of mitochondria preceded cell shrinkage by 20–35 min. Rubisco cleavage, DNA laddering, and victorin binding to the P protein occurred concomitantly with cell shrinkage. During and following cell shrinkage, tonoplast rupture did not occur, and membranes, including the plasma membrane and tonoplast, retained integrity. Ethylene signaling was implicated upstream of a victorin‐induced loss in mitochondrial motility and the collapse in Δψm. Results suggest that the victorin‐induced collapse in Δψm is a consequence of an MPT and that the timing of the victorin‐induced MPT is poised to influence the cell death response. The retention of plasma membrane and tonoplast integrity during cell shrinkage supports the interpretation that victorin induces an apoptotic‐like cell death response.
Automatic quantification of morphological traits via three‐dimensional measurement of Arabidopsis
Authors: Kaminuma Eli , Heida Naohiko , Tsumoto Yuko , Yamamoto Naoki , Goto Nobuharu , Okamoto Naoki , Konagaya Akihiko , Matsui Minami , Toyoda Tetsuro
Published: April 2004

Many mutants have been isolated from the model plant Arabidopsis thaliana, and recent important genetic resources, such as T‐DNA knockout lines, facilitate the speed of identifying new mutants. However, present phenotypic analysis of mutant screens depends mainly on qualitative descriptions after visual observation of morphological traits. We propose a novel method of phenotypic analysis based on precise three‐dimensional (3D) measurement by a laser range finder (LRF) and automatic data processing. We measured the 3D surfaces of young plants of two Arabidopsis ecotypes and successfully defined two new traits, the direction of the blade surface and epinasty of the blade, quantitatively. The proposed method enables us to obtain quantitative and precise descriptions of plant morphologies compared to conventional 2D measurement. The method will open a way to find new traits from mutant pools or natural ecotypes based on 3D data.
The tobacco bZIP transcription factor BZI‐1 binds theGH3promoterin vivoand modulates auxin‐induced transcription
Authors: Heinekamp Thorsten , Strathmann Anne , Kuhlmann Markus , Froissard Marine , Müller Axel , Perrot‐Rechenmann Catherine , Dröge‐Laser Wolfgang
Published: April 2004

In order to establish the biological function of the tobaccobasic leucinezipper (bZIP) transcription factor BZI‐1 in hormone signalling, we have analysed transgenic plants which were altered with respect to the protein level or the activation potential of BZI‐1. Overexpression of a dominant‐negative derivative of BZI‐1, lacking the N‐terminal activation domain, resulted in plants displaying reduced internode size, enhanced lateral shoot formation and small, curly leaves. The response to auxin monitored with reference to root organogenesis, epinastic leaf curvature and transcription of the auxin‐inducedGH3gene was reduced.In vitro, BZI‐1 specifically binds toACGTelements (ACEs) present in theGH3promoter.In vivo, binding to theGH3promoter was confirmed by chromatin immunoprecipitation (ChIP). Overexpression of BZI‐1 in transgenic plants did not lead to a significant activation of theGH3target gene. In contrast, plants expressing a VP16 (Herpes simplex virion protein 16)‐BZI‐1 fusion protein showed enhanced auxin‐inducedGH3transcription. However, VP16‐BZI‐1 is insufficient to triggerGH3expression independently of the auxin stimulus. Whereas auxin responsiveness has been shown to be mediated by ARF (auxin response factor) transcription factors, we discuss a function of BZI‐1 assisting in fine‐tuning of auxin‐induced transcription.
Molecular cloning of the potato Gro1‐4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach
Authors: Paal Jürgen , Henselewski Heike , Muth Jost , Meksem Khalid , Menéndez Cristina M. , Salamini Francesco , Ballvora Agim , Gebhardt Christiane
Published: April 2004

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode–plant interactions and assist in breeding nematode‐resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co‐localized with a resistance‐gene‐like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1‐4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1‐4 encodes a protein of 1136 amino acids that contains Toll‐interleukin 1 receptor (TIR), nucleotide‐binding (NB), leucine‐rich repeat (LRR) homology domains and a C‐terminal domain with unknown function. The deduced Gro1‐4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon‐like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT‐PCR products showed that Gro1‐4 is expressed, among other members of the family including putative pseudogenes, in non‐infected roots of nematode‐resistant plants. RT‐PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.
The tandem complex of BEL and KNOX partners is required for transcriptional repression of ga20ox1
Authors: Chen Hao , Banerjee Anjan K. , Hannapel David J.
Published: April 2004

Two interacting three amino acid loop extension (TALE) proteins of potato, StBEL5 (Solanum tuberosom BEL 5) and POTH1 (potato homeobox 1), mediate developmental processes by regulating phytohormone levels. Overexpression of either partner alone increased tuber yields by lowering gibberellin (GA) levels and increasing cytokinins. Gel shift assays demonstrated that StBEL5 and POTH1 bind to the regulatory region of ga20 oxidase1 (ga20ox1) from potato, a gene encoding a key enzyme in the GA biosynthetic pathway. In tandem, StBEL5 and POTH1 had a greater binding affinity for the ga20ox1 promoter than either protein alone. The StBEL5–POTH1 heterodimer bound specifically to a composite 10‐bp sequence, containing two TGAC cores. Using a transcription assay, StBEL5 and POTH1 suppressed the activity of the ga20ox1 promoter by more than 50%. Dominant negative constructs containing the protein‐binding domains of StBEL5 or POTH1 blocked the repression activity of StBEL5 or POTH1, respectively. The mutated ga20ox1 promoter that could be bound by the StBEL5 or POTH1 proteins individually but not by the StBEL5–POTH1 heterodimer also abolished the repression activity of StBEL5, POTH1, and the StBEL5–POTH1 heterodimer. These results indicate that the tandem interaction of StBEL5 and POTH1 is essential for regulation of the expression of their target gene.
Real‐time RT‐PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root‐ and shoot‐specific genes
Authors: Czechowski Tomasz , Bari Rajendra P. , Stitt Mark , Scheible Wolf‐Rüdiger , Udvardi Michael K.
Published: April 2004

To overcome the detection limits inherent to DNA array‐based methods of transcriptome analysis, we developed a real‐time reverse transcription (RT)‐PCR‐based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene‐specific primer pairs and SYBR® Green to monitor amplification of double‐stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild‐type plants. Only 4% of reactions produced non‐specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR® Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real‐time RT‐PCR revealed 35 root‐specific and 52 shoot‐specific TF genes, most of which have not been identified as organ‐specific previously. Finally, many of the TF transcripts detected by RT‐PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.
The Arabidopsis transcription factor HY5 integrates light and hormone signaling pathways
Authors: Cluis Corinne P. , Mouchel Céline F. , Hardtke Christian S.
Published: April 2004

The role of the Arabidopsis transcription factor LONG HYPOCOTYL 5 (HY5) in promoting photomorphogenic development has been extensively characterized. Although the current model for HY5 action largely explains its role in this process, it does not adequately address the root phenotype observed in hy5 mutants. In our search for common mechanisms underlying all hy5 traits, we found that they are partly the result of an altered balance of signaling through the plant hormones auxin and cytokinin. hy5 mutants are resistant to cytokinin application, and double mutant analyses indicate that a decrease in auxin signaling moderates hy5 phenotypes. Microarray analyses and semiquantitative RT‐PCR indicate that two negative regulators of auxin signaling, AUXIN RESISTANT 2 (AXR2)/INDOLE ACETIC ACID 7 (IAA7) and SOLITARY ROOT (SLR)/IAA14, are underexpressed in hy5 mutants. The promoters of these genes contain a putative HY5 binding site, and in line with this observation, HY5 can bind to the promoter of AXR2 in vitro. Increased AXR2 expression in a hy5 background partially rescues the elongated hypocotyl phenotype. In summary, it appears that auxin signaling is elevated in hy5 mutants because HY5 promotes the expression of negative regulators of auxin signaling, thereby linking hormone and light signaling pathways.
Complex Noun Phrases and Linkers
Authors: Den Dikken Marcel , Singhapreecha Pornsiri
Published: April 2004

Abstract.  Complex noun phrases crosslinguistically contain meaningless elements whose sole function is to serve as linkers of predicates to their subjects—thus, Mandarin Chinese de links inverted AP predicates to their subjects as well as possessors, PPs, relative clauses, and noun‐complement clauses (e.g., the claim that John was asleep). Taking a comparative analysis of French de and Thai thîi as its point of departure, this paper argues for a maximally generalized account of the noun phrases in which linkers occur, in terms of DP‐internal Predicate Inversion. This approach prompts an analysis of relative‐clause constructions that recognizes relative clauses as predicates of DP‐internal small clauses, combining the attractions of the traditional approach and the Vergnaud/Kayne raising approach by assigning relative clauses an internal structure similar to the traditional one while giving it the external distribution of a predicate by treating it as the predicate of a noun‐phrase‐internal small clause. For noun‐complement clauses, the approach leads to a revitalization of Stowell's (1981) predication approach to the relationship between the head noun and the clause, the latter serving as the former's subject in a DP‐contained small‐clause structure. A uniform linker approach to French de, Thai thîi, Mandarin Chinese de, Japanese no, and so on (a) emphasizes the pervasiveness of Predicate Inversion in the noun phrase, (b) confirms the role of linkers as purely functional aides to the inversion operation, (c) furthers our understanding of the structure and derivation of complex noun phrases, and (d) presents a particularly interesting window on microparametric variation in the syntax of noun phrases.
Symmetry in Structure Building
Authors: Hallman Peter
Published: April 2004

Abstract.  This paper defends three interconnected claims: (a) selection is the only licensing procedure available to UG, specifically, checking is an instance of selection; (b) selection obtains in the mutual c‐command configuration; and (c) though a head does not mutually c‐command its own specifier, it mutually c‐commands the specifier of its complement. A head may therefore license the specifier of its complement (as well as its complement) but not its own specifier (it is not local enough). This effectively eliminates the spec‐head configuration from the repertoire of syntactic configurations, in favor of a unified notion of locality strictly identifiable with mutual c‐command, a symmetric configuration. The discussion shows that a theory that collapses these distinctions remains empirically discriminating. The resulting theory is therefore genuinely reductionist.
Early embryogenesis of the pinewood nematode Bursaphelenchus xylophilus
Authors: Hasegawa Koichi , Futai Kazuyoshi , Miwa Satsuki , Miwa Johji
Published: April 2004

The early embryogenesis and cell lineage of the pinewood nematode Bursaphelenchus xylophilus was followed from a single‐cell zygote to a 46‐cell embryo under Nomarski optics, and elongation of the microtubules was studied by immunostaining. As a B. xylophilus oocyte matures, it passes through a passage connecting the oviduct with the quadricolumella, the distal part of the uterus, and reaches the quadricolumella where it stays for a few minutes and is fertilized. After fertilization, the germinal vesicle disappears, an eggshell is formed, and the male and female pronuclei appear. The pronuclei move toward each other and fuse at the center of the egg. Around this time, the microtubule‐organizing center appears. The presumptive region of sperm entry into the oocyte becomes the future anterior portion of the embryo. This anterior–posterior axis determination is opposite to that of Caenorhabditis elegans, where the sperm entry site becomes the posterior portion of the embryo. The optimal growth temperatures of these two nematodes also differ in that temperatures of about 30°C afford the fastest growth rate and highest hatching frequency in B. xylophilus. Otherwise, the lineage resembles that of C. elegans with respect to timing, positioning and the axis orientation of each cell division.
Calcineurin inhibitors block dorsal‐side signaling that affect late‐stage development of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis
Authors: Yoshida Yoko , Kim Shokaku , Chiba Kazuhiro , Kawai Shinya , Tachikawa Hiroyuki , Takahashi Nobuhiro
Published: April 2004

Calcineurin, a calcium/calmodulin‐dependent serine/threonine protein phosphatase, is a key constituent of signaling pathways involved in antigen‐dependent T‐cell activation and development of the mammalian heart. In addition, calcineurin constitutes a part of the Wnt/calcium‐signaling pathway that regulates early stages of dorsoventral axis formation in Xenopus embryos. Although some of the Wnt family members are involved in organ formation at relatively late stages of Xenopus development, the involvement of calcineurin in the development of those organs remains unclear. In the present study, we demonstrate that calcineurin inhibitors (cyclosporine A, FK506, and FK520), but not non‐calcineurin inhibitors (rapamycin and GPI1046) that bind the same intracellular receptor as that for FK506, induce edema and gut coiling disruption and exhibit teratogenesis in the kidney, heart, gut, liver, and somitic tissue during Xenopus development. The same effects were observed by injecting the calcineurin inhibitors into the dorsal side, but not ventral side, of blastomeres at the 4‐cell stage, although the inhibitors did not affect dorsoventral axis formation. These results suggest that calcineurin is involved in dorsal‐side signaling that leads to the formation of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis.
Tracing cell fate in brain formation during embryogenesis of the ascidian Halocynthia roretzi
Authors: Taniguchi Kazuto , Nishida Hiroki
Published: April 2004

Ascidian eggs develop into tadpole larvae. They have a simple central nervous system (CNS) at the dorsal midline. The CNS is formed through neural tube formation at the neurula stage, as in vertebrates. The total number of cells in the CNS is approximately 300. In Halocynthia roretzi, the anterior part of the CNS, which consists of the brain (sensory vesicle) and the visceral ganglion, is exclusively derived from 10 blastomeres at the 110‐cell stage. The anterior CNS is relatively complex and shows remarkable left–right asymmetry, with the lumen of the sensory vesicle, the otolith, and the ocellus on the right side, and the presumed hydrostatic pressure organ on the left side. We labeled these 10 precursor blastomeres – six in the animal hemisphere (a‐line) and four in the vegetal hemisphere (A‐line) – with lineage tracer, and examined the fates in swimming larvae. The clonal organization of the anterior CNS is essentially invariant among individuals, although slight variation in the clonal boundary was observed. There was no extensive mixing between descendants of each precursor. We observed no evidence of cell migration except for two neuronal cells derived from a8.25 blastomeres. The eventual fates of the bilateral blastomeres produced extensive left–right asymmetry. The results suggest that the anterior neural tube rotates in a clockwise direction when viewed from the posterior pole. Staged observation indicated that this rotation takes place during the last 5 h of embryogenesis. We describe detailed positions of descendants of each precursor blastomere. In particular, specific cells of sensory structures were identified by their morphology and staining with specific antibodies and probes. The otolith and ocellus pigment cells were derived from left and right a8.25 blastomeres. Lens cells of the ocellus have a right A8.8 origin, and most of the photoreceptor cells originated from the right A8.7. The presumed pressure organ was formed by descendants of left and right a8.19 and left a8.17 blastomeres. The description of cell lineages of the CNS would facilitate future research to analyze the mechanisms of development of the simple CNS of ascidian tadpole larvae.