bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of expression of the cyanide-insensitive terminal oxidase in Pseudomonas aeruginosa
Authors: Cooper Megan , Tavankar Gholam Reza , Williams Huw D.
Published: May 2003

The regulation of the cyanide-insensitive oxidase (CIO) inPseudomonas aeruginosa, a bacterium that can synthesize HCN, is reported. The expression of acioA–lacZtranscriptional fusion, CioA protein levels and CIO activity were low in exponential phase but induced about fivefold upon entry into stationary phase. Varying the O2transfer coefficient from 11·5 h−1to 87·4 h−1had no effect on CIO expression and no correlation was observed between CIO induction and the dissolved O2levels in the growth medium. However, a mutant deleted for the O2-sensitive transcriptional regulator ANR derepressed CIO expression in an O2-sensitive manner, with the highest induction occurring under low-O2conditions. Therefore, CIO expression can respond to a signal generated by low O2levels, but this response is normally kept in check by ANR repression. ANR may play an important role in preventing overexpression of the CIO in relation to other terminal oxidases. A component present in spent culture medium was able to induce CIO expression. However, experiments with purifiedN-butanoyl-l-homoserine lactone orN-(3-oxododecanoyl)homoserine lactone ruled out a role for these quorum-sensing molecules in the control of CIO expression. Cyanide was a potent inducer of the CIO at physiologically relevant concentrations and experiments using spent culture medium from a ΔhcnBmutant, which is unable to synthesize cyanide, showed that cyanide was the inducing factor present inP. aeruginosaspent culture medium. However, the finding that in a ΔhcnBmutantcioA–lacZexpression was induced normally upon entry into stationary phase indicated that cyanide was not the endogenous inducer of the terminal oxidase. The authors suggest that the failure of O2to have an effect on CIO expression in the wild-type can be explained either by the requirement for an additional, stationary-phase-specific inducing signal or by the loss of an exponential-phase-specific repressing signal.
The LicT protein acts as both a positive and a negative regulator of loci within the bgl regulon of Streptococcus mutans
Authors: Cote Christopher K. , Honeyman Allen L.
Published: May 2003

An open reading frame (ORF) that would encode a putative antiterminator protein (LicT) of the BglG family was identified in the genomic DNA sequence of Streptococcus mutans. A DNA sequence that would encode a potential ribonucleic antiterminator (RAT) site in the mRNA at which the putative antitermination protein LicT would bind was located immediately downstream from this ORF. These putative antitermination components are upstream of a glucose-independent β-glucoside-utilization system that is responsible for aesculin utilization by S. mutans NG8 in the presence of glucose. It was hypothesized that these putative regulatory components were an important mechanism that was involved with the controlled expression of the S. mutans
bglP locus. A strain of S. mutans containing a licT : : Ω-Kan2 insertional mutation was created. This strain could not hydrolyse aesculin in the presence of glucose. The transcriptional activity associated with other genes from the bgl regulon was determined in the licT : : Ω-Kan2 genetic background using lacZ transcriptional fusions and β-galactosidase assays to determine the effect of LicT on these loci. The LicT protein had no significant effect on the expression of the bglC promoter, a regulator of the bglA locus. However, it is essential for the optimal expression of bglP. These data correlate with the phenotype observed on aesculin plates for the S. mutans wild-type strain NG8 and the licT : : Ω-Kan2 strain. Thus, the glucose-independent β-glucoside-specific phosphotransferase system (PTS) regulon in S. mutans relies on LicT for BglP expression and, in turn, aesculin transport in the presence of glucose. Interestingly, LicT also seems to negatively regulate the expression of the bglA promoter region. In addition, the presence of the S. mutans licT gene has been shown to be able to activate a cryptic β-glucoside-specific operon found in Escherichia coli.
Inactivation of mshB, a key gene in the mycothiol biosynthesis pathway in Mycobacterium smegmatis
Authors: Rawat Mamta , Kovacevic Svetozar , Billman-Jacobe Helen , Av-Gay Yossef
Published: May 2003

ThemshBgene encodingN-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption ofmshBinMycobacterium smegmatisresulted in decreased production of mycothiol (5–10 % of the parent strain mc2155) but did not abolish mycothiol synthesis completely. Complementation of the MshB−mutants with themshBgene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providingN-acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression ofmcain the MshB−mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB−mutant did not exhibit increased resistance to isoniazid. However, the MshB−mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA−mutants also exhibit ethionamide resistance but that a MshC−mutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.
Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways
Authors: Wojda Iwona , Alonso-Monge Rebeca , Bebelman Jan-Paul , Mager Willem H. , Siderius Marco
Published: May 2003

In the yeastSaccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway. HOG pathway mutant strains display osmosensitive phenotypes. Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 °C). A notable exception is theste11ssk2ssk22mutant, which displays hypersensitivity to osmotic stress at 37 °C. This paper reports that overexpression ofFPS1orGPD1(encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress ofste11ssk2ssk22at 37 °C. Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not. Rather, evidence was obtained that the glycerol level may have an indirect effect, viz. by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity. In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains. PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions. Expression ofbck1-20or overexpression ofWSC3in wild-type cells resulted in restoration of PKC signalling. Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature. The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast.
A computer investigation of chemically mediated detachment in bacterial biofilms
Authors: Hunt Stephen M. , Hamilton Martin A. , Sears John T. , Harkin Gary , Reno Jason
Published: May 2003

A three-dimensional computer model was used to evaluate the effect of chemically mediated detachment on biofilm development in a negligible-shear environment. The model, BacLAB, combines conventional diffusion-reaction equations for chemicals with a cellular automata algorithm to simulate bacterial growth, movement and detachment. BacLAB simulates the life cycle of a bacterial biofilm from its initial colonization of a surface to the development of a mature biofilm with cell areal densities comparable to those in the laboratory. A base model founded on well established transport equations that are easily adaptable to investigate conjectures at the biological level has been created. In this study, the conjecture of a detachment mechanism involving a bacterially produced chemical detachment factor in which high local concentrations of this detachment factor cause the bacteria to detach from the biofilm was examined. The results show that the often observed ‘mushroom’-shaped structure can occur if detachment events create voids so that the remaining attached cells look like mushrooms.
SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2-encoded type III secretion system
Authors: Ruiz-Albert Javier , Mundy Rosanna , Yu Xiu-Jun , Beuzón Carmen R. , Holden David W.
Published: May 2003

The type III secretion system (TTSS) encoded by theSalmonellapathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages and for systemic infection in mice. Many TTSS secreted proteins, including effectors and components of the translocon, require chaperones which promote their stability, prevent their premature interactions or facilitate their secretion. In this study, the function of the first gene (sseA) of one of the SPI-2 operons (sseA–G) was investigated. This operon includes genes that encode translocon components (SseB, SseC and SseD), translocated proteins (SseF and SseG) and putative chaperones (SscA and SscB).sseAencodes a 12·5 kDa protein with a C-terminal region with the potential to form a coiled-coil structure, but no sequence similarity to other proteins. Mutation ofsseAresults in severe virulence attenuation and an intracellular replication defect. It is shown here that SseA is not a secreted protein, but is required for SPI-2-dependent translocation of two effector proteins (SifA and PipB). Furthermore, the translocon components SseB and SseD were not detected in ansseAmutant strain. By using a yeast two-hybrid assay and column binding experiments, it is demonstrated that SseA interacts directly with SseB and SseD. These results indicate that SseA is a chaperone for SseB and SseD. The inability of ansseAmutant to assemble the SPI-2 TTSS translocon accounts for its high level of virulence attenuationin vivo. To the authors' knowledge, this is the first chaperone described for the SPI-2 TTSS.
Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport
Authors: Dorsey Caleb W. , Tolmasky Marcelo E. , Crosa Jorge H. , Actis Luis A.
Published: May 2003

TheAcinetobacter baumannii8399 clinical isolate secretes dihydroxybenzoic acid (DHBA) and a high-affinity catechol siderophore, which is different from other bacterial iron chelators already characterized. Complementation assays with enterobactin-deficientEscherichia colistrains led to the isolation of a cosmid clone containingA. baumannii8399 genes required for the biosynthesis and activation of DHBA. Accordingly, the cloned fragment harbours adhbACEBpolycistronic operon encoding predicted proteins highly similar to several bacterial proteins required for DHBA biosynthesis from chorismic acid. Genes encoding deduced proteins related to theE. coliFes and theBacillus subtilisDhbF proteins, and a putativeYersinia pestisphosphopantetheinyl transferase, all of them involved in the assembly and utilization of catechol siderophores in other bacteria, were found next to thedhbACEBlocus. ThisA. baumannii8399 gene cluster also contained theom73,p45andp114predicted genes encoding proteins potentially involved in transport of ferric siderophore complexes. The deduced products of thep114andp45genes are putative membrane proteins that belong to the RND and MFS efflux pump proteins, respectively. Interestingly, P45 is highly related to theE. coliP43 (EntS) protein that participates in the secretion of enterobactin. Although P114 is similar to other bacterial efflux pump proteins involved in antibiotic resistance, its genetic arrangement within thisA. baumannii8399 locus is different from that described in other bacteria. The product ofom73is a Fur- and iron-regulated surface-exposed outer-membrane protein. These characteristics together with the presence of a predicted TonB box and its high similarity to other siderophore receptors indicate that OM73 plays such a role inA. baumannii8399. The 184 ntom73–p114intergenic region contains promoter elements that could drive the expression of these divergently transcribed genes, all of which are in close proximity to almost perfect Fur boxes. This arrangement explains the iron- and Fur-regulated expression ofom73, and provides strong evidence for a similar regulation for the expression ofp114.
Molecular analysis of mutS expression and mutation in natural isolates of pathogenic Escherichia coli
Authors: Li Baoguang , Tsui Ho-Ching T. , LeClerc J. Eugene , Dey Manashi , Winkler Malcolm E. , Cebula Thomas A.
Published: May 2003

Deficiencies in the MutS protein disrupt methyl-directed mismatch repair (MMR), generating a mutator phenotype typified by high mutation rates and promiscuous recombination. How such deficiencies might arise in the natural environment was determined by analysing pathogenic strains ofEscherichia coli. Quantitative Western immunoblotting showed that the amount of MutS in a wild-type strain of the enterohaemorrhagic pathogenE. coliO157 : H7 decreased about 26-fold in stationary-phase cells as compared with the amount present during exponential-phase growth. The depletion of MutS in O157 : H7 is significantly greater than that observed for a laboratory-attenuatedE. coliK-12 strain. In the case of stable mutators,mutSdefects in strains identified among natural isolates were analysed, including twoE. coliO157 : H7 strains, a diarrhoeagenicE. coliO55 : H7 strain, and a uropathogenic strain from theE. colireference (ECOR) collection. No MutS could be detected in the four strains by Western immunoblot analyses. RNase T2 protection assays showed that the strains were either deficient inmutStranscripts or produced transcripts truncated at the 3′ end. Nucleotide sequence analysis revealed extensive deletions in themutSregion of three strains, ranging from 7·5 to 17·3 kb relative toE. coliK-12 sequence, while the ECOR mutator contained a premature stop codon in addition to other nucleotide changes in themutScoding sequence. These results provide insights into the status of themutSgene and its product in pathogenic strains ofE. coli.
Predicting Gene Ontology Biological Process From Temporal Gene Expression Patterns
Authors: Lægreid Astrid , Hvidsten Torgeir R. , Midelfart Herman , Komorowski Jan , Sandvik Arne K.
Published: May 2003

The aim of the present study was to generate hypotheses on the involvement of uncharacterized genes in biological processes. To this end, supervised learning was used to analyze microarray-derived time-series gene expression data. Our method was objectively evaluated on known genes using cross-validation and provided high-precision Gene Ontology biological process classifications for 211 of the 213 uncharacterized genes in the data set used. In addition, new roles in biological process were hypothesized for known genes. Our method uses biological knowledge expressed by Gene Ontology and generates a rule model associating this knowledge with minimal characteristic features of temporal gene expression profiles. This model allows learning and classification of multiple biological process roles for each gene and can predict participation of genes in a biological process even though the genes of this class exhibit a wide variety of gene expression profiles including inverse coregulation. A considerable number of the hypothesized new roles for known genes were confirmed by literature search. In addition, many biological process roles hypothesized for uncharacterized genes were found to agree with assumptions based on homology information. To our knowledge, a gene classifier of similar scope and functionality has not been reported earlier.

[Supplemental material is available online atwww.genome.org. All annotations, reclassifications of known genes, and classifications of uncharacterized genes are available online athttp://www.lcb.uu.se/∼hvidsten/fibroblast.]
Genomic Gene Clustering Analysis of Pathways in Eukaryotes
Authors: Lee Jennifer M. , Sonnhammer Erik L.L.
Published: May 2003

Genomic clustering of genes in a pathway is commonly found in prokaryotes due to transcriptional operons, but these are not present in most eukaryotes. Yet, there might be clustering to a lesser extent of pathway members in eukaryotic genomes, that assist coregulation of a set of functionally cooperating genes. We analyzed five sequenced eukaryotic genomes for clustering of genes assigned to the same pathway in the KEGG database. Between 98% and 30% of the analyzed pathways in a genome were found to exhibit significantly higher clustering levels than expected by chance. In descending order by the level of clustering, the genomes studied were Saccharomyces cerevisiae,Homo sapiens, Caenorhabditis elegans,Arabidopsis thaliana, and Drosophila melanogaster. Surprisingly, there is not much agreement between genomes in terms of which pathways are most clustered. Only seven of 69 pathways found in all species were significantly clustered in all five of them. This species-specific pattern of pathway clustering may reflect adaptations or evolutionary events unique to a particular lineage. We note that although operons are common in C. elegans, only 58% of the pathways showed significant clustering, which is less than in human. Virtually all pathways in S. cerevisiae showed significant clustering.
Complex Evolution of 7E Olfactory Receptor Genes in Segmental Duplications
Authors: Newman Tera , Trask Barbara J.
Published: May 2003

Large segmental duplications (SDs) constitute at least 3.6% of the human genome and have increased its size, complexity, and diversity. SDs can mediate ectopic sequence exchange resulting in gross chromosomal rearrangements that could contribute to speciation and disease. We have identified and evaluated a subset of human SDs that harbor an 88-member subfamily of olfactory receptor (OR)-like genes called the 7Es. At least 92% of these genes appear to be pseudogenes when compared to other OR genes. The 7E-containing SDs (7E SDs) have duplicated to at least 35 regions of the genome via intra- and interchromosomal duplication events. In contrast to many human SDs, the 7E SDs are not biased towards pericentromeric or subtelomeric regions. We find evidence for gene conversion among 7E genes and larger sequence exchange between 7E SDs, supporting the hypothesis that long, highly similar stretches of DNA facilitate ectopic interactions. The complex structure and history of the 7E SDs necessitates extension of the current model of large-scale DNA duplication. Despite their appearance as pseudogenes, some 7E genes exhibit a signature of purifying selection, and at least one 7E gene is expressed.

[Supplemental material is available online atwww.genome.org.]
Unbiased Whole-Genome Amplification Directly From Clinical Samples
Authors: Hosono Seiyu , Faruqi A. Fawad , Dean Frank B. , Du Yuefen , Sun Zhenyu , Wu Xiaohong , Du Jing , Kingsmore Stephen F. , Egholm Michael , Lasken Roger S.
Published: May 2003

Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods.
Genome Size Evolution in Pufferfish: A Comparative Analysis of Diodontid and Tetraodontid Pufferfish Genomes
Authors: Neafsey Daniel E. , Palumbi Stephen R.
Published: May 2003

Smooth pufferfish of the family Tetraodontidae have the smallest vertebrate genomes yet measured. They have a haploid genome size of ∼400 million bp (Mb), which is almost eight times smaller than the human genome. Given that spiny pufferfish from the sister family Diodontidae and a fish from the outgroup Molidae have genomes twice as large as smooth puffers, it appears that the genome size of smooth puffers has contracted in the last 50–70 million years since their divergence from the spiny puffers. Here we use renaturation kinetics to compare the repetitive nature of the smooth and spiny puffer genomes. We also estimate the rates of small (<400 bp) insertions and deletions in smooth and spiny puffers using defunct non-LTR retrotransposons. We find a significantly greater abundance of a transposon-like repetitive DNA class in spiny puffers relative to smooth puffers, in addition to nearly identical indel rates. We comment on the role that large insertions may play in the evolution of genome size in these two groups.

[The sequence data from this study have been submitted to GenBank under accession nos. AY212336–AY212504.]
Selecting Open Reading Frames From DNA
Authors: Zacchi Paola , Sblattero Daniele , Florian Fiorella , Marzari Roberto , Bradbury Andrew R.M.
Published: May 2003

We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the β-lactamase gene flanked bylox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the β-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems.
In Situ-Synthesized Novel Microarray Optimized for Mouse Stem Cell and Early Developmental Expression Profiling
Authors: Carter Mark G. , Hamatani Toshio , Sharov Alexei A. , Carmack Condie E. , Qian Yong , Aiba Kazuhiro , Ko Naomi T. , Dudekula Dawood B. , Brzoska Pius M. , Hwang S. Stuart , Ko Minoru S.H.
Published: May 2003

Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research.

[Supplemental material is available online atwww.genome.organd at the NIA Mouse cDNA Project Web site,http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html.]
Software for Automated Analysis of DNA Fingerprinting Gels
Authors: Fuhrmann Daniel R. , Krzywinski Martin I. , Chiu Readman , Saeedi Parvaneh , Schein Jacqueline E. , Bosdet Ian E. , Chinwalla Asif , Hillier LaDeana W. , Waterston Robert H. , McPherson John D. , Jones Steven J.M. , Marra Marco A.
Published: May 2003

Here we describe software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting gels. We present a mathematical model for the location and shape of the restriction fragments as a function of fragment size, with model parameters determined empirically from “marker” lanes containing molecular size standards. Automated identification of restriction fragments involves several steps, including: image preprocessing, to put the data in a form consistent with a linear model; marker lane analysis, for determination of the model parameters; and data lane analysis, a procedure for detecting restriction fragment multiplets while simultaneously determining the amplitude curve that describes restriction fragment amplitude as a function of mobility. In validation experiments conducted on fingerprinted and sequenced Bacterial Artificial Chromosome (BAC) clones, sensitivity and specificity of restriction fragment identification exceeded 96% on restriction fragments ranging in size from 600 base pairs (bp) to 30,000 bp. The integrated suite of software tools, written in MATLAB and collectively called BandLeader, is in use at the BC Cancer Agency Genome Sciences Centre (GSC) and the Washington University Genome Sequencing Center, and has been provided to the Wellcome Trust Sanger Institute and the Whitehead Institute. Employed in a production mode at the GSC, BandLeader has been used to perform automated restriction fragment identification for more than 850,000 BAC clones for mouse, rat, bovine, and poplar fingerprint mapping projects.
Genome-Wide In Silico Identification of Transcriptional Regulators Controlling the Cell Cycle in Human Cells
Authors: Elkon Ran , Linhart Chaim , Sharan Roded , Shamir Ron , Shiloh Yosef
Published: May 2003

Dissection of regulatory networks that control gene transcription is one of the greatest challenges of functional genomics. Using human genomic sequences, models for binding sites of known transcription factors, and gene expression data, we demonstrate that the reverse engineering approach, which infers regulatory mechanisms from gene expression patterns, can reveal transcriptional networks in human cells. To date, such methodologies were successfully demonstrated only in prokaryotes and low eukaryotes. We developed computational methods for identifying putative binding sites of transcription factors and for evaluating the statistical significance of their prevalence in a given set of promoters. Focusing on transcriptional mechanisms that control cell cycle progression, our computational analyses revealed eight transcription factors whose binding sites are significantly overrepresented in promoters of genes whose expression is cell-cycle-dependent. The enrichment of some of these factors is specific to certain phases of the cell cycle. In addition, several pairs of these transcription factors show a significant co-occurrence rate in cell-cycle-regulated promoters. Each such pair indicates functional cooperation between its members in regulating the transcriptional program associated with cell cycle progression. The methods presented here are general and can be applied to the analysis of transcriptional networks controlling any biological process.

[Supplemental material is available online atwww.genome.org, including full lists of genes whose promoters were found to contain high scoring sites for any of the enriched transcription factors reported in Tables 1 and 3.]
Analysis of the ArabidopsisMADS AFFECTING FLOWERINGGene Family:MAF2Prevents Vernalization by Short Periods of Cold [W]
Authors: Ratcliffe Oliver J. , Kumimoto Roderick W. , Wong Becky J. , Riechmann José Luis
Published: May 2003

The Arabidopsis FLOWERING LOCUS C (FLC) gene is a key floral repressor in the maintenance of a vernalization response. In vernalization-sensitive genetic backgrounds, FLC levels are high, and they decline after exposure to long cold periods. Four FLC paralogs (MAF2 [MADS AFFECTING FLOWERING2] to MAF5) are arranged in a tandem array on the bottom of Arabidopsis chromosome V. We used a reverse genetics approach to analyze their functions. Loss-of-function and gain-of-function studies indicate that MAF2 acts as a floral repressor. In particular, maf2 mutant plants display a pronounced vernalization response when subjected to relatively short cold periods, which are insufficient to elicit a strong flowering response in the wild type, despite producing a large reduction in FLC levels. MAF2 expression is less sensitive to vernalization than that of FLC, and its repressor activity is exerted independently or downstream of FLC transcription. Thus, MAF2 can prevent premature vernalization in response to brief cold spells. Overexpression of MAF3 or MAF4 produces alterations in flowering time that suggest that these genes also act as floral repressors and might contribute to the maintenance of a vernalization requirement. However, the final gene in the cluster, MAF5, is upregulated by vernalization. Therefore, MAF5 could play an opposite role to FLC in the vernalization response.
The ArabidopsisSLEEPY1Gene Encodes a Putative F-Box Subunit of an SCF E3 Ubiquitin Ligase[W]
Authors: McGinnis Karen M. , Thomas Stephen G. , Soule Jonathan D. , Strader Lucia C. , Zale Janice M. , Sun Tai-ping , Steber Camille M.
Published: May 2003

The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCFSLY1-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.
Rapid Genome Divergence at Orthologous Low Molecular Weight Glutenin Loci of the A and Am Genomes of Wheat[W]
Authors: Wicker Thomas , Yahiaoui Nabila , Guyot Romain , Schlagenhauf Edith , Liu Zhong-Da , Dubcovsky Jorge , Keller Beat
Published: May 2003

To study genome evolution in wheat, we have sequenced and compared two large physical contigs of 285 and 142 kb covering orthologous low molecular weight (LMW) glutenin loci on chromosome 1AS of a diploid wheat species (Triticum monococcum subsp monococcum) and a tetraploid wheat species (Triticum turgidum subsp durum). Sequence conservation between the two species was restricted to small regions containing the orthologous LMW glutenin genes, whereas >90% of the compared sequences were not conserved. Dramatic sequence rearrangements occurred in the regions rich in repetitive elements. Dating of long terminal repeat retrotransposon insertions revealed different insertion events occurring during the last 5.5 million years in both species. These insertions are partially responsible for the lack of homology between the intergenic regions. In addition, the gene space was conserved only partially, because different predicted genes were identified on both contigs. Duplications and deletions of large fragments that might be attributable to illegitimate recombination also have contributed to the differentiation of this region in both species. The striking differences in the intergenic landscape between the A and Am genomes that diverged 1 to 3 million years ago provide evidence for a dynamic and rapid genome evolution in wheat species.
Evolution to aperiodic penetrative convection in odd shaped rectangular enclosures
Authors: Hasan Nadeem , Baig Faisal M.
Published: May 2003

The purpose of this numerical study is to analyse the character of transition from laminar to chaotic convection in a fluid layer bounded by no‐slip walls in two space dimensions for varying aspect ratio odd‐shaped enclosures consisting of two rectangular chambers, with a linking horizontal enclosure. For a medium Prandtl number fluid (Pr=7), the numerical solution of two‐dimensional Navier‐Stokes momentum and energy equations with Bousinessq approximation has been carried out. It has been found that there are finite Rayleigh numbers Ra1, Ra2 and Ra3 for the onset of single, two and multiple frequency oscillatory motion at different spatial locations in the enclosure. As Ra is further increased period doubling is observed. The onset of strong chaos appears when Ra=Ra3. This system does not revert to steady state convection at high Ra as observed by other researchers for the case of Rayleigh‐Benard convection. Moreover, the period doubling transition process is consistent with the scenario of Ruelle, Takens and Newhouse. As Ra increases, the power spectrum, and time series of various dynamical variable signals, etc. all show an increasing degree of characteristics of chaos.
Interaction between condensate film and moist air in a cross flow
Authors: Besednjak Dean , Poredosˇ Alojz , Sˇkerget Leopold
Published: May 2003

A three‐dimensional numerical analysis was carried out to study in detail the combined heat and mass transfer processes between a moist air flow and a cooled surface when film condensation occurs. A cross‐flow was considered between the air flow and the film flow. A turbulent flow was modelled using the Wilcox k−ω turbulence model. The shape of the interface between the air and the film was treated as a moving boundary, and it was calculated with the assumptions that the interface ways remain an interface, the stress at the interface is continuous and that there is no slip at the interface. Numerical results were obtained by solving simultaneous coupled equations of the air, film and solid. The results show that the condensate film flow has a significant effect on the extended surface temperature distribution and consequently on its efficiency. It is shown that the simultaneous influence of gravity and the air flow on the condensate film results in an asymmetric velocity profile in the film as well as in the asymmetric shape of the film.
Effect of Biodiesel Utilization of Wear of Vital Parts in Compression Ignition Engine
Authors: Agarwal A. K. , Bijwe J. , Das L. M.
Published: May 2003

The combustion related properties of vegetable oils are somewhat similar to diesel oil. Neat vegetable oils or their blends with diesel, however, pose various long-term problems in compression ignition engines, e.g., poor atomization characteristics, ring-sticking, injector coking, injector deposits, injector pump failure, and lube oil dilution by crank-case polymerization. These undesirable features of vegetable oils are because of their inherent properties like high viscosity, low volatility, and polyunsaturated character. Linseed oil methyl ester (LOME) was prepared using methanol for long-term engine operations. The physical and combustion-related properties of the fuels thus developed were found to be closer to that of the diesel oil. A blend of 20 percent was selected as optimum biodiesel blend. Two similar new engines were completely disassembled and subjected to dimensioning of various vital moving parts and then subjected to long-term endurance tests on 20 percent biodiesel blend and diesel oil, respectively. After completion of the test, both the engines were again disassembled for physical inspection and wear measurement of various vital parts. The physical wear of various vital parts, injector coking, carbon deposits on piston, and ring sticking were found to be substantially lower in case of 20 percent biodiesel-fuelled engine. The lubricating oil samples drawn from both engines were subjected to atomic absorption spectroscopy for measurement of various wear metal traces present. AAS tests confirmed substantially lower wear and thus improved life for biodiesel operated engines.
Influence of the Injection Parameters on the Efficiency and Power Output of a Hydrogen Fueled Engine
Authors: Sierens R. , Verhelst S.
Published: May 2003

The advantages of hydrogen fueled internal combustion engines are well known, certainly concerning the ultra-low noxious emissions (only NOx is to be considered). Disadvantages are the backfire phenomenon and the gaseous state of hydrogen at atmospheric conditions. A complete control of the mixture formation is necessary and therefore a test engine with sequential port injection was chosen. The tests are carried out on a single-cylinder CFR engine with the intention to use the results to optimize a 6 and 8-cylinder engine with multipoint injection. Different positions of the injector against the intake air duct are examined (represented as different junctions). A numerical simulation CFD code (FLUENT) is used under “stationary” conditions (continuous injection) for all geometries and under “real” conditions (sequential injection) for one situation. For each of the geometries the influences of the start of injection, the air/fuel equivalence ratio, injection pressure, and ignition timing on the power output and efficiency of the engine are analyzed. A comparison and discussion is given for all results. It is clearly shown that the start of injection for a certain engine speed and inlet geometry influences the volumetric efficiency and thus the power output of the engine due to the interaction between the injected hydrogen and the inlet pressure waves. Furthermore, the small influence of the injection pressure and the contradictory benefits of the different junctions between power output and fuel efficiency are measured. With retarded injection, so that cool air decreases the temperature of the “hot-spots” in the combustion chamber before the fuel is injected, backfire safe operation is possible.
Parametric Characterization of High-Pressure Diesel Fuel Injection Systems
Authors: Wang T.-C. , Han J.-S. , Xie X.-B. , Lai M.-C. , Henein N. A. , Schwarz E. , Bryzik W.
Published: May 2003

The focus of the study described herein is the characterization of the high-pressure hydraulic electronic unit injector (HEUI) and of the electronic unit injector (EUI) diesel injection systems. The characterization items include injection pressure, injection rate, injector response time, needle lift, start up injection transient, and dynamic discharge coefficient of the nozzles. Macroscopic and microscopic spray visualizations were also performed. The effects of injection conditions and nozzle configurations on injection characteristics were reviewed. Nozzle sac pressure was measured to correlate with the up-stream injection pressure. A LabVIEW data acquisition and controls system was implemented to operate the injection systems and to acquire and analyze data. For an HEUI system, based on the results of the study, it can be concluded that common-rail pressure and length of the injection rate-shaping pipe determine the injection pressure, while the pressure rising rate and injection duration determine the peak injection pressure; it was also found that the nozzle flow area, common-rail pressure, and the length of the rate-shaping pipe are the dominating parameters that control the injection rate, and the rate shape is affected mainly by common-rail pressure, especially the pressure rising rate and length of the rate-shaping pipe. Both injection pressure and ambient pressure affected the spray tip penetration significantly. The penetration increased corresponding to the increase of injection pressure or decrease of ambient pressure. The variation of spray penetration depends on the type of injection system, nozzle configuration, and ambient pressure. The large penetration variation observed on the HEUI sprays could be caused by eccentricity of the VCO (valve-covered-orifices) nozzle. The variation of the mini-sac nozzle was 50% less than that of the VCO nozzle. The near-field spray behavior was shown to be highly transient and strongly depended on injector design, nozzle configuration, needle lift and oscillation, and injection pressure.
Analysis of Oil Film Thickness on a Piston Ring of Diesel Engine: Effect of Oil Film Temperature
Authors: Harigaya Y. , Suzuki M. , Takiguchi M.
Published: May 2003

This paper describes an analysis of oil film thickness on a piston ring of a diesel engine. The analysis of the oil film thickness has been performed by using Reynolds equation and unsteady, two-dimensional energy equation with heat generated from viscous dissipation. The mean oil film temperature was determined from the calculation of the temperature distribution in the oil field which was calculated using the energy equation. The oil film viscosity was then estimated using the mean oil film temperature. The effect of oil film temperature on the oil film thickness of a piston ring was examined. This model has been verified with published experimental results. Moreover, the heat flow at ring and liner surfaces was examined. Results show that the oil film thickness could be calculated using the viscosity estimated from the mean oil film temperature. The calculated values generally agree with the measured values. For higher engine speed conditions, the maximum values of the calculated oil film thickness are larger than the measured values.
Modeling the Initial Growth of the Plasma and Flame Kernel in SI Engines
Authors: Willems H. , Sierens R.
Published: May 2003

The initial size and growth of the plasma and flame kernel just after spark discharge in S.I. engines determines if the flame becomes self-sustainable or extinguishes. On the other hand, the development of the kernel during the initial phases has non-negligible influences on the further combustion. For example, cyclic variations often find their origin in the beginning of combustion and determine the working limits of the engine and the driving behavior of the vehicle. These factors demonstrate the crucial importance of the knowledge of the initial growth of the plasma and flame kernel in S.I. engines. A complete model is developed for the growth of the initial plasma and flame kernel in S.I. engines, which takes into account the fundamental properties of the ignition system (electrical energy and power, geometry of the spark plug, heat losses to the electrodes and the cylinder wall), the combustible mixture (pressure, temperature, equivalence ratio, fraction of residual gasses, kind of fuel), and the flow (average flow velocity, turbulence intensity, stretch, characteristic time and length scales). The proposed model distinguishes three phases: the pre-breakdown, the plasma, and the initial combustion phase. The model of the first two phases is proposed in a previous article of the same authors [1], the latter is exposed in this article. A thermodynamic model based on flamelet models and which takes stretch into account, is used to describe the initial combustion phase. The difference between heat losses to the electrodes and the cylinder wall is considered. The burning velocity varies from the order of the laminar velocity to the fully developed burning velocity. The evolution is determined as well by the life time as by the size of the kernel. The stretch (caused by turbulence and by the growth of the kernel), the nonadiabatic character of the flame, and instabilities have influence on the laminar burning velocity. Validation of this model is done using measurements of the expansion in a propane-air mixture executed by Pischinger [2] at M.I.T. The agreement seems very good.
Characteristics of Homogeneous Charge Compression Ignition (HCCI) Engine Operation for Variations in Compression Ratio, Speed, and Intake Temperature While Using n-Butane as a Fuel
Authors: Iida M. , Hayashi M. , Foster D. E. , Martin J. K.
Published: May 2003

In this paper, some basic properties of homogeneous charge compression ignition operation are reported. The effect of inlet temperature, compression ratio and engine speed on the homogeneous charge compression ignition (HCCI) operating ranges were evaluated in a CFR engine using n-butane as a fuel. The minimum and maximum loads for HCCI operation were determined using criteria of coefficient of variation of the indicated mean effective pressure and the derivative of in-cylinder pressure, respectively. Exhaust emissions, particularly hydrocarbons, were measured using a Fourier transform infrared spectrometer. The concentration of intermediate hydrocarbon species rapidly decreased as the magnitude of the energy release increased. Hydrocarbon emission at the maximum HCCI load mainly consists of the fuel itself, which is probably emitted from colder areas in the combustion chamber. Finally, the relationship between IMEPCOV and ISFC is discussed.
A Nonequilibrium Turbulence Dissipation Correction and Its Influence on Pollution Predictions for DI Diesel Engines
Authors: Tanner F. X. , Zhu G.-S. , Reitz R. D.
Published: May 2003

A correction for the turbulence dissipation rate, based on nonequilibrium turbulence considerations from rapid distortion theory, has been derived and implemented in combination with the RNG k-ε model in a KIVA-based code. This correction reflects the time delay between changes in the turbulent kinetic energy due to changes in the mean flow and its turbulence dissipation rate, and it is shown that this time delay is controlled by the turbulence Reynolds number. The model correction has been validated with experimental data in the compression and expansion phase of a small diesel engine operated in motored mode. Combustion simulations of two heavy-duty DI diesel engines have been performed with the RNG k-ε model and the dissipation rate correction. The focus of these computations has been on the nitric oxide formation and the net soot production. These simulations have been compared with experimental data and their preditions are explained in terms of the turbulence dissipation effect on the transport coefficients for mass and heat diffusion. It has been found, that the dissipation correction yields consistent results with observations reported in previous studies.