bluuebunny/crossref_metadata_2025 huggingface dataset:
The C-terminal Region of Mitochondrial Single-subunit RNA Polymerases Contains Species-specific Determinants for Maintenance of Intact Mitochondrial Genomes
Authors: Lisowsky Thomas , Wilkens Detlef , Stein Torsten , Hedtke Boris , Börner Thomas , Weihe Andreas
Published: July 2002

Functional conservation of mitochondrial RNA polymerases was investigated in vivo by heterologous complementation studies in yeast. It turned out that neither the full-length mitochondrial RNA polymerase of Arabidopsis thaliana, nor a set of chimeric fusion constructs from plant and yeast RNA polymerases can substitute for the yeast mitochondrial core enzyme Rpo41p when expressed in Δrpo41 yeast mutants. Mitochondria from mutant cells, expressing the heterologous mitochondrial RNA polymerases, were devoid of any mitochondrial genomes. One important exception was observed when the carboxyl-terminal domain of Rpo41p was exchanged with its plant counterpart. Although this fusion protein could not restore respiratory function, stable maintenance of mitochondrial petite genomes (ρ−)−was supported. A carboxyl-terminally truncated Rpo41p exhibited a comparable activity, in spite of the fact that it was found to be transcriptionally inactive. Finally, we tested the carboxyl-terminal domain for complementation intrans. For this purpose the last 377 amino acid residues of yeast mitochondrial Rpo41p were fused to its mitochondrial import sequence. Coexpression of this fusion protein with C-terminally truncated Rpo41p complemented the Δrpo41 defect. These data reveal the importance of the carboxyl-terminal extension of Rpo41p for stable maintenance of intact mitochondrial genomes and for distinct species-specific intramolecular protein–protein interactions.
Recognition of a Subset of Signal Sequences by Ssh1p, a Sec61p-related Protein in the Membrane of Endoplasmic Reticulum of YeastSaccharomyces cerevisiae
Authors: Wittke Sandra , Dünnwald Martin , Albertsen Markus , Johnsson Nils
Published: July 2002

Ssh1p of Saccharomyces cerevisiae is related in sequence to Sec61p, a general receptor for signal sequences and the major subunit of the channel that guides proteins across the membrane of the endoplasmic reticulum. The split-ubiquitin technique was used to determine whether Ssh1p serves as an additional receptor for signal sequences in vivo. We measured the interactions between the Nub-labeled Ssh1p and Cub-translocation substrates bearing four different signal sequences. The so-determined interaction profile of Ssh1p was compared with the signal sequence interaction profile of the correspondingly modified Nub-Sec61p. The assay reveals interactions of Ssh1p with the signal sequences of Kar2p and invertase, whereas Sec61p additionally interacts with the signal sequences of Mfα1 and carboxypeptidase Y. The measured physical proximity between Ssh1p and the β-subunit of the signal sequence recognition particle receptor confirms our hypothesis that Ssh1p is directly involved in the cotranslational translocation of proteins across the membrane of the endoplasmic reticulum.
Genomic Screen for Vacuolar Protein Sorting Genes inSaccharomyces cerevisiae
Authors: Bonangelino Cecilia J. , Chavez Edna M. , Bonifacino Juan S.
Published: July 2002

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains ofSaccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature α-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30°C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.
Large-Scale Isolation of Cajal Bodies from HeLa Cells
Authors: Lam Yun Wah , Lyon Carol E. , Lamond Angus I.
Published: July 2002

The Cajal body (CB) is a conserved, dynamic nuclear structure that is implicated in various cellular processes, such as the maturation of splicing small nuclear ribonucleoproteins and the assembly of transcription complexes. Here, we report the first procedure for the large-scale purification of CBs from HeLa cell nuclei, resulting in an ∼750-fold enrichment of the CB marker protein p80-coilin. Immunofluorescence, immunoblotting, and mass spectrometric analyses showed that the composition of the isolated CBs was similar to that of CBs in situ. The morphology and structure of the isolated CBs, as judged by transmission and scanning electron microscopy analysis, are also similar to those of CBs in situ. This protocol demonstrates the feasibility of isolating intact distinct classes of subnuclear bodies from cultured cells in sufficient yield and purity to allow detailed characterization of their molecular composition, structure, and properties.
Rac Regulates Endothelial Morphogenesis and Capillary Assembly
Authors: Connolly John O. , Simpson Nandi , Hewlett Lindsay , Hall Alan
Published: July 2002

Endothelial cells undergo branching morphogenesis to form capillary tubes. We have utilized an in vitro Matrigel overlay assay to analyze the role of the cytoskeleton and Rho GTPases during this process. The addition of matrix first induces changes in cell morphology characterized by the formation of dynamic cellular protrusions and the assembly of discrete aggregates or cords of aligned cells resembling primitive capillary-like structures, but without a recognizable lumen. This is followed by cell migration leading to the formation of a complex interconnecting network of capillary tubes with readily identifiable lumens. Inhibition of actin polymerization or actin-myosin contraction inhibits cell migration but has no effect on the initial changes in endothelial cell morphology. However, inhibition of microtubule dynamics prevents both the initial cell shape changes as well as cell migration. We find that the small GTPase Rac is essential for the matrix-induced changes in endothelial cell morphology, whereas p21-activated kinase, an effector of Rac, is required for cell motility. We conclude that Rac integrates signaling through both the actin and microtubule cytoskeletons to promote capillary tube assembly.
Neuronal Ceroid Lipofuscinoses Are Connected at Molecular Level: Interaction of CLN5 Protein with CLN2 and CLN3
Authors: Vesa Jouni , Chin Mark H. , Oelgeschläger Kathrin , Isosomppi Juha , DellAngelica Esteban C. , Jalanko Anu , Peltonen Leena
Published: July 2002

Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative storage diseases characterized by mental retardation, visual failure, and brain atrophy as well as accumulation of storage material in multiple cell types. The diseases are caused by mutations in the ubiquitously expressed genes, of which six are known. Herein, we report that three NCL disease forms with similar tissue pathology are connected at the molecular level: CLN5 polypeptides directly interact with the CLN2 and CLN3 proteins based on coimmunoprecipitation and in vitro binding assays. Furthermore, disease mutations in CLN5 abolished interaction with CLN2, while not affecting association with CLN3. The molecular characterization of CLN5 revealed that it was synthesized as four precursor forms, due to usage of alternative initiator methionines in translation. All forms were targeted to lysosomes and the longest form, translated from the first potential methionine, was associated with membranes. Interactions between CLN polypeptides were shown to occur with this longest, membrane-bound form of CLN5. Both intracellular targeting and posttranslational glycosylation of the polypeptides carrying human disease mutations were similar to wild-type CLN5.
A Conserved DrosophilaTransportin-Serine/Arginine-rich (SR) Protein Permits Nuclear Import ofDrosophila SR Protein Splicing Factors and Their Antagonist Repressor Splicing Factor 1
Authors: Allemand Eric , Dokudovskaya Svetlana , Bordonné Rémy , Tazi Jamal
Published: July 2002

Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted us to examine the mechanism by whichDrosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. Herein, we report the identification and characterization of a Drosophilaimportin β-family protein (dTRN-SR), homologous to TRN-SR2, that specifically interacts with both SR proteins and RSF1. dTRN-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with dTRN-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, we demonstrate that complementation with dTRN-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans.
Global analysis of tandem aromatic octapeptide repeats:
The significance of the aromatic–glycine motif
Authors: Gazit Ehud
Published: July 2002

Motivation: Tandem peptide repeats play a key role in self-assembly and aggregation processes. A notable example is the occurrence of tandem peptide repeats in prionic proteins and their role in the aggregation process that leads to the formation of the prion. One of the structural characteristics that is evident from the comparison of mammalian and yeast prion proteins is the presence of aromatic residues in their tandem repeats. These residues are accompanied by glycine residues before and/or after the aromatic amino acid. Such aromatic–glycine conjugates are also present in the tandem repeats of the large family of the bacterial ice nucleation proteins. To study the significance of such aromatic–glycine occurrences, a global analysis of all the aromatic octapeptide repeats in the Swiss-Prot and TrEMBL databases was conducted. The search pattern was formulated to compare the number of conjugates of each of the 20 natural amino acids before or after the different aromatic residues.

Results: The presence of aromatic-glycine conjugates appears to be significantly higher than aromatic conjugates to any other amino acid. Furthermore, all the six various combination of glycine occurrences before or after the three aromatic residues are present. No such pattern was observed for any other amino acid. The significance of the findings is being discussed in the context of the physicochemical properties of aromatic–glycine conjugates and its possible role in the facilitation of aggregates formation.

Contact: ehudg@post.tau.ac.il
Synonymous–non-synonymous mutation rates between sequences
containing ambiguous nucleotides (Syn-SCAN)
Authors: Gonzales Matthew J. , Dugan Jonathan M. , Shafer Robert W.
Published: July 2002

Summary: Direct PCR sequencing on genetic material containing allelic mixtures results in sequences containing ambiguous nucleotides. Because codons exhibiting allelic mixtures present evidence of evolutionary pressure, it is important to include this information in the assessment of codon synonymy. We developed a program, ‘Synonymous–Nonsynonymous Mutation Rates between Sequences Containing Ambiguous Nucleotides’ (Syn-SCAN), that calculates synonymous and non-synonymous substitution rates using a model that includes allelic mixtures.

Availability: Syn-SCAN is implemented on the web and can be downloaded from http://hivdb.stanford.edu

Contact: rshafer@cmgm.stanford.edu

* To whom correspondence should be addressed.
TangleSolve: topological analysis of site-specific recombination
Authors: Saka Yuki , Vázquez Mariel
Published: July 2002

Summary: TangleSolve is a program for analysing site-specific recombination using the tangle model. The program offers an easy-to-use graphical user interface and a visualization tool. Biologists working in topological enzymology can use this program to compute and visualize site-specific recombination mechanisms that accommodate their experimental data. TangleSolve can also prove useful as a teaching aid for mathematical biology and computational molecular biology courses.

Availability: http://bio.math.berkeley.edu/TangleSolve/

Contact: mariel@math.berkeley.edu

* To whom correspondence should be addressed.
A bioinformatic strategy to rapidly characterize cDNA libraries
Authors: Ostermeier G. Charles , Dix David J. , Krawetz Stephen A.
Published: July 2002

Motivation: Complementary DNA libraries can define the genetic constituents of specific cells and/or tissues. Their sequencing will illuminate the transcriptome but it is a monumental task requiring considerable resources.

Results: We have employed a computational search in conjunction with a microarray-based strategy to alleviate the impediments of deriving a consensus of records that describe testis gene expression. This strategy identified 5681 unique testes-expressed genes of which 3265 were previously portrayed in the UniGene database. Interestingly, a total of 2416 novel testes-expressed genes were identified. This clearly demonstrates that microarrays can be used to rapidly discover a large number of new transcripts.

Contact: steve@compbio.med.wayne.edu

* To whom correspondence should be addressed at: Charlotte B. Failing Professor, 253 C.S. Mott Center, 275 East Hancock, Detroit, MI, 48201, USA.
Comparative genomics study of inverted repeats in bacteria
Authors: Lillo Fabrizio , Basile Salvatore , Mantegna Rosario N.
Published: July 2002

Motivation: Comparative genomics provides a powerful way to investigate regularities and differences observed at DNA level across species. Here we study the number and location of inverted repeats occurring in complete genomes of bacteria. Inverted repeats are compatible with the formation of hairpin structures in the messenger RNA. Some of these structures are known to be rho-independent intrinsic terminators.

Results: We investigate the number of inverted repeats observed in 37 complete genomes of bacteria. The number of inverted repeats observed is much higher than expected using Markovian models of DNA sequences in most of the eubacteria. By using the information annotated in the genomes we discover that in most of the eubacteria the inverted repeats of stem length longer than 8 nucleotides preferentially locate near the 3′ end of the nearest coding regions. We also show that IRs characterized by large values of the stem length locate preferentially in short non-coding regions bounded by two 3′ ends of convergent genes. By using the program TransTerm recently introduced to predict transcription terminators in bacterial genomes, we conclude that only a part of the observed inverted repeats fullfils the model requirements characterizing rho-independent termination in several genomes.

Availability: http://lagash.dft.unipa.it/IR.html

Contact: mantegna@unipa.it
CX, an algorithm that identifies protruding atoms in proteins
Authors: Pintar Alessandro , Carugo Oliviero , Pongor Sándor
Published: July 2002

Motivation: A simple and fast algorithm is described that calculates a measure of protrusion (cx) for atoms in protein structures, directly useable with the common molecular graphics programs.

Results: A sphere of predetermined radius is centered around each non-hydrogen atom, and the volume occupied by the protein and the free volume within the sphere (internal and external volumes, respectively) are calculated. Atoms in protruding regions have a high ratio (cx) between the external and the internal volume. The program reads a PDB file, and writes the output in the same format, with cx values in the B factor field. Output structure files can be directly displayed with standard molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB Viewer and colored according to cx values. We show the potential use of this program in the analysis of two protein–protein complexes and in the prediction of limited proteolysis sites in native proteins.

Availability: The algorithm is implemented in a standalone program written in C and its source is freely available at ftp.icgeb.trieste.it/pub/CX or on request from the authors.

Contact: pintar@icgeb.trieste.it; carugo@icgeb.trieste.it; pongor@icgeb.trieste.it

* To whom correspondence should be addressed.
Calculations of protein volumes: sensitivity analysis and
parameter database
Authors: Tsai Jerry , Gerstein Mark
Published: July 2002

Motivation: The precise sizes of protein atoms in terms of occupied packing volume are of great importance. We have previously presented standard volumes for protein residues based on calculations with Voronoi-like polyhedra. To understand the applicability and limitations of our set, we investigated, in detail, the sensitivity of the volume calculations to a number of factors: (i) the van der Waals radii set, (ii) the criteria for including buried atoms in the calculations or atom selection, (iii) the method of positioning the dividing plane in polyhedra construction, and (iv) the set of structures used in the averaging.

Results: We find that different radii sets have only moderate affects to the distribution and mean of volumes. Atom selection and dividing plane methods cause larger changes in protein atoms volumes. More significantly, we show how the variation in volumes appears to be clearly related to the quality of the structures analyzed, with higher quality structures giving consistently smaller average volumes with less variance.

Availability/Supplementary Information: Programs and associated data files are available from http://bioinfo.mbb.yale.edu/geometry and http://molmovdb.org. In particular, we make available an extensive database of many different sets of protein geometric parameters.

Contact: JerryTsai@TAMU.edu; Mark.Gerstein@Yale.edu

* To whom correspondence should be addressed.
CUSTOMER TYPE AND BANK EFFICIENCY IN POLAND: IMPLICATIONS FOR EMERGING MARKET BANKING
Authors: Nikiel Ewa M. , Opiela Timothy P.
Published: July 2002

Bank efficiency studies on emerging markets tend to show that foreign banks are more cost‐, profit‐, and operationally efficient than statE‐owned or domestic private banks. They also show that large banks are more efficient than small banks. Using a parametric approach to measuring efficiency, this article finds that foreign banks servicing foreign and business customers are more cost‐efficient and less profit‐efficient than other banks in Poland. Additionally, evidence of cost economies and profit diseconomies of scale are found. These results have implications for regulatory policies focused on promoting efficiency in the banking systems of emerging markets.
TRADE LIBERALIZATION AND THE MANUFACTURING SECTOR: THE CASE OF THE SMALL DEVELOPING COUNTRY
Authors: Lewis‐Bynoe Denny , Griffith Jennifer , Moore Winston
Published: July 2002

Historically, the lack of competition in developing countries has resulted in highly concentrated domestic industries that suffer from diseconomies of scale but prosper behind high walls of protection. Liberalization is expected to reverse this trend but at what cost? This article utilizes an import demand framework to examine the potential impact of trade liberalization on the manufacturing sector in the Caribbean using the case of Barbados. The results indicate that the manufacturing industry could encounter tremendous price competition, which could compromise the future survival of these industries. The study recommends that industries reorganize production processes to increase efficiency, which will allow them to compete effectively in the new global trading environment. These results may also be applicable to the wider Caribbean.
Structure and function of antifreeze proteins
Authors: Davies Peter L. , Baardsnes Jason , Kuiper Michael J. , Walker Virginia K.
Published: July 2002

High–resolution three–dimensional structures are now available for four of seven non–homologous fish and insect antifreeze proteins (AFPs). For each of these structures, the ice–binding site of the AFP has been defined by site–directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice–binding proteins shows that they have the following attributes in common. The binding sites are relatively flat and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic—more so than that portion of the protein exposed to the solvent. Surface–surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts.
The shoot apical meristem: the dynamics of a stable structure
Authors: Traas Jan , Vernoux Teva
Published: July 2002

The shoot apical meristem (SAM) is a group of proliferating, embryonic–type cells that generates the aerial parts of the plant. SAMs are highly organized and stable structures that can function for years or even centuries. This is in apparent contradiction to the behaviour of their constituent cells, which continuously proliferate and differentiate. To reconcile the dynamic nature of the cells with the stability of the overall system the existence of elaborate signalling networks has been proposed. This is supported by recent work suggesting that the exchange of signals between cells, rather than a rigidly predetermined genetic program, is required for the establishment and functioning of an organized meristem. Together these interactions form a stable network, set up during embryogenesis, that assures the coordination of cell behaviour throughout development. Besides meristem–specific signalling cascades such as the CLAVATA receptor kinase pathway, which controls meristem size, these interactions involve plant hormones. In particular, cytokinins and auxins are implicated in the maintenance of meristem identity and phyllotaxis, respectively.
Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity
Authors: Lee Catherine S. , Perreault Nathalie , Brestelli John E. , Kaestner Klaus H.
Published: July 2002

The notch signaling pathway is essential for the endocrine cell fate in various tissues including the enteroendocrine system of the gastrointestinal tract. Enteroendocrine cells are one of the four major cell types found in the gastric epithelium of the glandular stomach. To understand the molecular basis of enteroendocrine cell development, we have used gene targeting in mouse embryonic stem cells to derive anEGFP-marked null allele of the bHLH transcription factor,neurogenin 3 (ngn3). In ngn3−/−mice, glucagon secreting A-cells, somatostatin secreting D-cells, and gastrin secreting G-cells are absent from the epithelium of the glandular stomach, whereas the number of serotonin-expressing enterochromaffin (EC) cells is decreased dramatically. In addition,ngn3−/− mice display intestinal metaplasia of the gastric epithelium. Thus, ngn3 is required for the differentiation of enteroendocrine cells in the stomach and the maintenance of gastric epithelial cell identity.
Spo13 regulates cohesin cleavage
Authors: Lee Brian H. , Amon Angelika , Prinz Susanne
Published: July 2002

A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during meiosis I and from centromeric regions during meiosis II. The budding yeast protein Spo13 plays a key role in preventing centromeric cohesin from being lost during meiosis I. We have determined the molecular basis for the metaphase arrest obtained when SPO13 is overexpressed during the mitotic cell cycle. Overexpression of SPO13 inhibits anaphase onset by at least two mechanisms. First, Spo13 causes a transient delay in degradation of the anaphase inhibitor Pds1. Second, Spo13 inhibits cleavage of the cohesin subunit Scc1/Mcd1 or its meiosis-specific homolog, Rec8, by the separase Esp1. The finding that Spo13 did not prevent cleavage of another Esp1 substrate, Slk19, suggests that overexpression ofSPO13 is sufficient to prevent cohesin cleavage by protecting specific substrates from separase activity.
A New Group-Training Procedure for Habituation Demonstrates That Presynaptic Glutamate Release Contributes to Long-Term Memory in Caenorhabditis elegans
Authors: Rose Jacqueline K. , Kaun Karla R. , Rankin Catharine H.
Published: July 2002

In the experiments reported here we have developed a new group-training protocol for assessing long-term memory for habituation in Caenorhabditis elegans. We have replicated all of the major findings of the original single-worm protocol using the new protocol: (1) distributed training produced long-term retention of training, massed training did not; (2) distributed training at long interstimulus intervals (ISIs) produced long-term retention, short ISIs did not; and (3) long-term memory for distributed training is protein synthesis-dependent as it could be blocked by heat shock during the inter-block interval. In addition, we have shown that long-term memory for habituation is graded, depending on the number of blocks of stimuli in training. The inter-block interval must be >40 min for long-term retention of training to occur. Finally, we have tested long-term memory for habituation training in a strain of worms with a mutation in a vesicular glutamate transporter in the sensory neurons that transduce tap (eat-4). The results from these eat-4 worms indicate that glutamate release from the sensory neurons has an important role in the formation of long-term memory for habituation.
Mapping of Olfactory Memory Circuits: Region-Specific c-fos Activation After Odor-Reward Associative Learning or After Its Retrieval
Authors: Tronel Sophie , Sara Susan J.
Published: July 2002

Although there is growing knowledge about intracellular mechanisms underlying neuronal plasticity and memory consolidation and reconsolidation after retrieval, information concerning the interaction among brain areas during formation and retrieval of memory is relatively sparse and fragmented. Addressing this question requires simultaneous monitoring of activity in multiple brain regions during learning, the post-acquisition consolidation period, and retrieval and subsequent reconsolidation. Immunoreaction to the immediate early genec-fos is a powerful tool to mark neuronal activation of specific populations of neurons. Using this method, we are able to report, for the first time, post-training activation of a network of closely related brain regions, particularly in the frontal cortex and the basolateral amygdala (BLA), that is specific to the learning of an odor-reward association. On the other hand, retrieval of a well-established associative memory trace does not seem to differentially activate the same regions. The amygdala, in particular, is not engaged after retrieval, whereas the lateral habenula (LHab) shows strong activation that is restricted to animals having previously learned the association. Although intracellular mechanisms may be similar during consolidation and reconsolidation, this study indicates that different brain circuits are involved in the two processes, at least with respect to a rapidly learned olfactory task.
The Role of Ethylene and Wound Signaling in Resistance of Tomato to Botrytis cinerea
Authors: Dı́az José , ten Have Arjen , van Kan Jan A.L.
Published: July 2002

Ethylene, jasmonate, and salicylate play important roles in plant defense responses to pathogens. To investigate the contributions of these compounds in resistance of tomato (Lycopersicon esculentum) to the fungal pathogen Botrytis cinerea, three types of experiments were conducted: (a) quantitative disease assays with plants pretreated with ethylene, inhibitors of ethylene perception, or salicylate; (b) quantitative disease assays with mutants or transgenes affected in the production of or the response to either ethylene or jasmonate; and (c) expression analysis of defense-related genes before and after inoculation of plants with B. cinerea. Plants pretreated with ethylene showed a decreased susceptibility toward B. cinerea, whereas pretreatment with 1-methylcyclopropene, an inhibitor of ethylene perception, resulted in increased susceptibility. Ethylene pretreatment induced expression of several pathogenesis-related protein genes before B. cinerea infection. Proteinase inhibitor I expression was repressed by ethylene and induced by 1-methylcyclopropene. Ethylene also induced resistance in the mutantNever ripe. RNA analysis showed that Never ripe retained some ethylene sensitivity. The mutantEpinastic, constitutively activated in a subset of ethylene responses, and a transgenic line producing negligible ethylene were also tested. The results confirmed that ethylene responses are important for resistance of tomato to B. cinerea. The mutant Defenseless, impaired in jasmonate biosynthesis, showed increased susceptibility to B. cinerea. A transgenic line with reduced prosystemin expression showed similar susceptibility as Defenseless, whereas a prosystemin-overexpressing transgene was highly resistant. Ethylene and wound signaling acted independently on resistance. Salicylate and ethylene acted synergistically on defense gene expression, but antagonistically on resistance.
PROLIFERATING INFLORESCENCE MERISTEM, a MADS-Box Gene That Regulates Floral Meristem Identity in Pea
Authors: Taylor Scott A. , Hofer Julie M.I. , Murfet Ian C. , Sollinger John D. , Singer Susan R. , Knox Maggie R. , Ellis T.H. Noel
Published: July 2002

SQUAMOSA and APETALA1 are floral meristem identity genes from snapdragon (Antirrhinum  majus) and Arabidopsis, respectively. Here, we characterize the floral meristem identity mutation proliferating inflorescence meristem(pim) from pea (Pisum sativum) and show that it corresponds to a defect in the PEAM4 gene, a homolog of SQUAMOSA and APETALA1. ThePEAM4 coding region was deleted in thepim-1 allele, and this deletion cosegregated with thepim-1 mutant phenotype. The pim-2 allele carried a nucleotide substitution at a predicted 5′ splice site that resulted in mis-splicing of pim-2 mRNA. PCR products corresponding to unspliced and exon-skipped mRNA species were observed. The pim-1 and pim-2 mutations delayed floral meristem specification and altered floral morphology significantly but had no observable effect on vegetative development. These floral-specific mutant phenotypes and the restriction ofPIM gene expression to flowers contrast with other known floral meristem genes in pea that additionally affect vegetative development. The identification of PIM provides an opportunity to compare pathways to flowering in species with different inflorescence architectures.
The Effects of Abiotic Factors on Induced Volatile Emissions in Corn Plants
Authors: Gouinguené Sandrine P. , Turlings Ted C.J.
Published: July 2002

Many plants respond to herbivory by releasing a specific blend of volatiles that is attractive to natural enemies of the herbivores. In corn (Zea mays), this induced odor blend is mainly composed of terpenoids and indole. The induced signal varies with plant species and genotype, but little is known about the variation due to abiotic factors. Here, we tested the effect of soil humidity, air humidity, temperature, light, and fertilization rate on the emission of induced volatiles in young corn plants. Each factor was tested separately under constant conditions for the other factors. Plants released more when standing in dry soil than in wet soil, whereas for air humidity, the optimal release was found at around 60% relative humidity. Temperatures between 22°C and 27°C led to a higher emission than lower or higher temperatures. Light intensity had a dramatic effect. The emission of volatiles did not occur in the dark and increased steadily with an increase in the light intensity. An experiment with an unnatural light-dark cycle showed that the release was fully photophase dependent. Fertilization also had a strong positive effect; the emission of volatiles was minimal when plants were grown under low nutrition, even when results were corrected for plant biomass. Changes in all abiotic factors caused small but significant changes in the relative ratios among the different compounds (quality) in the induced odor blends, except for air humidity. Hence, climatic conditions and nutrient availability can be important factors in determining the intensity and variability in the release of induced plant volatiles.
Arabidopsis Map-Based Cloning in the Post-Genome Era
Authors: Jander Georg , Norris Susan R. , Rounsley Steven D. , Bush David F. , Levin Irena M. , Last Robert L.
Published: July 2002

Map-based cloning is an iterative approach that identifies the underlying genetic cause of a mutant phenotype. The major strength of this approach is the ability to tap into a nearly unlimited resource of natural and induced genetic variation without prior assumptions or knowledge of specific genes. One begins with an interesting mutant and allows plant biology to reveal what gene or genes are involved. Three major advances in the past 2 years have made map-based cloning in Arabidopsis fairly routine: sequencing of the Arabidopsis genome, the availability of more than 50,000 markers in the Cereon Arabidopsis Polymorphism Collection, and improvements in the methods used for detecting DNA polymorphisms. Here, we describe the Cereon Collection and show how it can be used in a generic approach to mutation mapping in Arabidopsis. We present the map-based cloning of theVTC2 gene as a specific example of this approach.
RASPBERRY3Gene Encodes a Novel Protein Important for Embryo Development
Authors: Apuya Nestor R. , Yadegari Ramin , Fischer Robert L. , Harada John J. , Goldberg Robert B.
Published: July 2002

We identified a new gene that is interrupted by T-DNA in an Arabidopsis embryo mutant called raspberry3.  raspberry3 has “raspberry-like” cellular protuberances with an enlarged suspensor characteristic of otherraspberry embryo mutants, and is arrested morphologically at the globular stage of embryo development. The predicted RASPBERRY3 protein has domains found in proteins present in prokaryotes and algae chloroplasts. Computer prediction analysis suggests that the RASPBERRY3protein may be localized in the chloroplast. Complementation analysis supports the possibility that the RASPBERRY3 protein may be involved in chloroplast development. Our experiments demonstrate the important role of the chloroplast, directly or indirectly, in embryo morphogenesis and development.
Molecular and Physiological Analysis of Arabidopsis Mutants Defective in Cytosolic or Chloroplastic Aspartate Aminotransferase
Authors: Miesak Barbara H. , Coruzzi Gloria M.
Published: July 2002

Arabidopsis mutants deficient in cytosolic (AAT2) or chloroplastic (AAT3) aspartate (Asp) aminotransferase were characterized at the molecular and physiological levels. All of the ethyl methane sulfonate- or nitrosomethylurea-generated mutants are missense mutations, as determined by sequencing of theASP2 gene from the cytosolic aat2 mutants (aat2-1, aat2-2, aat2-4, and aat2-5) and the ASP5 gene from the chloroplastic aat3 mutants (aat3-1,aat3-2, and aat3-4). A T-DNA insertion mutant in cytosolic AAT2 (aat2-T) was also identified. All the cytosolic aat2 and chloroplasticaat3 mutants have less than 6% AAT2 and less than 3% AAT3 activity, respectively, as determined by the native gel assay; however, none are nulls. The metabolic and physiological affect of these mutations in AAT isoenzymes was determined by measuring growth and amino acid levels in the aat mutants. Twoaat2 mutants (aat2-2 andaat2-T) show reduced root length on Murashige and Skoog medium. For aat2-2, this growth defect is exaggerated by Asp supplementation, suggesting a defect in Asp metabolism. Amino acid analysis of the aat mutants showed alterations in levels of Asp and/or Asp-derived amino acids in several aat2alleles. Two aat2 mutants show dramatic decreases in Asp and asparagine levels in leaves and/or siliques. As such, the cytosolic AAT2 isoenzyme appears to serve a nonredundant function in plant nitrogen metabolism of Asp and Asp-derived amino acids.
Arabidopsis Mutants Deficient in Diacylglycerol Acyltransferase Display Increased Sensitivity to Abscisic Acid, Sugars, and Osmotic Stress during Germination and Seedling Development
Authors: Lu Chaofu , Hills Matthew J.
Published: July 2002

Arabidopsis seeds store triacylglycerol (TAG) as the major carbon reserve, which is used to support postgerminative seedling growth. Diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG synthesis, and two isoforms of DGAT have previously been identified in Arabidopsis. It has been shown that DGAT1 plays an important role in seed development because Arabidopsis with mutations at the TAG1 locus accumulate less seed oil. There is also evidence showing that DGAT1 is active after seed germination. The aim of this study is to investigate the effect of mutations of DGAT1 on postembryonic development in Arabidopsis. We carried out detailed analyses of two tag1 mutants in different ecotypic backgrounds of Arabidopsis. Results show that during germination and seedling growth, seed storage TAG degradation was not affected in thetag1 mutants. However, sugar content of the mutant seedlings is altered, and activities of the hexokinases are significantly increased in the tag1 mutant seedlings. The tag1 mutants are also more sensitive to abscisic acid, glucose, and osmotic strength of the medium in germination and seedling growth.
Effect of Short-Term N2 Deficiency on Expression of the Ureide Pathway in Cowpea Root Nodules
Authors: Smith Penelope M.C. , Winter Heike , Storer Paul J. , Bussell John D. , Schuller Kathryn A. , Atkins Craig A.
Published: July 2002

Root systems of 28-d-old cowpea (Vigna unguiculata L. Walp cv Vita 3: Bradyrhizobiumsp. strain CB756) plants bearing nitrogen-fixing nodules in sand culture were exposed to an atmosphere of Ar:O2 (80:20, v/v) for 48 h and then returned to air. Root systems of control plants were maintained in air throughout. Nodules were harvested at the same times in control and Ar:O2-treated root systems. Activities of two enzymes of de novo purine synthesis, glycinamide ribonucleotide transformylase (GART; EC 2.1.2.2), aminoimidazole ribonucleotide synthetase (AIRS; EC 6.3.3.1), uricase (EC 1.7.3.3), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) were measured together with the protein level of each using immune-specific polyclonal antibodies. AIRS activity and protein both declined to very low levels within 6 h in Ar:O2 together with a decline in transcript level of pur5, the encoding gene. GART activity, protein, and transcript (pur3) levels were relatively stable. Uricase activity declined in Ar:O2 as rapidly as AIRS activity but the protein was stable. PEPC activity showed evidence of increased sensitivity to inhibition by malate but the protein level was stable. The data indicate that the flux of fixed N from bacteroids (N2-fixing nodule bacteria) is in some way associated with transcriptional control over pur5and possibly also catabolism of AIRS protein. In contrast, there is limited posttranslational control over GART and PEPC and close posttranslational control over uricase activity. The significance of these different levels of regulation is discussed in relation to the overall control of enhanced expression of plant enzymes in the cowpea symbiosis.
Signals Involved in Arabidopsis Resistance toTrichoplusia ni Caterpillars Induced by Virulent and Avirulent Strains of the Phytopathogen Pseudomonas syringae
Authors: Cui Jianping , Jander Georg , Racki Lisa R. , Kim Paul D. , Pierce Naomi E. , Ausubel Frederick M.
Published: July 2002

Plants have evolved different but interconnected strategies to defend themselves against herbivorous insects and microbial pathogens. We used an Arabidopsis/Pseudomonas syringaepathosystem to investigate the impact of pathogen-induced defense responses on cabbage looper (Trichoplusia ni) larval feeding. Arabidopsis mutants [npr1,pad4, eds5, andsid2(eds16)] or transgenic plants (nahG) that are more susceptible to microbial pathogens and are compromised in salicylic acid (SA)-dependent defense responses exhibited reduced levels of feeding by T. ni compared with wild-type plants. Consistent with these results, Arabidopsis mutants that are more resistant to microbial pathogens and have elevated levels of SA (cpr1 and cpr6) exhibited enhanced levels of T. ni feeding. These experiments suggested an inverse relationship between an active SA defense pathway and insect feeding. In contrast to these results, there was increased resistance toT. ni in wild-type Arabidopsis ecotype Columbia plants that were infected with P. syringae pv.maculicola strain ES4326 (Psm ES4326) expressing the avirulence genes avrRpt2 oravrB, which elicit a hypersensitive response, high levels of SA accumulation, and systemic acquired resistance to bacterial infection. Similar results were obtained with other ecotypes, including Landsberg erecta, Cape Verdi Islands, and Shakdara. When infected with PsmES4326(avrRpt2) or PsmES4326(avrB), nahG transgenic andnpr1 mutant plants (which are more susceptible to virulent and avirulent P. syringae strains) failed to show the increased insect resistance exhibited by wild-type plants. It was surprising that wild-type plants, as well as nahGand npr1 plants, infected with Psm ES4326 not expressing avrRpt2 or avrB, which elicits disease, became more susceptible to T. ni. Our results suggest two potentially novel systemic signaling pathways: a systemic response elicited by HR that leads to enhanced T. ni resistance and overrides the SA-mediated increase in T. ni susceptibility, and a SA-independent systemic response induced by virulent pathogens that leads to enhanced susceptibility to T. ni.
Selective Activation of the Developmentally Regulated Ha hsp17.6 G1 Promoter by Heat Stress Transcription Factors
Authors: Rojas Anabel , Almoguera Concepción , Carranco Raúl , Scharf Klaus-Dieter , Jordano Juan
Published: July 2002

Using two well-characterized heat stress transcription factors (Hsfs) from tomato (Lycopersicon peruvianum; LpHsfA1 and LpHsfA2), we analyzed the transcriptional activation of theHa hsp17.6 G1 promoter in sunflower (Helianthus annuus) embryos. In this system, we observed transient promoter activation only with LpHsfA2. In contrast, both factors were able to activate mutant versions of the promoter with improved consensus Hsf-binding sites. Exclusive activation by LpHsfA2 was also observed in yeast (Saccharomyces cerevisiae) without other Hsfs and with a minimal Cyc1 promoter fused to theHa hsp17.6 G1 heat stress cis-element. Furthermore, the same promoter mutations reproduced the loss of activation selectivity, as observed in sunflower embryos. The results of in vitro binding experiments rule out differential DNA binding of the two factors as the explanation for the observed differential activation capacity. We conclude that the specific sequence of this heat stress cis-element is crucial for Hsf promoter selectivity, and that this selectivity could involve preferential transcriptional activation following DNA binding. In sunflower embryos, we also observed synergistic transcriptional activation by co-expression of LpHsfA1 and LpHsfA2. Mutational analyses of the Ha hsp17.6 G1 promoter, combined with in vitro binding assays, suggest that mixed oligomers of the two factors may be involved in promoter activation. We discuss the relevance of our observations for mechanisms of developmental regulation of plant heat stress protein genes.
The Liverwort Contains a Lectin That Is Structurally and Evolutionary Related to the Monocot Mannose-Binding Lectins
Authors: Peumans Willy J. , Barre Annick , Bras Julien , Rougé Pierre , Proost Paul , Van Damme Els J.M.
Published: July 2002

A mannose (Man)-binding lectin has been isolated and characterized from the thallus of the liverwort Marchantia polymorpha. N-terminal sequencing indicated that the M. polymorpha agglutinin (Marpola) shares sequence similarity with the superfamily of monocot Man-binding lectins. Searches in the databases yielded expressed sequence tags encoding Marpola. Sequence analysis, molecular modeling, and docking experiments revealed striking structural similarities between Marpola and the monocot Man-binding lectins. Activity and specificity studies further indicated that Marpola is a much stronger agglutinin than the Galanthus nivalis agglutinin and exhibits a preference for methylated Man and glucose, which is unprecedented within the family of monocot Man-binding lectins. The discovery of Marpola allows us, for the first time, to corroborate the evolutionary relationship between a lectin from a lower plant and a well-established lectin family from flowering plants. In addition, the identification of Marpola sheds a new light on the molecular evolution of the superfamily of monocot Man-binding lectins. Beside evolutionary considerations, the occurrence of aG. nivalis agglutinin homolog in a lower plant necessitates the rethinking of the physiological role of the whole family of monocot Man-binding lectins.