Mol Cell Biol, 2005 Feb, 25(3), 933 - 44 RAD51-Dependent Break-Induced Replication Differs in Kinetics and Checkpoint Responses from RAD51-Mediated Gene Conversion; Malkova A et al.; Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time . However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms . Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome . Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G(2)/M DNA damage checkpoint . RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process . RAD51-dependent BIR occurs efficiently in G(2)-arrested cells . Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB. Am J Gastroenterol, 2005 Jan, 100(1), 84 - 92 Variants of CARD15 are associated with an aggressive clinical course of Crohn's disease--an IG-IBD study; Annese V et al.; BACKGROUND: Three major variants of the CARD15 gene confer susceptibility to Crohn's disease (CD) . Whether or not these variants correlate with specific clinical features of the disease is under evaluation . AIM: We investigated the possible association of CARD15 variants with specific clinical characteristics, including the occurrence of anti-Saccharomyces cerevisiae antibodies (ASCA) and antineutrophil cytoplasmic antibodies (ANCA), in a large cohort of inflammatory bowel disease (IBD) patients and their unaffected relatives . METHODS: Three hundred and sixteen CD patients (156 with positive family history), 408 ulcerative colitis (UC) patients (206 with positive family history), 588 unaffected relatives, and 205 unrelated healthy controls (HC) were studied . Single nucleotide polymorphisms (SNPs) R702W, G908R, and L1007finsC of the CARD15 gene were investigated and correlated to age at diagnosis, gender, family history, localization, extraintestinal manifestations, previous resective surgery, stenosing/fistulizing pattern, ANCA, and ASCA . RESULTS: Compared to HC, the frequencies of all three variants in CD were significantly increased: 8.7% versus 4.1% for R702W (p < 0.006), 7.3% versus 2.7% for G908R (p < 0.002), 9.3% versus 0.7% for L1007finsC (p < 0.00001) . At least one risk allele was found in 38.2% (p < 0.0001, compared to HC), 13.7% (NS), and 15.1% of CD, UC, and HC, respectively . The L1007finsC risk allele was also significantly increased in unaffected relatives of familial (9.5%; p < 0.00001), and sporadic CD (9%; p < 0.00001), compared to HC (0.7%) . Sixteen healthy relatives, carriers of two risk alleles, were asymptomatic after 5-8 yr of follow-up . CD carriers of at least one variant were younger (p= 0.03), more likely to have ileal localization (p= 0.0001), stenosing pattern (p= 0.01), previous resective surgery (p= 0.0001), and presence of ASCA (p= 0.0001) . No difference in SNPs frequency between familial and sporadic cases of CD was found . CONCLUSION: In our population, both familial and sporadic CD patients carrying at least one major variant of CARD15 had an aggressive clinical course. Bioinformatics . 2005 Jan 12; {Epub ahead of print} Detecting clusters of different geometrical shapes in microarray gene expression data; Kim DW et al.; MOTIVATION: Clustering has been used as a popular technique for finding groups of genes that show similar expression patterns under multiple experimental conditions . Many clustering methods have been proposed for clustering gene expression data, including the hierarchical clustering, k-means clustering, and self-organizing map . However, the conventional methods are limited to identify different shapes of clusters because they use a fixed distance norm when calculating the distance between genes . The fixed distance norm imposes a fixed geometrical shape on the clusters regardless of the actual data distribution . Thus, different distance norms are required for handling the different shapes of clusters . RESULTS: We present the Gustafson-Kessel (GK) clustering method for microarray gene expression data . To detect clusters of different shapes in a data set, we use an adaptive distance norm that is calculated by a fuzzy covariance matrix (F) of each cluster in which the eigenstructure of F is used as an indicator of the shape of the cluster . Moreover, the GK method is less prone to falling into local minima than the k-means and self-organizing map because it makes decisions through the use of membership degrees of a gene to clusters . The algorithmic procedure is accomplished by the alternating optimization technique, which iteratively improves a sequence of sets of clusters until no further improvement is possible . To test the performance of the GK method, we applied the GK method and well-known conventional methods to three recently published yeast data sets, and compared the performance of each method using the Saccharomyces Genome Database annotations . The clustering results of the GK method are more significantly relevant to the biological annotations than those of the other methods, demonstrating its effectiveness and potential for clustering gene expression data . AVAILABILITY: The software was developed using Java language, and can be executed on the platforms that JVM (Java Virtual Machine) is running . It is available from the authors upon request . Supplementary data are available at http://dragon.kaist.ac.kr/gk. Protein J, 2004 Oct, 23(7), 453 - 60 High-activity barley alpha-amylase by directed evolution; Wong DW et al.; Barley alpha-amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae . The gene coding for the wild-type enzyme was subjected to directed evolution . Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate . The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity . After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system . Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B . Two of these mutations . Q44H and R303K, result in amino acids highly conserved in cereal alpha-amylases . R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule. Genome Biol . 2004;5(12):R98 . Epub 2004. MONKEY: identifying conserved transcription-factor binding sites in multiple alignments using a binding site-specific evolutionary model; Moses AM et al.; We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments . MONKEY employs probabilistic models of factor specificity and binding-site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit . Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function. EMBO J, 2004 Dec 8, 23(24), 4847 - 56 Epub 2004 Dec 8. Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing; Dziembowski A et al.; Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b . While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome . Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex . TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex . Subunits of this complex were not essential for viability . However, they are required for efficient splicing in vitro and in vivo . Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus . The corresponding complex was named pre-mRNA REtention and Splicing (RES) . The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved. Int Rev Cytol, 2004, 241, 53 - 153 Microtubule-associated proteins and their essential roles during mitosis; Maiato H et al.; Microtubules play essential roles during mitosis, including chromosome capture, congression, and segregation . In addition, microtubules are also required for successful cytokinesis . At the heart of these processes is the ability of microtubules to do work, a property that derives from their intrinsic dynamic behavior . However, if microtubule dynamics were not properly regulated, it is certain that microtubules alone could not accomplish any of these tasks . In vivo, the regulation of microtubule dynamics is the responsibility of microtubule-associated proteins . Among these, we can distinguish several classes according to their function: (1) promotion and stabilization of microtubule polymerization, (2) destabilization or severance of microtubules, (3) functioning as linkers between various structures, or (4) motility-related functions . Here we discuss how the various properties of microtubule-associated proteins can be used to assemble an efficient mitotic apparatus capable of ensuring the bona fide transmission of the genetic information in animal cells. Appl Microbiol Biotechnol . 2004 Nov 5; {Epub ahead of print} A novel fungal omega3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4; Sakuradani E et al.; A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4 n-6) but also eicosapentaenoic acid (EPA; 20:5 n-3) below a cultural temperature of 20show $132# degrees show $132#C . Here, we describe the isolation and characterization of a gene ( maw3) that encodes a novel omega3-desaturase from M . alpina 1S-4 . Based on the conserved sequence information for M . alpina 1S-4 Delta12-desaturase and Saccharomyces kluyveri omega3-desaturase, the omega3-desaturase gene from M . alpina 1S-4 was cloned . Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M . alpina 1S-4 Delta12-desaturase, whereas it exhibited 36% identity with Sac . kluyveri omega3-desaturase . The cloned cDNA was confirmed to encode the omega3-desaturase by its expression in the yeast Sac . cerevisiae . Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs . The substrate specificity of the M . alpina 1S-4 omega3-desaturase differs from those of the known fungal omega3-desaturases from Sac . kluyveri and Saprolegnia diclina . Plant, cyanobacterial and Sac . kluyveri omega3-desaturases desaturate 18-carbon n-6 PUFAs, Spr . diclina omega3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans omega3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs . The substrate specificity of M . alpina 1S-4 omega3-desaturase is rather similar to that of C . elegans omega3-desaturase, but the M . alpina omega3-desaturase can more effectively convert AA into EPA when expressed in yeast . The M . alpina 1S-4 omega3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates. Cell, 2004 Oct 29, 119(3), 355 - 68 Homologous recombination generates T-loop-sized deletions at human telomeres; Wang RC et al.; The t-loop structure of mammalian telomeres is thought to repress nonhomologous end joining (NHEJ) at natural chromosome ends . Telomere NHEJ occurs upon loss of TRF2, a telomeric protein implicated in t-loop formation . Here we describe a mutant allele of TRF2, TRF2DeltaB, that suppressed NHEJ but induced catastrophic deletions of telomeric DNA . The deletion events were stochastic and occurred rapidly, generating dramatically shortened telomeres that were accompanied by a DNA damage response and induction of senescence . TRF2DeltaB-induced deletions depended on XRCC3, a protein implicated in Holliday junction resolution, and created t-loop-sized telomeric circles . These telomeric circles were also detected in unperturbed cells and suggested that t-loop deletion by homologous recombination (HR) might contribute to telomere attrition . Human ALT cells had abundant telomeric circles, pointing to frequent t-loop HR events that could promote rolling circle replication of telomeres in the absence of telomerase . These findings show that t-loop deletion by HR influences the integrity and dynamics of mammalian telomeres. Bioinformatics . 2004 Oct 12; {Epub ahead of print} Understanding protein dispensability through machine-learning analysis of high-throughput data; Chen Y et al.; MOTIVATION: Protein dispensability is fundamental to understanding of gene function and evolution . Recent advances in generating high-throughput data such as genomic sequence data, protein-protein interaction data, gene-expression data, and growth-rate data of mutants allow us to investigate protein dispensability systematically at the genome scale . RESULTS: In our studies, protein dispensability is represented as a fitness score that is measured by the growth rate of gene-deletion mutants . Through analyses of high-throughput data in yeast Saccharomyces cerevisia, we found that a protein's dispensability had significant correlations with its evolutionary rate and duplication rate, as well as its connectivity in protein-protein interaction network and gene-expression correlation network . Neural network and support vector machine were applied to predict protein dispensability through high-throughput data . Our studies shed some lights on global characteristics of protein dispensability and evolution . AVAILABILITY: The original datasets for protein dispensability analysis and prediction, together with related scripts, are available at http://digbio.missouri.edu/~ychen/ProDispen/. Postepy Hig Med Dosw (Online), 2004 Aug 20, 58, 312 - 20 {RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response}; Lewandowicz AM et al.; RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins . By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction . Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins) . The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies . In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways . They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation . They also take an essential part in inflammation . High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors . The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response FEMS Microbiol Lett, 2004 Oct 1, 239(1), 95 - 101 Glutamic protease distribution is limited to filamentous fungi; Sims AH et al.; Glutamic proteases are a distinct, and recently re-classified, group of peptidases that are thought to be found only in fungi . We have identified and analysed the distribution of over 20 putative glutamic proteases from all fungal species whose genomes have been sequenced so far . Although absent from the Saccharomycetales class, glutamic proteases appear to be present in all other ascomycetes species examined . A large number of coding regions for glutamic proteases were also found clustered together in the Phanerochaete chrysosporium genome, despite apparently being absent from three other species of Basidiomycota. Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1883 - 90 Metschnikowia chrysoperlae sp . nov., Candida picachoensis sp . nov . and Candida pimensis sp . nov., isolated from the green lacewings Chrysoperla comanche and Chrysoperla carnea (Neuroptera: Chrysopidae); Suh SO et al.; Fourteen yeast isolates comprising three taxa were cultured from digestive tracts of adult Chrysoperla species (Neuroptera: Chrysopidae) and their eggs . The yeast taxa were distinguished based on an estimated molecular phylogeny, DNA sequences and traditional taxonomic criteria . The new yeasts are closely related to Metschnikowia pulcherrima but are sufficiently distinguished by sequence comparison of rRNA gene sequences to consider them as novel species . Here, three novel species are described and their relationships with other taxa in the Saccharomycetes are discussed . Metschnikowia chrysoperlae sp . nov . (type strain, NRRL Y-27615T = CBS 9803T) produced needle-shaped ascospores and was the only teleomorph found . Large numbers of chlamydospores similar to those observed in M . pulcherrima were also produced . The other two novel species are asexual yeasts, Candida picachoensis sp . nov . (type strain, NRRL Y-27607T = CBS 9804T) and Candida pimensis sp . nov . (type strain, NRRL Y-27619T = CBS 9805T), sister taxa of M . chrysoperlae and M . pulcherrima . A specialized relationship between yeasts and lacewing hosts may exist, because the yeasts were isolated consistently from lacewings only . Although M . chrysoperlae was isolated from eggs and adult lacewings, suggesting the possibility of vertical transmission, no yeast was isolated from larvae. Mol Biol Evol, 2005 Jan, 22(1), 174 - 177 Epub 2004 Sep 15. Adjusting for Selection on Synonymous Sites in Estimates of Evolutionary Distance; Hirsh AE et al.; Evolution at silent sites is often used to estimate the pace of selectively neutral processes or to infer differences in divergence times of genes . However, silent sites are subject to selection in favor of preferred codons, and the strength of such selection varies dramatically across genes . Here, we use the relationship between codon bias and synonymous divergence observed in four species of the genus Saccharomyces to provide a simple correction for selection on silent sites. Nature, 2004 Sep 2, 431(7004), 99 - 104 Transcriptional regulatory code of a eukaryotic genome; Harbison CT et al.; DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression . Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites . We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species . The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed . We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators. J Biol Chem, 2004 Oct 29, 279(44), 46182 - 90 Epub 2004 Aug 18. The Sac3 homologue shd1 is involved in mitotic progression in mammalian cells; Khuda SE et al.; Saccharomyces Sac3 required for actin assembly was shown to be involved in DNA replication . Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression . SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to alpha-tubulin, at M phase . RNA interference suppression of endogenous shd1 caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei . Conversely, increased expression of SHD1 by DNA transfection with shd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells . These results demonstrated that shd1 is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function. FEMS Microbiol Lett, 2004 Aug 15, 237(2), 243 - 8 Stress responses of linear plasmids from Debaryomyces hansenii; Fukuda K et al.; The triplet linear plasmids pDHL1/2/3 from the salt-tolerant yeast Debaryomyces hansenii TK are localized in the cytoplasm and characterized by a unique feature that they require environmental stressors (0.3 M NaCl or solutes such as sorbitol with equivalent osmolarity) for stable replication and maintenance . The degree of osmolarity dependence of pDHLs was greatly affected by growth temperature of the host cells: the stability of pDHLs was maintained in the absence of osmolarity in cells growing at 25 degrees C, and required osmorarity equivalent to 0.3-1.0 M NaCl on shifting to 30-35 degrees C . Although to less extent, similar osmolarity dependence at high temperatures was observed with another system of D . hansenii linear plasmids . Short-term conditioning of cells to heat or high osmolarity resulted in significant improvement in the plasmid stability, suggesting possible involvement of stress proteins and/or high glycerol level in the stabilization process. J Mol Biol, 2004 Sep 3, 342(1), 19 - 30 Consensus folding of aligned sequences as a new measure for the detection of functional RNAs by comparative genomics; Washietl S et al.; Facing the ever-growing list of newly discovered classes of functional RNAs, it can be expected that further types of functional RNAs are still hidden in recently completed genomes . The computational identification of such RNA genes is, therefore, of major importance . While most known functional RNAs have characteristic secondary structures, their free energies are generally not statistically significant enough to distinguish RNA genes from the genomic background . Additional information is required . Considering the wide availability of new genomic data of closely related species, comparative studies seem to be the most promising approach . Here, we show that prediction of consensus structures of aligned sequences can be a significant measure to detect functional RNAs . We report a new method to test multiple sequence alignments for the existence of an unusually structured and conserved fold . We show for alignments of six types of well-known functional RNA that an energy score consisting of free energy and a covariation term significantly improves sensitivity compared to single sequence predictions . We further test our method on a number of non-coding RNAs from Caenorhabditis elegans/Caenorhabditis briggsae and seven Saccharomyces species . Most RNAs can be detected with high significance . We provide a Perl implementation that can be used readily to score single alignments and discuss how the methods described here can be extended to allow for efficient genome-wide screens. Inflamm Bowel Dis, 2004 May, 10(3), 270 - 3 Scalloping of duodenal mucosa in Crohn's disease; Culliford A et al.; Scalloping of the duodenal mucosal folds is an endoscopic finding of small bowel mucosal pathology that is generally due to villous atrophy . Though it can be seen in many disease processes, it is most commonly associated with celiac disease . We report three patients with scalloping of duodenal folds and histologic confirmation of villous atrophy due to Crohn's disease . All patients had negative celiac serologies and two had positive markers for Crohn's disease (anti-Saccharomyces cerevisiae antibodies) . Patients had either ileitis or ileocolitis in addition to duodenal abnormalities . These cases illustrate that scalloping can occur in the duodenum in Crohn's disease. Bioinformatics, 2004 Jul 10, 20(10), 1506 - 11 Bifurcation analysis of the regulatory modules of the mammalian G1/S transition; Swat M et al.; MOTIVATION: Mathematical models of the cell cycle can contribute to an understanding of its basic mechanisms . Modern simulation tools make the analysis of key components and their interactions very effective . This paper focuses on the role of small modules and feedbacks in the gene-protein network governing the G1/S transition in mammalian cells . Mutations in this network may lead to uncontrolled cell proliferation . Bifurcation analysis helps to identify the key components of this extremely complex interaction network . RESULTS: We identify various positive and negative feedback loops in the network controlling the G1/S transition . It is shown that the positive feedback regulation of E2F1 and a double activator-inhibitor module can lead to bistability . Extensions of the core module preserve the essential features such as bistability . The complete model exhibits a transcritical bifurcation in addition to bistability . We relate these bifurcations to the cell cycle checkpoint and the G1/S phase transition point . Thus, core modules can explain major features of the complex G1/S network and have a robust decision taking function. Genome Biol . 2004;5(6):R43 . Epub 2004 May 28. TXTGate: profiling gene groups with text-based information; Glenisson P et al.; We implemented a framework called TXTGate that combines literature indices of selected public biological resources in a flexible text-mining system designed towards the analysis of groups of genes . By means of tailored vocabularies, term- as well as gene-centric views are offered on selected textual fields and MEDLINE abstracts used in LocusLink and the Saccharomyces Genome Database . Subclustering and links to external resources allow for in-depth analysis of the resulting term profiles. Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 9033 - 8 Epub 2004 Jun 02. Coevolution of gene expression among interacting proteins; Fraser HB et al.; Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions . Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids . Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components . Here, we show that the expression levels of physically interacting proteins coevolve . We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species . We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence . These results demonstrate that gene expression levels can coevolve, adding another dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time . Our results also suggest that expression coevolution can be used for computational prediction of protein-protein interactions. Methods Mol Biol, 2004, 284, 217 - 27 Assaying phosphoinositide phosphatases; Taylor GS et al.; The roles of phosphoinositide second messengers as signaling molecules in a vast array of cellular processes including cell growth, metabolism, vesicular transport, programmed cell death, and responses to extracellular signals are only beginning to be understood . The recent identification of novel phosphoinositide signaling molecules underscores the need for methodology with which to characterize the enzymes responsible for regulating cellular phosphoinositide levels . One of the ways in which cells control these lipids is through dephosphorylation by phosphoinositide phosphatases, which oppose and regulate the actions of phosphoinositide kinases . We describe herein two rapid and simple assays for characterizing phosphoinositide phosphatases that can be used to provide a basis for understanding the activity and specificity of these enzymes. Int J Food Microbiol, 2004 Jul 1, 94(1), 55 - 66 Contribution of a selected fungal population to proteolysis on dry-cured ham; Martin A et al.; The proteolytic changes taking place in dry-cured hams lead to increases in free amino acids . Such free amino acids not only contribute to flavour, but also serve as precursors of volatile compounds . Several months of ripening time are required to allow the particular flavour to develop . The fungal population allowed to grow on the surface of some types of dry-cured could play a key role on proteolysis, as it has been shown for dry-cured sausages . The purpose of this work was to study the possible contribution of fungi to proteolysis in dry-cured ham . For this, a strain each of non-toxigenic Penicillium chrysogenum (Pg222) and Debaryomyces hansenii (Dh345), selected for their proteolytic activity on myofibrillar proteins, were inoculated as starter cultures . Changes in the high ionic strength-soluble proteins of an external muscle (adductor) revealed in only 6 months higher proteolysis in the inoculated hams when compared to non-inoculated control hams . Proteolytic strains among the wild fungal population on non-inoculated control hams prevented from obtaining similar differences at the end of processing . However, inoculation with Pg222 and Dh345 led to higher levels for most free amino acids at the external muscle in fully dry-cured hams . In addition, the concentration for some of the more polar free amino acids (i.e . Asp, Glu, Ser and Gln) in inoculated hams was higher at external than at internal (biceps femoris) muscles . These promising results deserve further studies to know the impact of a selected fungal population on the volatile compounds and sensory properties of dry-cured ham. Trends Cell Biol, 1996 Oct, 6(10), 371 - 5 The subunit-exchange model of histone acetylation; Roth SY et al.; Increased histone acetylation has long been linked to gene activation, but little is known about how acetylation levels are regulated, largely because the histone acetyltrans ferase activities (HATs) responsible for this modification have been cloned only recently . Comparison of the biochemical nature of the Tetrahymena HAT A complex with the genetic and biochemical properties of the Saccharomyces Gcn5p-Ado complex leads us to propose that histone acetylase assemblies may be modular in nature and that this modularity may be an intimate part of the association of these enzymes with chromatin . The 'subunit-exchange' model provides a mechanism for the regulation and targeting of both histone acetylases and deacetylases and has implications for the control of cell growth, proliferation and tumorigenesis. Bioinformatics, 2004 Sep 22, 20(14), 2242 - 50 Epub 2004 May 06. Gene co-expression network topology provides a framework for molecular characterization of cellular state; Carter SL et al.; MOTIVATION: Gene expression data have become an instrumental resource in describing the molecular state associated with various cellular phenotypes and responses to environmental perturbations . The utility of expression profiling has been demonstrated in partitioning clinical states, predicting the class of unknown samples and in assigning putative functional roles to previously uncharacterized genes based on profile similarity . However, gene expression profiling has had only limited success in identifying therapeutic targets . This is partly due to the fact that current methods based on fold-change focus only on single genes in isolation, and thus cannot convey causal information . In this paper, we present a technique for analysis of expression data in a graph-theoretic framework that relies on associations between genes . We describe the global organization of these networks and biological correlates of their structure . We go on to present a novel technique for the molecular characterization of disparate cellular states that adds a new dimension to the fold-based methods and conclude with an example application to a human medulloblastoma dataset . RESULTS: We have shown that expression networks generated from large model-organism expression datasets are scale-free and that the average clustering coefficient of these networks is several orders of magnitude higher than would be expected for similarly sized scale-free networks, suggesting an inherent hierarchical modularity similar to that previously identified in other biological networks . Furthermore, we have shown that these properties are robust with respect to the parameters of network construction . We have demonstrated an enrichment of genes having lethal knockout phenotypes in the high-degree (i.e . hub) nodes in networks generated from aggregate condition datasets; using process-focused Saccharomyces cerivisiae datasets we have demonstrated additional high-degree enrichments of condition-specific genes encoding proteins known to be involved in or important for the processes interrogated by the microarrays . These results demonstrate the utility of network analysis applied to expression data in identifying genes that are regulated in a state-specific manner . We concluded by showing that a sample application to a human clinical dataset prominently identified a known therapeutic target . AVAILABILITY: Software implementing the methods for network generation presented in this paper is available for academic use by request from the authors in the form of compiled linux binary executables. Chem Biol, 2004 Feb, 11(2), 211 - 23 A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors; Becker F et al.; In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors . Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats . For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography . The Y3H system described here may prove generally useful in the discovery of candidate drug targets. Genetics, 2004 Mar, 166(3), 1177 - 86 The identification of Pcl1-interacting proteins that genetically interact with Cla4 may indicate a link between G1 progression and mitotic exit; Keniry ME et al.; In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes . Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes . However, loss of both proteins is lethal, suggesting some functional overlap . To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4 Delta strain . These mutations define >60 genes . Recently, both Ste20 and Cla4 have been implicated in mitotic exit . Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4 . We further show that the Pho85-coupled G(1) cyclins Pcl1 and Pcl2 contribute to this Pho85 role . We performed a two-hybrid screen with Pcl1 . Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1 . Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1.Pho85 complexes . NCP1, HMS1, and EPA1 also genetically interact with CLA4 . Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins . We propose a model in which the G(1) Pcl1.Pho85 complex regulates mitotic exit machinery. Mol Cell Proteomics, 2004 Jul, 3(7), 704 - 14 Epub 2004 Apr 07. Identification of the linker-SH2 domain of STAT as the origin of the SH2 domain using two-dimensional structural alignment; Gao Q et al.; The availability of large volumes of genomic sequences presents an unprecedented proteomic challenge to characterize the structure and function of various protein motifs . Primary structural alignment is often unable to accurately identify a given motif due to sequence divergence; however, with the aid of secondary structural prediction for analysis, it becomes feasible to explore protein motifs on a proteome-wide scale . Here we report the use of secondary structural alignment to characterize the Src homology 2 (SH2) domains of both conventional and divergent sequences and divide them into two groups, Src-type and STAT-type . In addition to the basic "alphabetabetabetaalpha" structure (betaBeta), the Src-type SH2 domain contains an extra beta-strand (betaE or betaE-betaF motif) . Alternatively, the linker domain-conjugated SH2 domain in STAT contains the alphaB' motif . Combining BLAST data from betaBeta core motif sequences with predicted secondary structural alignment, we have screened for SH2 domains in various eukaryotic model systems including Arabidopsis, Dictyostelium, and Saccharomyces . Two novel genes carrying the linker-SH2 domain of STAT were discovered and subsequently cloned from Arabidopsis . These genes, designated as STAT-type linker-SH2 domain factors (STATL), are found in a wide array of vascular and nonvascular plants, suggesting that the linker-SH2 domain evolved prior to the divergence of plants and animals . Using this approach, we expanded the number of putative SH2 domain-bearing genes in Dictyostelium and comparatively studied the secondary structural profiles of both typical and atypical SH2 domains . Our results indicate that the linker-SH2 domain of the transcription factor STAT is one of the most ancient and fully developed functional domains, serving as a template for the continuing evolution of the SH2 domain essential for phosphotyrosine signal transduction. J Clin Microbiol, 2004 Apr, 42(4), 1832 - 6 Genotyping and antifungal susceptibility profile of Dipodascus capitatus isolates causing disseminated infection in seven hematological patients of a tertiary hospital; Gadea I et al.; Seven cases of disseminated infection due to Dipodascus capitatus are reported . Infections occurred in a hematological unit of a tertiary hospital during a period of 5 years . Five cases were refractory to antifungal therapy . Antifungal susceptibility testing of seven isolates was performed, and strains were typed by PCR fingerprinting with the core sequence of phage M13 and by random amplification of polymorphic DNA with two primers, Ap12h and W-80A . A very short range of MICs of each antifungal agent was observed . The MICs of amphotericin B ranged between 0.50 and 2 microg/ml . Strains were susceptible in vitro to flucytosine and susceptible (dose-dependent) to fluconazole and itraconazole . Voriconazole exhibited an activity in vitro comparable to that of itraconazole . Typing techniques allowed seven additional isolates of D . capitatus neither geographically nor temporally related to be classified into two different genomic patterns . The genomic type of the seven strains from the hematological unit was identical regardless of typing technique utilized . It would indicate that the seven cases of disseminated infection could be related epidemiologically. Cell, 2004 Apr 2, 117(1), 29 - 45 Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis; Borner GV et al.; Yeast mutants lacking meiotic proteins Zip1, Zip2, Zip3, Mer3, and/or Msh5 (ZMMs) were analyzed for recombination, synaptonemal complex (SC), and meiotic progression . At 33 degrees C, recombination-initiating double-strand breaks (DSBs) and noncrossover products (NCRs) form normally while formation of single-end invasion strand exchange intermediates (SEIs), double Holliday junctions, crossover products (CRs), and SC are coordinately defective . Thus, during wild-type meiosis, recombinational interactions are differentiated into CR and NCR types very early, prior to onset of stable strand exchange and independent of SC . By implication, crossover interference does not require SC formation . We suggest that SC formation may require interference . Subsequently, CR-designated DSBs undergo a tightly coupled, ZMM-promoted transition that yields SEI-containing recombination complexes embedded in patches of SC . zmm mutant phenotypes differ strikingly at 33 degrees C and 23 degrees C, implicating higher temperature as a positive effector of recombination and identifying a checkpoint that monitors local CR-specific events, not SC formation, at late leptotene. J Exp Clin Cancer Res, 2003 Dec, 22(4), 581 - 9 Free radical scavenging, antitumor and anticarcinogenic activity of gossypin; Babu BH et al.; Antioxidant, antitumor and anticarcinogenic activity of gossypin (3,5,8,3',4'-pentahydroxy-7-O glucosyl flavone) was carried out . The compound needed for 50% inhibition of superoxide, hydroxyl and nitric oxide radicals was 3 microg/ml, 41 microg/ml and 12 microg/ml, respectively . Gossypin also impart 50% inhibition at concentrations 37 microg/ml and 43 microg/ml, respectively for in vitro lipid peroxidation . The compound shows IC50 of 30 microM, 42.5 microM and 45.1 microM concentrations in L 929, HT 29 and K 562 cell lines in 72 hrs MTT assay . The compound shows a zone of inhibition of 8 mm in topo I and topo II inhibition assay in Saccharomyces ceriviseae mutant cultures . It reduces the tumor burden in solid tumor harboring animals (p < 0.001) and effectively inhibits the formation of new blood vessels on tumor mass . 20 mg/kg b.wt of gossypin increase the life span of ascites tumor harboring animals (100%) and (164.7%) respectively by oral and intraperitoneal administration of the drug . Gossypin reduced the incidence of papilloma formation and papilloma/mouse in DMBA/ croton oil induced skin papilloma. J Biol Chem, 2004 Jun 11, 279(24), 24957 - 64 Epub 2004 Mar 26. C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats; Jones JC et al.; The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 . In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5 . A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern . We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3'-end formation . Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5 . We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2 . When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD . We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates. FEMS Yeast Res, 2004 Mar, 4(6), 605 - 7 Metschnikowia kunwiensis comb . nov., the teleomorph of Candida kunwiensis; Brysch-Herzberg M; The teleomorph of Candida kunwiensis Hong, Bae, Herzberg, Titze, Lachance, Metschnikowia kunwiensis, is described . Repeated attempts to obtain ascospore formation succeeded using modified V8 sporulation media and extended incubation times . The asci are ovoid, with only a small protrusion caused by the spore(s) . The species is diplontic, possibly homothallic, with one or two ascospores per ascus . Aside from having atypical ovoid asci, the acicular shape of the spores is characteristic of the genus Metschnikowia . The type strain is CBS 9676(T). FEMS Yeast Res, 2004 Mar, 4(6), 587 - 96 Speciation in the large-spored Metschnikowia clade and establishment of a new species, Metschnikowia borealis comb . nov; Marinoni G et al.; The reproductive boundaries among species in the large-spored Metschnikowia clade were studied by prototrophic recombinant selection, electrophoretic karyotyping, mitochondrial DNA restriction analysis, and DNA sequence analysis . Inviable ascospores arose from crosses between the two varieties of Metschnikowia continentalis, indicating that they should be recognized as separate species . Prototrophic recombinants were recovered from crosses between auxotrophic mutants of Metschnikowia borealis, M . continentalis, Metschnikowia lochheadii, Metschnikowia sp . UWO(PS)00-154.1, and Candida ipomoeae, showing that some genetic exchange is possible in spite of the sterility of the asci formed in interspecific crosses . Metschnikowia hawaiiensis, although capable of ascus formation when its h(-) mating type is crossed with the h(+) mating type of the other species, did not give rise to recombinants . In the other species, some recombinants acquired the ability to form asci directly from single cells . These often contained the chromosomes of both parents, suggesting formation of allodiploid hybrids . Other recombinants behaved as haploids and were similar to one parent except for having inherited the selectable wild-type allele from the other parent . In most, but not all cases, inheritance of the mitochondrial genome was uniparental and correlated with the inheritance of the nuclear chromosome complement . In some cases, what appeared to be a recombinant mitochondrial genome was observed . Phylogenies derived from the sequences of various DNA regions were not congruent, indicating that hybridization may have taken place in nature as the large-spored species diverged from their common ancestor . Further evidence that C . ipomoeae arose from a natural recombination event was obtained, but a pair of Metschnikowia species that might represent derived forms of the parents could not be identified conclusively . C . ipomoeae and most of its closely related Metschnikowia species contained a group-II intron in the mitochondrial small-subunit ribosomal gene . The intron was absent in M . borealis, M . hawaiiensis, and other species in the genus Metschnikowia. Appl Environ Microbiol, 2004 Mar, 70(3), 1347 - 55 Molecular detection and identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in spoiled wines; Cocolin L et al.; In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B . anomalus . Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used . No amplification product was obtained when DNA from other Brettanomyces spp . or wine yeasts were used as the templates . The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B . bruxellensis and B . anomalus, and each species could be identified on the basis of the different restriction profiles . After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed . Total agreement between traditional identification and molecular identification was observed . The protocol developed was also used for direct detection of B . bruxellensis and B . anomalus in wines suspected to be spoiled by Brettanomyces spp . Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B . bruxellensis . Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels. BMC Evol Biol . 2004 Jan 28;4(1):2. A molecular timescale of eukaryote evolution and the rise of complex multicellular life; Hedges SB et al.; BACKGROUND: The pattern and timing of the rise in complex multicellular life during Earth's history has not been established . Great disparity persists between the pattern suggested by the fossil record and that estimated by molecular clocks, especially for plants, animals, fungi, and the deepest branches of the eukaryote tree . Here, we used all available protein sequence data and molecular clock methods to place constraints on the increase in complexity through time . RESULTS: Our phylogenetic analyses revealed that (i) animals are more closely related to fungi than to plants, (ii) red algae are closer to plants than to animals or fungi, (iii) choanoflagellates are closer to animals than to fungi or plants, (iv) diplomonads, euglenozoans, and alveolates each are basal to plants+animals+fungi, and (v) diplomonads are basal to other eukaryotes (including alveolates and euglenozoans) . Divergence times were estimated from global and local clock methods using 20-188 proteins per node, with data treated separately (multigene) and concatenated (supergene) . Different time estimation methods yielded similar results (within 5%): vertebrate-arthropod (964 million years ago, Ma), Cnidaria-Bilateria (1,298 Ma), Porifera-Eumetozoa (1,351 Ma), Pyrenomycetes-Plectomycetes (551 Ma), Candida-Saccharomyces (723 Ma), Hemiascomycetes-filamentous Ascomycota (982 Ma), Basidiomycota-Ascomycota (968 Ma), Mucorales-Basidiomycota (947 Ma), Fungi-Animalia (1,513 Ma), mosses-vascular plants (707 Ma), Chlorophyta-Tracheophyta (968 Ma), Rhodophyta-Chlorophyta+Embryophyta (1,428 Ma), Plantae-Animalia (1,609 Ma), Alveolata-plants+animals+fungi (1,973 Ma), Euglenozoa-plants+animals+fungi (1,961 Ma), and Giardia-plants+animals+fungi (2,309 Ma) . By extrapolation, mitochondria arose approximately 2300-1800 Ma and plastids arose 1600-1500 Ma . Estimates of the maximum number of cell types of common ancestors, combined with divergence times, showed an increase from two cell types at 2500 Ma to approximately 10 types at 1500 Ma and 50 cell types at approximately 1000 Ma . CONCLUSIONS: The results suggest that oxygen levels in the environment, and the ability of eukaryotes to extract energy from oxygen, as well as produce oxygen, were key factors in the rise of complex multicellular life . Mitochondria and organisms with more than 2-3 cell types appeared soon after the initial increase in oxygen levels at 2300 Ma . The addition of plastids at 1500 Ma, allowing eukaryotes to produce oxygen, preceded the major rise in complexity. Genome Biol . 2004;5(3):R21 . Epub 2004 Feb 13. MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins; Maurer-Stroh S et al.; We evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences . Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated . We give experimental evidence for in vitro myristoylation of selected predictions . Furthermore, we classify five membrane-attachment factors that occur most frequently in combination with, or even replacing, myristoyl anchors, as some protein family examples show. Pac Symp Biocomput . 2004;:324-35. Phylogenetic motif detection by expectation-maximization on evolutionary mixtures; Moses AM et al.; The preferential conservation of transcription factor binding sites implies that non-coding sequence data from related species will prove a powerful asset to motif discovery . We present a unified probabilistic framework for motif discovery that incorporates evolutionary information . We treat aligned DNA sequence as a mixture of evolutionary models, for motif and background, and, following the example of the MEME program, provide an algorithm to estimate the parameters by Expectation-Maximization . We examine a variety of evolutionary models and show that our approach can take advantage of phylogenic information to avoid false positives and discover motifs upstream of groups of characterized target genes . We compare our method to traditional motif finding on only conserved regions . An implementation will be made available at http://rana.lbl.gov. Med Mycol, 2004 Feb, 42(1), 87 - 92 Co-isolation of Trichosporon inkin and Candida parapsilosis from a scalp white piedra case; Taj-Aldeen SJ et al.; White piedra is a rare fungal infection of the hair shaft characterized by small, firm, irregular white-brown nodules . The infection is caused by basidiomycetous yeasts in the genus Trichosporon . We report a case of a 28-year-old female patient who acquired the infection in Qatar . In this case, the scalp was the only site affected, but infection at that site was extensive . The hair had a Saccharomyces-like yeast odor and appeared to be beaded, with light-brown nodules of varying sizes up to 2 mm long . Trichosporon sp . accompanied by Candida parapsilosis grew out along hair shafts planted in primary isolation media . Molecular identification of the Trichosporon carried out by analyzing the 26S ribosomal gene gave a 100% match with Trichosporon inkin, a major cause of pubic white piedra . The patient was treated with daily applications of ketoconazole shampoo followed by econazole shampoo and cream, and was considered clinically and mycologically cured after 2 months . Novel findings in the present case are the first identification of T . inkin as an agent of scalp white piedra, and the heavy outgrowth of C . parapsilosis from the concretions, although in the latter case it is not clear if the co-occurring yeast was etiologically contributory to the pathogenesis of the white piedra. Med Mycol, 2004 Feb, 42(1), 27 - 34 Delineation of Clavispora lusitaniae clinical isolates by polymerase chain reaction-single strand conformation polymorphism analysis of the ITSI region: a retrospective study comparing five typing methods; Arabatzis M et al.; Strain delineation of the emerging opportunistic pathogen Clavispora lusitaniae was studied using 12 strains, including two strains of known opposite mating type, CBS 6936 (h+) and CBS 5094 (h-), and 10 strains isolated between 1998 and 2001 from immunocompromized patients . This retrospective study assessed the occurrence of C . lusitaniae subtypes within and among hospitals, and in outpatients who were regularly screened for fungal infections in the course of radio-chemotherapy . Strain typing was accomplished for the first time using single strand conformation polymorphism (SSCP) analysis of amplicons of the ribosomal DNA internal transcribed spacer (ITS) 1 and 2 regions . The results were compared with those produced by three pulsed-field gel electrophoresis (PFGE) methods and PCR fingerprinting with the minisatellite-specific primer M13 . Karyotyping separated 7-9 chromosomes, not 6-8 as previously reported . Pulsotyping of SfiI and NotI digested chromosomes grouped isolates in five and four distinct clusters, respectively . All methods revealed strain heterogeneity, though not as extensive as previously recorded . SSCP analysis of the ITS1 region generated five subtypes, based on a sequencing-confirmed nucleotide polymorphism . The discriminatory power of this method was high . All strains displayed a homogeneous SSCP pattern for the ITS2 region . ITS1 PCR-SSCP appears to allow rapid and reliable delineation of C . lusitaniae strains . Pending examination of a larger sample size and interlaboratory study, this protocol can be recommended for rapid prospective identification of hospital outbreaks. Am J Hum Genet, 2004 Mar, 74(3), 545 - 51 Epub 2004 Feb 17. Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I; Kranz C et al.; This study describes the discovery of a new inherited disorder of glycosylation named "CDG-Ik." CDG-Ik (congenital disorder of glycoslyation type Ik) is based on a defect of human mannosyltransferase I (MT-I {MIM 605907}), an enzyme necessary for the elongation of dolichol-linked chitobiose during N-glycan biosynthesis . Mutations in semiconserved regions in the corresponding gene, HMT-1 (yeast homologue, Alg1), in two patients caused drastically reduced enzyme activity, leading to a severe disease with death in early infancy . One patient had a homozygous point mutation (c.773C-->T, S258L), whereas the other patient was compound heterozygous for the mutations c.773C-->T and c.1025A-->C (E342P) . Glycosylation and growth of Alg1-deficient PRY56 yeast cells, showing a temperature-sensitive phenotype, could be restored by the human wild-type allele, whereas only slight restoration was observed after transformation with the patients' alleles. Fungal Genet Biol, 2004 Mar, 41(3), 285 - 92 Comparative sequence analysis of Sordaria macrospora and Neurospora crassa as a means to improve genome annotation; Nowrousian M et al.; One of the most challenging parts of large scale sequencing projects is the identification of functional elements encoded in a genome . Recently, studies of genomes of up to six different Saccharomyces species have demonstrated that a comparative analysis of genome sequences from closely related species is a powerful approach to identify open reading frames and other functional regions within genomes {Science 301 (2003) 71, Nature 423 (2003) 241} . Here, we present a comparison of selected sequences from Sordaria macrospora to their corresponding Neurospora crassa orthologous regions . Our analysis indicates that due to the high degree of sequence similarity and conservation of overall genomic organization, S . macrospora sequence information can be used to simplify the annotation of the N . crassa genome. J Biol Chem, 2004 Apr 2, 279(14), 14245 - 55 Epub 2004 Jan 26. Cell cycle-dependent phosphorylation of the DNA polymerase epsilon subunit, Dpb2, by the Cdc28 cyclin-dependent protein kinase; Kesti T et al.; DNA polymerase epsilon (Polepsilon), one of the three major eukaryotic replicative polymerases, is comprised of the essential catalytic subunit, called Pol2 in budding yeast, and three accessory subunits, only one of which, Dpb2, is essential . Polepsilon is recruited to replication origins during late G(1) phase prior to activation of replication . In this work we show that the budding yeast Dpb2 is phosphorylated in a cell cycle-dependent manner during late G(1) phase . Phosphorylation results in the appearance of a lower mobility species . The appearance of that species in vivo is dependent upon the Cdc28 cyclin-dependent protein kinase (CDK), which can directly phosphorylate Dpb2 in vitro . Either G(1) cyclin (Cln) or B-type cyclin (Clb)-associated CDK is sufficient for phosphorylation . Mapping of phosphorylation sites by mass spectrometry using a novel gel-based proteolysis protocol shows that, of the three consensus CDK phosphorylation sites, at least two, Ser-144 and Ser-616, are phosphorylated in vivo . The Cdc28 CDK phosphorylates only Ser-144 in vitro . Using site-directed mutagenesis, we show that Ser-144 is sufficient for the formation of the lower mobility form of Dpb2 in vivo . In contrast, Ser-616 appears not to be phosphorylated by Cdc28 . Finally, inactivation of all three CDK consensus sites in Dpb2 results in a synthetic phenotype with the pol2-11 mutation, leading to decreased spore viability, slow growth, and increased thermosensitivity . We suggest that phosphorylation of Dpb2 during late G(1) phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Polepsilon J Mol Evol, 2003 Dec, 57(6), 694 - 701 Mammalian mutation pressure, synonymous codon choice, and mRNA degradation; Duan J et al.; The usage of synonymous codons (SCs) in mammalian genes is highly correlated with local base composition and is therefore thought to be determined by mutation pressure . The usage is nonetheless structured . For instance, mammals share with Saccharomyces and Drosophila most preferences for the C-ending over the G-ending codon (or vice versa) within each fourfold-degenerate SC family and the fact that their SCs are placed along coding regions in ways that minimize the number of T|A and C|G dinucleotides ("|" being the codon boundary) . TA and CG underrepresentations are observed everywhere in the mammalian genome affecting the SC usage, the amino acid composition of proteins, and the primary structure of introns and noncoding DNA . While the rarity of CG is ascribed to the high mutability of this dinucleotide, the rarity of TA in coding regions is considered adaptive because UA dinucleotides are cleaved by endoribonucleases . Here we present in vivo experimental evidence indicating that the number of T|A and/or C|G dinucleotides of a human gene can affect strongly the expression level and degradation of its mRNA . Our results are consistent with indirect evidence produced by other workers and with the detailed work that has been devoted to characterize UA cleavage in vitro and in vivo . We conclude that SC choice can influence strongly mRNA function and gene expression through effects not directly related to the codon-anticodon interaction . These effects should constrain heavily the nucleotide motif composition of the most abundant mRNAs in the transcriptome, in particular, their SC usage, a usage that must be reflected by cellular tRNA concentrations and thus defines for all other genes which SCs are translated fastest and most accurately . Furthermore, the need to avoid such effects genome-wide appears serious enough to have favored the evolution of biases in context-dependent mutation that reduce the occurrence of intrinsically unfavorable motifs, and/or, when possible, to have induced the molecular machinery mediating such effects to rely opportunistically on already existing motif rarities and abundances . This may explain why nucleotide motif preferences are very similar in transcribed and nontranscribed mammalian DNA even though the preferences appear to be adaptive only in transcribed DNA. Mol Biol Cell, 2004 Apr, 15(4), 1724 - 35 Epub 2004 Jan 23. S-phase checkpoint genes safeguard high-fidelity sister chromatid cohesion; Warren CD et al.; Cohesion establishment and maintenance are carried out by proteins that modify the activity of Cohesin, an essential complex that holds sister chromatids together . Constituents of the replication fork, such as the DNA polymerase alpha-binding protein Ctf4, contribute to cohesion in ways that are poorly understood . To identify additional cohesion components, we analyzed a ctf4Delta synthetic lethal screen performed on microarrays . We focused on a subset of ctf4Delta-interacting genes with genetic instability of their own . Our analyses revealed that 17 previously studied genes are also necessary for the maintenance of robust association of sisters in metaphase . Among these were subunits of the MRX complex, which forms a molecular structure similar to Cohesin . Further investigation indicated that the MRX complex did not contribute to metaphase cohesion independent of Cohesin, although an additional role may be contributed by XRS2 . In general, results from the screen indicated a sister chromatid cohesion role for a specific subset of genes that function in DNA replication and repair . This subset is particularly enriched for genes that support the S-phase checkpoint . We suggest that these genes promote and protect a chromatin environment conducive to robust cohesion. J Biol Chem, 2004 Apr 9, 279(15), 15621 - 9 Epub 2004 Jan 21. Dictyostelium macroautophagy mutants vary in the severity of their developmental defects; Otto GP et al.; Macroautophagy is the major mechanism that eukaryotes use to recycle cellular components during stressful conditions . We have shown previously that the Atg12-Atg5 conjugation system, required for autophagosome formation in yeast, is necessary for Dictyostelium development . A second conjugation reaction, Aut7/Atg8 lipidation with phosphatidylethanolamine, as well as a protein kinase complex and a phosphatidylinositol 3-kinase complex are also required for macroautophagy in yeast . In this study, we characterize mutations in the putative Dictyostelium discoideum orthologues of budding yeast genes that are involved in one of each of these functions, ATG1, ATG6, and ATG8 . All three genes are required for macroautophagy in Dictyostelium . Mutant amoebae display reduced survival during nitrogen starvation and reduced protein degradation during development . Mutations in the three genes produce aberrant development with defects of varying severity . As with other Dictyostelium macroautophagy mutants, development of atg1-1, atg6(-), and atg8(-) is more aberrant in plaques on bacterial lawns than on nitrocellulose filters . The most severe defect is observed in the atg1-1 mutant, which does not aggregate on bacterial lawns and arrests as loose mounds on nitrocellulose filters . The atg6(-) and atg8(-) mutants display almost normal development on nitrocellulose filters, producing multi-tipped aggregates that mature into small fruiting bodies . The distribution of a green fluorescent protein fusion of the autophagosome marker, Atg8, is aberrant in both atg1-1 and atg6(-) mutants. Bioinformatics, 2004 Jan 22, 20(2), 180 - 5 A comparative phylogenetic approach for dating whole genome duplication events; Chapman BA et al.; MOTIVATION: Whole genome duplications have played a major role in determining the structure of eukaryotic genomes . Current evidence revealing large blocks of duplicated chromatin yields new insights into the evolutionary history of species, but also presents a major challenge for researchers attempting to utilize comparative genomics techniques . Understanding the timing of duplication events relative to divergence among taxa is critical to accurate and comprehensive cross-species comparisons . RESULTS: We describe a large-scale approach to estimate the timing of duplication events in a phylogenetic context . The methodology has been previously utilized for analysis of Arabidopsis and Saccharomyces duplication events . This new implementation provides a more flexible and reusable framework for these analyses . Scripts written in the Python programming language drive a number of freely available bioinformatics programs, creating a no-cost tool for researchers . The usefulness of the approach is demonstrated through genome-scale analysis of Arabidopsis and Oryza (rice) duplications . AVAILABILITY: Software and documentation are freely available from http://plantgenome.agtec.uga.edu/bioinformatics/dating/ Clin Infect Dis, 2004 Feb 1, 38(3), 335 - 41 Epub 2004 Jan 14. Blastoschizomyces capitatus infection in patients with leukemia: report of 26 cases; Martino R et al.; Twenty-six cases of Blastoschizomyces capitatus infection were diagnosed in 25 patients at 7 tertiary care hematology units in Spain over a 10-year period . Most patients (92%) had acute leukemia and developed infection during a period of severe and prolonged neutropenia . Two patients had esophagitis, and the rest had invasive infection . Fungemia (20 cases) was a common finding, with frequent visceral dissemination . The 30-day mortality associated with this infection was 52%, compared with 57% among patients with systemic infection . In a univariate analysis, the following 3 variables had a positive impact on 30-day survival: removal of the central venous catheter within 5 days after the onset of infection (P=.02), a good performance status (P=.003), and receipt of systemic prophylactic or empirical antifungal therapy before infection onset (P=.006) . Outcome for neutropenic patients with B . capitatus infection is still poor . Rapid removal of the central venous catheter and novel antifungal therapies are recommended for treatment of this rare infection. BMC Genomics . 2004 Jan 13;5(1):5. Identification and comparative analysis of components from the signal recognition particle in protozoa and fungi; Rosenblad MA et al.; BACKGROUND: The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane . The SRP of metazoans is well characterized and composed of an RNA molecule and six polypeptides . The particle is organized into the S and Alu domains . The Alu domain has a translational arrest function and consists of the SRP9 and SRP14 proteins bound to the terminal regions of the SRP RNA . So far, our understanding of the SRP and its evolution in lower eukaryotes such as protozoa and yeasts has been limited . However, genome sequences of such organisms have recently become available, and we have now analyzed this information with respect to genes encoding SRP components . RESULTS: A number of SRP RNA and SRP protein genes were identified by an analysis of genomes of protozoa and fungi . The sequences and secondary structures of the Alu portion of the RNA were found to be highly variable . Furthermore, proteins SRP9/14 appeared to be absent in certain species . Comparative analysis of the SRP RNAs from different Saccharomyces species resulted in models which contain features shared between all SRP RNAs, but also a new secondary structure element in SRP RNA helix 5 . Protein SRP21, previously thought to be present only in Saccharomyces, was shown to be a constituent of additional fungal genomes . Furthermore, SRP21 was found to be related to metazoan and plant SRP9, suggesting that the two proteins are functionally related . CONCLUSIONS: Analysis of a number of not previously annotated SRP components show that the SRP Alu domain is subject to a more rapid evolution than the other parts of the molecule . For instance, the RNA portion is highly variable and the protein SRP9 seems to have evolved into the SRP21 protein in fungi . In addition, we identified a secondary structure element in the Saccharomyces RNA that has been inserted close to the Alu region . Together, these results provide important clues as to the structure, function and evolution of SRP. Curr Biol, 2004 Jan 6, 14(1), R33 - 4 Vesicle transport: a close collaboration of Rabs and effectors; Spang A; COPII vesicles transport proteins destined for secretion from the ER to the Golgi apparatus . A recent study has shown that, in budding yeast, the formation of COPII vesicles requires Yip1p, an effector protein of a Rab GTPase. Bioinformatics, 2004 Jan 1, 20(1), 5 - 20 Identifying periodically expressed transcripts in microarray time series data; Wichert S et al.; MOTIVATION: Microarray experiments are now routinely used to collect large-scale time series data, for example to monitor gene expression during the cell cycle . Statistical analysis of this data poses many challenges, one being that it is hard to identify correctly the subset of genes with a clear periodic signature . This has lead to a controversial argument with regard to the suitability of both available methods and current microarray data . METHODS: We introduce two simple but efficient statistical methods for signal detection and gene selection in gene expression time series data . First, we suggest the average periodogram as an exploratory device for graphical assessment of the presence of periodic transcripts in the data . Second, we describe an exact statistical test to identify periodically expressed genes that allows one to distinguish periodic from purely random processes . This identification method is based on the so-called g-statistic and uses the false discovery rate approach to multiple testing . RESULTS: Using simulated data it is shown that the suggested method is capable of identifying cell-cycle-activated genes in a gene expression data set even if the number of the cyclic genes is very small and regardless the presence of a dominant non-periodic component in the data . Subsequently, we re-examine 12 large microarray time series data sets (in part controversially discussed) from yeast, human fibroblast, human HeLa and bacterial cells . Based on the statistical analysis it is found that a majority of these data sets contained little or no statistical significant evidence for genes with periodic variation linked to cell cycle regulation . On the other hand, for the remaining data the method extends the catalog of previously known cell-cycle-specific transcripts by identifying additional periodic genes not found by other methods . The problem of distinguishing periodicity due to generic cell cycle activity and to artifacts from synchronization is also discussed . AVAILABILITY: The approach has been implemented in the R package GeneTS available from under the terms of the GNU General Public License. Adv Space Res, 2003, 31(10), 2181 - 6 Gravitaxis and graviperception in flagellates; Hader DP et al.; There is strong evidence that gravitactic orientation in flagellates and ciliates is mediated by an active physiological gravireceptor rather than by passive alignment of the cells in the water column . In flagellates the threshold for graviorientation was found to be at 0.12 x g on a slow rotating centrifuge during the IML-2 mission on the Shuttle Columbia and a subsequent parabolic rocket flight (TEXUS) . During the IML-2 mission no adaptation to microgravity was observed over the duration of the space flight, while gravitaxis was lost in a terrestrial closed environmental system over the period of almost two years . Sedimenting statoliths are not likely to be involved in graviperception because of the small size of the cells and their rotation around the longitudinal axis during forward locomotion . Instead the whole cytoplasmic content of the cell, being heavier than the surrounding aqueous medium (1.05 g/ml), exerts a pressure on the lower membrane . This force activates stretch-sensitive calcium specific ion channels which can be inhibited by the addition of gadolinium which therefore abolishes gravitaxis . The channels seem to mainly allow calcium ions to pass since gravitaxis is blocked by the addition of the calcium ionophore A23187 and by vanadate which blocks the Ca-ATPase in the cytoplasmic membrane . Recently, a gene for a mechanosensitive channel, originally sequenced for Saccharomyces, was identified in Euglena by PCR . The increase in intracellular free calcium during reorientation can be visualized by the fluorophore Calcium Crimson using laser excitation and image intensification . This result was confirmed during recent parabolic flights . The gated calcium changes the membrane potential across the membrane which may be the trigger for the reorientation of the flagellum . cAMP plays a role as a secondary messenger . Photosynthetic flagellates are suitable candidates for life support systems since they absorb CO2 and produce oxygen . Preliminary experiments are discussed . c2003 COSPAR . Published by Elsevier Ltd . All rights reserved. Curr Biol, 2003 Dec 16, 13(24), 2190 - 5 EST analysis of the cnidarian Acropora millepora reveals extensive gene loss and rapid sequence divergence in the model invertebrates; Kortschak RD et al.; A significant proportion of mammalian genes are not represented in the genomes of Drosophila, Caenorhabditis or Saccharomyces, and many of these are assumed to have been vertebrate innovations . To test this assumption, we conducted a preliminary EST project on the anthozoan cnidarian, Acropora millepora, a basal metazoan . More than 10% of the Acropora ESTs with strong metazoan matches to the databases had clear human homologs but were not represented in the Drosophila or Caenorhabditis genomes; this category includes a surprising diversity of transcription factors and metabolic proteins that were previously assumed to be restricted to vertebrates . Consistent with higher rates of divergence in the model invertebrates, three-way comparisons show that most Acropora ESTs match human sequences much more strongly than they do any Drosophila or Caenorhabditis sequence . Gene loss has thus been much more extensive in the model invertebrate lineages than previously assumed and, as a consequence, some genes formerly thought to be vertebrate inventions must have been present in the common metazoan ancestor . The complexity of the Acropora genome is paradoxical, given that this organism contains apparently few tissue types and the simplest extant nervous system consisting of a morphologically homogeneous nerve net. Proteomics, 2003 Dec, 3(12), 2330 - 8 Proteomic analysis of Candida magnoliae strains by two-dimensional gel electrophoresis and mass spectrometry; Lee DY et al.; Candida magnoliae which has been newly isolated from honey comb is an osmotolerant yeast to produce erythritol as a major product . Erythritol is a noncariogenic, low calorie sweetener and safe for diabetics . Strain development by chemical mutation to obtain the improved erythritol yield and productivity relative to the parental strain made it necessary to elucidate the physiological differences between the wild and mutant strains . Proteomic analyses of C . magnoliae wild and mutant strains with two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry were carried out to identify intracellular proteins and to estimate the effects of newly characterized metabolic enzymes on the yeast cell growth and erythritol production . Most of the molecular mass of intracellular proteins were distributed in the range of pI 4-8 and molecular mass of approximately 130 kDa . Six out of nine protein spots expressed at different levels between the wild and mutant strains were analyzed with nanoelectrospray tandem mass spectrometry and identified by comparing amino acid sequences with the National Center for Biotechnology Information and Saccharomyces Genome Databases . Except for Ygr086cp, these proteins were believed to be the metabolic enzymes involved in the citric acid cycle (citrate synthase, succinyl-CoA ligase and fumarase) and the glycolysis pathway (pyruvate decarboxylase and enolase) . Up-regulated enzymes in the citric acid cycle could explain high growth of the C . magnoliae mutant strain owing to the increased NADH and ATP formation . Down-regulated enolase and up-regulated fumarase in the mutant strain seemed to play a role in the improved bioconversion of erythrose-4-phosphate to erythritol compared with the wild strain. J Gen Appl Microbiol, 2003 Oct, 49(5), 267 - 70 Ribosomal DNA sequencing and reinstatement of the genus Arthroascus von Arx; Naumov GI et al.; Sequence analysis of the D1/D2 domain of 26S rDNA was conducted upon seven Arthroascus strains from different geographic localities . The European and Asian species Arthroascus schoenii was documented from the North-American continent and from the Island of Hawaii . We discuss the heterogeneity of the genus Saccharomycopsis sensu Kurtzman and Robnett 1995 . On the basis of molecular and genetic data the genus Arthroascus von Arx is reinstated. Genome, 2003 Dec, 46(6), 947 - 52 Unfolding large-scale maps; Jenkins G; This is an account of the development and use of genetic maps, from humble beginnings at the hands of Thomas Hunt Morgan, to the sophistication of genome sequencing . The review charters the emergence of molecular marker maps exploiting DNA polymorphism, the renaissance of cytogenetics through the use of fluorescence in situ hybridisation, and the discovery and isolation of genes by map-based cloning . The historical significance of sequencing of DNA prefaces a section describing the sequencing of genomes, the ascendancy of particular model organisms, and the utility and limitations of comparative genomic and functional genomic approaches to further our understanding of the control of biological processes . Emphasis is given throughout the treatise as to how the structure and biological behaviour of the DNA molecule underpin the technological development and biological applications of maps. J Cell Sci, 2003 Dec 15, 116(Pt 24), 4883 - 90 Wee1-dependent mechanisms required for coordination of cell growth and cell division; Kellogg DR; Wee1-related kinases function in a highly conserved mechanism that controls the timing of entry into mitosis . Loss of Wee1 function causes fission yeast and budding yeast cells to enter mitosis before sufficient growth has occurred, leading to formation of daughter cells that are smaller than normal . Early work in fission yeast suggested that Wee1 is part of a cell-size checkpoint that prevents entry into mitosis before cells have reached a critical size . Recent experiments in fission yeast and budding yeast have provided new support for this idea . In addition, studies in budding yeast have revealed the existence of highly intricate signaling networks that are required for regulation of Swe1, the budding yeast homolog of Wee1 . Further understanding of these signaling networks may provide important clues to how cell growth and cell division are coordinated. Curr Biol, 2003 Nov 11, 13(22), 1979 - 84 Spindle checkpoint component Mad2 contributes to biorientation of homologous chromosomes; Shonn MA et al.; Cell cycle checkpoints sense defects in chromosome metabolism, halt the cell cycle, and activate pathways that repair the defects . The spindle checkpoint arrests the cell cycle in response to defects in the interaction between microtubules and kinetochores (the proteinaceous complex assembled on centromeric DNA), but no repair function has been demonstrated for this checkpoint . We show that the roles of two spindle checkpoint components, Mad2 and Mad3, differ in meiosis I . In the absence of Mad2, meiosis I nondisjunction occurs at a high frequency and can be corrected by delaying the onset of anaphase . The absence of Mad3 does not induce nondisjunction, even though mad3Delta cells cannot arrest the cell cycle in response to kinetochores that lack either microtubules or tension on the linkage between chromosomes and microtubules . The two proteins have different roles in chromosome alignment . Compared to wild type and mad3Delta cells, mad2Delta mutants are slower to attach homologous chromosomes to opposite poles of the spindle . This observation suggests that Mad2 plays a role in reorienting chromosomes that are incorrectly attached to the spindle as well as delaying the cell cycle, whereas Mad3 is needed for the cell cycle delay, but not for chromosome reorientation. Curr Biol, 2003 Nov 11, 13(22), 1954 - 62 The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over; Rockmill B et al.; BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases . Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis . The sgs1 null mutant sporulates poorly and displays reduced spore viability . RESULTS: We have identified novel functions for Sgs1 in meiosis . Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation . In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis . Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion . The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase . A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation . CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions . The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis . Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange . We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase. Curr Biol, 2003 Nov 11, 13(22), 1930 - 40 A model for ATP hydrolysis-dependent binding of cohesin to DNA; Weitzer S et al.; BACKGROUND: Cohesion between sister chromatids is promoted by the chromosomal cohesin complex that forms a proteinaceous ring, large enough in principle to embrace two sister strands . The mechanism by which cohesin binds to DNA, and how sister chromatid cohesion is established, is unknown . RESULTS: Biochemical studies of cohesin have largely been limited to protein isolated from soluble cellular fractions . Here, we characterize cohesin purified from budding yeast chromatin, suggesting that chromosomal cohesin is sufficiently described by its known distinctive ring structure . We present evidence that the two Smc subunits of cohesin by themselves form a ring, closed at interacting ATPase head domains . A motif in the Smc1 subunit implicated in ATP hydrolysis is essential for loading cohesin onto DNA . In addition to functional ATPase heads, an intact cohesin ring structure is indispensable for DNA binding, suggesting that ATP hydrolysis may be coupled to DNA transport into the cohesin ring . DNA is released in anaphase when separase cleaves cohesin's Scc1 subunit . We show that a cleavage fragment of Scc1 disrupts the interaction between the two Smc heads, thereby opening the ring . CONCLUSIONS: We present a model for cohesin binding to chromatin by ATP hydrolysis-dependent transport of DNA into the cohesin ring . After DNA replication, two DNA strands may be trapped to promote sister chromatid cohesion . In anaphase, Scc1 cleavage opens the ring to release sister chromatids. J Biol Chem, 2004 Feb 27, 279(9), 8452 - 9 Epub 2003 Nov 10. Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes; Kanazawa T et al.; Autophagy, a major bulk proteolytic pathway, contributes to intracellular protein turnover, together with protein synthesis . Both are subject to dynamic control by amino acids and insulin . The mechanisms of signaling and cross-talk of their physiological anabolic effects remain elusive . Recent studies established that amino acids and insulin induce p70 S6 kinase (p70(S6k)) phosphorylation by mTOR, involved in translational control of protein synthesis . Here, the signaling mechanisms of amino acids and insulin in macroautophagy in relation to mTOR were investigated . In isolated rat hepatocytes, both regulatory amino acids (RegAA) and insulin coordinately activated p70(S6k) phosphorylation, which was completely blocked by rapamycin, an mTOR inhibitor . However, rapamycin blocked proteolytic suppression by insulin, but did not block inhibition by RegAA . These contrasting results suggest that insulin controls autophagy through the mTOR pathway, but amino acids do not . Furthermore, micropermeabilization with Saccharomyces aureus alpha-toxin completely deprived hepatocytes of proteolytic responsiveness to RegAA and insulin, but still maintained p70(S6k) phosphorylation by RegAA . In contrast, Leu(8)-MAP, a non-transportable leucine analogue, did not mimic the effect of leucine on p70(S6k) phosphorylation, but maintained the activity on proteolysis . Finally, BCH, a System L-specific amino acid, did not affect proteolytic suppression or mTOR activation by leucine . All the results indicate that mTOR is not common to the signaling mechanisms of amino acids and insulin in autophagy, and that the amino acid signaling starts extracellularly with their "receptor(s)," probably other than transporters, and is mediated through a novel route distinct from the mTOR pathway employed by insulin. Chem Commun (Camb), 2003 Oct 21, (20), 2636 - 7 Stereoinversion of beta- and gamma-substituted alpha-amino acids using a chemo-enzymatic oxidation-reduction procedure; Enright A et al.; Both D- and L-beta- and gamma-substituted alpha-amino acids can be interconverted to their respective L- and D- diastereoisomers by treatment with an enantioselective amino acid oxidase and a chemical reducing agent. J Cell Sci, 2003 Nov 15, 116(Pt 22), 4501 - 12 Cytoplasmic dynein in fungi: insights from nuclear migration; Yamamoto A et al.; Cytoplasmic dynein is a microtubule motor that mediates various biological processes, including nuclear migration and organelle transport, by moving on microtubules while associated with various cellular structures . The association of dynein with cellular structures and the activation of its motility are crucial steps in dynein-dependent processes . However, the mechanisms involved remain largely unknown . In fungi, dynein is required for nuclear migration . In budding yeast, nuclear migration is driven by the interaction of astral microtubules with the cell cortex; the interaction is mediated by dynein that is probably associated with the cortex . Recent studies suggest that budding yeast dynein is first recruited to microtubules, then delivered to the cortex by microtubules and finally activated by association with the cortex . Nuclear migration in many other fungi is probably driven by a similar mechanism . Recruitment of dynein to microtubules and its subsequent activation upon association with cellular structures are perhaps common to many dynein-dependent eukaryotic processes, including organelle transport. Nature, 2003 Oct 23, 425(6960), 798 - 804 Genome-scale approaches to resolving incongruence in molecular phylogenies; Rokas A et al.; One of the most pervasive challenges in molecular phylogenetics is the incongruence between phylogenies obtained using different data sets, such as individual genes . To systematically investigate the degree of incongruence, and potential methods for resolving it, we screened the genome sequences of eight yeast species and selected 106 widely distributed orthologous genes for phylogenetic analyses, singly and by concatenation . Our results suggest that data sets consisting of single or a small number of concatenated genes have a significant probability of supporting conflicting topologies . By contrast, analyses of the entire data set of concatenated genes yielded a single, fully resolved species tree with maximum support . Comparable results were obtained with a concatenation of a minimum of 20 genes; substantially more genes than commonly used but a small fraction of any genome . These results have important implications for resolving branches of the tree of life. Carbohydr Res, 2003 Oct 10, 338(21), 2221 - 5 Preparation and reactivity of a novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose); Yoshinaga K et al.; A novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose, GAF) was enzymatically prepared from 1,5-anhydro-D-fructose (1,5-AF) and cyclomaltoheptaose (beta-cyclodextrin) . Cyclodextrin glucanotransferase transferred various sizes of maltooligosaccharide to 1,5-AF . Glucoamylase digested the maltooligosyl chain of the products to a glucosyl residue giving a final product, GAF . An NMR analysis of GAF elucidated that the glucose residue was linked to C-3 of the 1,5-AF residue with an ether linkage . Reactivity on the aminocarbonyl reaction of GAF with bovine serum albumin was lower than that of 1,5-AF, but was higher than that of glucose. Dev Cell, 2003 Oct, 5(4), 528 - 30 Ac'septin' a signal: kinase regulation by septins; Moffat J et al.; Budding yeast monitor shape and the assembly of cytoskeletal structures and convey this information to regulators of cell division, but the molecular mechanisms responsible for monitoring and interpreting spatial information about the cytoskeleton remain poorly understood . A paper in the September issue of Molecular Cell shows that direct binding of components of the septin cytoskeleton may relieve autoinhibition of a conserved checkpoint kinase, creating a simple molecular device for sensing septin cytoskeleton organization. Clin Exp Allergy, 2003 Oct, 33(10), 1429 - 38 Sensitization to fungi: epidemiology, comparative skin tests, and IgE reactivity of fungal extracts; Mari A et al.; BACKGROUND: Several fungal species are known to cause severe respiratory and cutaneous allergic diseases . Extracts from several allergenic fungi are used for in vivo and in vitro tests, as standard preparations are still not available . OBJECTIVE: The aims are to define the pattern of in vivo and in vitro IgE reactivity to fungal species in an allergic population with respiratory symptoms; to determine the influence of different extract preparations on diagnostic results; and to evaluate whether there exists a relationship between the diagnostic pattern of reactivity and the pattern of specific IgE reactivity in immunoblots . METHODS: Skin prick tests were applied to a cohort of 4962 respiratory subjects, aged 3-80 years . Fungal extracts from Alternaria, Aspergillus, Candida, Cladosporium, Penicillium, Saccharomyces, and Trichophyton were used, along with extracts from pollens, mites, and animal dander . Demographical and diagnostic data were recorded . IgE detection was carried out with the same allergenic extracts plus Malassezia . Comparative skin tests and IgE detection were carried out using extracts from three commercial suppliers . IgE immunoblots were carried out with the same panel of commercial fungal extracts and were compared with in-house extracts . Data analysis was carried out by grouping the population on the basis of their reactivity to a single, to two or to more than two, mould species . RESULTS: Nineteen percent of the allergic population reacted to at least one fungal extract by means of the skin test . Alternaria and Candida accounted for the largest number of positive tests, and along with Trichophyton they were the main sensitizers in the subset of patients with an isolated sensitization . The prevalence of skin test reactivity increased for these three fungi in the subsets with two associated reactivities and, furthermore, in the subset showing reactivity to more than two mould species . In the latter group, a steady increase of the skin test reactivity was recorded for all the other fungal sources, suggesting a clustered reactivity . Comparative skin and IgE testing with different groups of subjects with a simple pattern of skin reactivity resulted in sensitivity differences between in vivo and in vitro tests, whereas discrepant results were recorded in the subsets of patients with multiple fungi sensitization . Although hampered by the limited reliability of fungal extracts, IgE immunoblots revealed differing patterns of reactivity when sera from the three subsets were used . This suggests a link between the diagnostic reactivity pattern and the IgE sensitization to extracts' components . Age and gender distribution differed among the Alternaria-, Candida-, and Trichophyton-sensitized subjects, but not in the subset with more than two fungi sensitizations . CONCLUSIONS: The preliminary assessment of a new classification of the mould-sensitized population has been reached . The limiting quality of fungal extracts requires future studies using an allergenic molecule-based approach . The diagnostic process and the definition of the reactivity pattern would thus be easy, and it could lead to a novel specific immunotherapy approach. J Clin Gastroenterol, 2003 Oct, 37(4), 315 - 29 Autoantibodies in the diagnosis and management of liver disease; Czaja AJ et al.; Autoantibodies are nonpathogenic manifestations of immune reactivity, and they may occur in acute and chronic liver diseases . Autoantibodies may be consequences rather than causes of the liver injury, and they should be regarded as diagnostic clues rather than etiologic markers . Conventional autoantibodies used in the categorization of autoimmune liver disease are antinuclear antibodies, smooth muscle antibodies, antibodies to liver/kidney microsome type 1, antimitochondrial antibodies, and atypical perinuclear anti-neutrophil cytoplasmic antibodies . Ancillary autoantibodies that enhance diagnostic specificity, have prognostic connotation, or direct treatment are antibodies to endomysium, tissue transglutaminase, histones, doubled-stranded DNA, and actin . Autoantibodies that have an emerging diagnostic and prognostic significance are antibodies to soluble liver antigen/liver pancreas, asialoglycoprotein receptor, liver cytosol type 1, and nuclear pore complex antigens . Autoantibodies of uncertain clinical value that remain under investigation are antibodies to chromatin, lactoferrin, and Saccharomyces cervisiae . Continued recognition and characterization of autoantibodies should improve diagnostic precision, provide prognostic indices, and elucidate target autoantigens . These advances may in turn clarify pathogenic mechanisms, facilitate the development of animal models, and generate novel site-specific therapies. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1665 - 70 Metschnikowia vanudenii sp . nov . and Metschnikowia lachancei sp . nov., from flowers and associated insects in North America; Gimenez-Jurado G et al.; Two new species of the ascosporic yeast genus Metschnikowia were isolated from nectaries and associated muscoid flies of flowers from the common milkweed (Asclepias syriaca) in North America, and are described as Metschnikowia vanudenii {type strain=PYCC 4650(T)=CBS 9134(T)=NRRL Y-27243(T)=UWO(PS) 86A4.1(T)} and Metschnikowia lachancei {type strain=PYCC 4605(T)=CBS 9131(T)=NRRL Y-27242(T)=UWO(PS) 7ASB2.3(T)} . As with the previously described Metschnikowia gruessii, M . vanudenii has vegetative cells with an 'aeroplane' or cross-like configuration, produces ovoid chlamydospores and forms ellipsoidopedunculate asci with two acicular ascospores . Metschnikowia lachancei is distinguished from other Metschnikowia species by formation of club-shaped asci with 1-2 thick clavate ascospores . The phylogenetic positions of the proposed new species within Metschnikowia were determined from sequence analysis of the D1/D2 domain of 26S rDNA . The new species show low nuclear DNA relatedness with neighbouring taxa. J Biol Chem, 2003 Oct 31, 278(44), 43178 - 87 Epub 2003 Aug 22. Human mitochondrial C1-tetrahydrofolate synthase: gene structure, tissue distribution of the mRNA, and immunolocalization in Chinese hamster ovary calls; Prasannan P et al.; C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase . The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces . Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase . The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon . The gene encodes a protein of 978 amino acids, including an N-terminal mitochondrial targeting sequence . The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme . Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain . Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue . The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8 . Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal . This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity . Both transcripts initiate at the same 5'-site, 107 nucleotides up-stream of the ATG start codon . The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells . Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein . Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase. Mol Cell Biol, 2003 Sep, 23(17), 6327 - 37 Specific inhibition of Elm1 kinase activity reveals functions required for early G1 events; Sreenivasan A et al.; In budding yeast, the Elm1 kinase is required for coordination of cell growth and cell division at G(2)/M . Elm1 is also required for efficient cytokinesis and for regulation of Swe1, the budding yeast homolog of the Wee1 kinase . To further characterize Elm1 function, we engineered an ELM1 allele that can be rapidly and selectively inhibited in vivo . We found that inhibition of Elm1 kinase activity during G(2) results in a phenotype similar to the phenotype caused by deletion of the ELM1 gene, as expected . However, inhibition of Elm1 kinase activity earlier in the cell cycle results in a prolonged G(1) delay . The G(1) requirement for Elm1 kinase activity occurs before bud emergence, polarization of the septins, and synthesis of G(1) cyclins . Inhibition of Elm1 kinase activity during early G(1) also causes defects in the organization of septins, and inhibition of Elm1 kinase activity in a strain lacking the redundant G(1) cyclins CLN1 and CLN2 is lethal . These results demonstrate that the Elm1 kinase plays an important role in G(1) events required for bud emergence and septin organization. Mol Cell Biol, 2003 Aug, 23(16), 5939 - 46 Replication checkpoint kinase Cds1 regulates recombinational repair protein Rad60; Boddy MN et al.; Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks . How Cds1 accomplishes this task is unknown . We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast . Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization . A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest . Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair . These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1 . We propose that control of Rad60 regulates recombination events at stalled forks. Int J Food Microbiol, 2003 Aug 1, 84(3), 327 - 38 Effect of Penicillium chrysogenum and Debaryomyces hansenii on the volatile compounds during controlled ripening of pork loins; Martin A et al.; During ripening of meat products such as dry-cured ham, the moulds and yeasts that proliferate on the surface may contribute to flavour development . However, their contribution to volatile components of dry-cured meat products is not known . One strain each of Penicillium chrysogenum and Debaryomyces hansenii, selected from dry-cured ham by their proteolytic activity, were tested to determine their effect on the volatile compounds during ripening . Sterile pork loins were inoculated and ripened for 106 days . Volatile compounds collected with a Solid Phase Micro-Extraction (SPME) fibre were analysed by GC/MS . Inoculation of pork loins with P . chrysogenum lead to a decrease in compounds attributed to lipid oxidation and to an increase of compounds derived from free amino acids . Inoculation with D . hansenii seemed to favour the formation of complex alcohols. Genome Biol . 2003;4(6):R40 . Epub 2003 May 30. A method to assess compositional bias in biological sequences and its application to prion-like glutamine/asparagine-rich domains in eukaryotic proteomes; Harrison PM et al.; We have derived a novel method to assess compositional biases in biological sequences, which is based on finding the lowest-probability subsequences for a given residue-type set . As a case study, the distribution of prion-like glutamine/asparagine-rich ((Q+N)-rich) domains (which are linked to amyloidogenesis) was assessed for budding and fission yeasts and four other eukaryotes . We find more than 170 prion-like (Q+N)-rich regions in budding yeast, and, strikingly, many fewer in fission yeast . Also, some residues, such as tryptophan or isoleucine, are unlikely to form biased regions in any eukaryotic proteome. Folia Microbiol (Praha), 2003, 48(2), 177 - 82 Colocalization of cortical microtubules and F-actin in Dipodascus magnusii using confocal laser scanning microscopy; Hasek J et al.; Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin . A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei . In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex . In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites . Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network . The actin system of D . magnusii consisted of patches and filaments . Patches were found to accumulate at the tips of growing cells . Bands of fine actin filaments were usually observed before F-actin rings were established . A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells . Cells with developing septa showed medial F-actin discs associated at both sides with microtubules . Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D . magnusii. J Biol Chem, 2003 Aug 15, 278(33), 31184 - 91 Epub 2003 Jun 03. Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase: an enzyme that preferentially regulates metabolism of very long chain ceramides; Mao C et al.; Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids . In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity . maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively . Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin . maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry . In vitro biochemical characterization determined that maCER1 hydrolyzed D-erythro-ceramide exclusively but not D-erythro-dihydroceramide or D-ribo-phytoceramide . Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+ . maCER1 was also inhibited by sphingosine, one of its products . Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells . Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of D-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P . Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids. Nat Rev Mol Cell Biol, 2003 Jun, 4(6), 468 - 78 A fuzzy mitochondrial fusion apparatus comes into focus; Mozdy AD et al.; Membrane fusion is fundamental to eukaryotic life . Unlike the predominant intracellular fusion machineries that fuse compartments bounded by a single membrane, the mitochondrial fusion machinery must sequentially fuse the outer and inner mitochondrial membranes . These coordinated fusion events rely on a transmembrane GTPase that is known as fuzzy onions or Fzo . Recent studies have revealed that Fzo has an evolutionarily conserved role in mitochondrial fusion, and they take the first strides in determining the molecular nature of such a role. Carbohydr Res, 2003 May 23, 338(11), 1175 - 82 A new mannoheptaose containing alpha and beta-(1-->2) linkages isolated from the mannan of Torulaspora delbrueckii: ELISA inhibition studies; Okawa Y et al.; Torulaspora delbrueckii starin IFO 0955 was examined with respect to its structural and serological properties of the cell wall mannan (Td-0955-M) . Td-0955-M revealed significant reactivities with sera from a commercially available factor serum kit (Candida Check) in ELISA . Td-0955-M was investigated for its chemical structure by acetolysis under conventional and mild conditions . NMR and GC techniques were used as analytical techniques . The mannooligosaccharide fractions eluted from a Bio-Gel P-2 column were found to consist of Man(alpha1-2)Man, M2, Man(alpha1-2)Man(alpha1-2)Man and Man(beta1-2)Man(alpha1-2)Man, M3, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M5, and a new mannoheptaose, which possesses the structure, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M7 . The results of the inhibition ELISA showed that the M7 oligosaccharide significantly inhibited the reactivities in the Td-0955-M-factor serum systems. Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 528 - 35 Epub 2003 May 06. The constitutive AHSB4 promoter--a novel component of the Arxula adeninivorans-based expression platform; Wartmann T et al.; An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized . The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes . The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones . The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions . The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment . Plasmids incorporating respective expression cassettes were used to transform the host strain A . adeninivorans LS3, which forms budding cells at 30 degrees C, and strain 135, which forms mycelia under these conditions . Transformants of both types were found to harbor a single copy of the heterologous DNA . Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4 . In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l(-1) culture medium were achieved . This productivity could be increased to 50 mg l(-1 )in strains harboring two copies of the expression cassette . The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions. FEMS Yeast Res, 2003 Apr, 3(2), 211 - 6 Physiological behaviour of Hanseniaspora guilliermondii in aerobic glucose-limited continuous cultures; Albergaria H et al.; The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures . By both cultivation techniques this yeast was found to be Crabtree-positive . Up to a dilution rate of 0.25 h(-1), glucose was completely metabolised into biomass, glycerol and carbon dioxide . Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids . Biomass yield during the purely oxidative growth was 0.49 g g(-1) and decreased to 0.26 g g(-1) for D=0.34 h(-1) . A maximal specific ethanol production rate of 1.36 mmol g(-1) h(-1) and a maximal ethanol yield of 0.05 g g(-1) were achieved at D=0.34 h(-1). FEMS Yeast Res, 2002 Jan, 1(4), 279 - 89 Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera; Cadez