Yeasts

Trends Plant Sci, 1999 Mar, 4(3), 117 - 120
Is hexokinase really a sugar sensor in plants?
Halford NG, Purcell PC, Hardie DG.
The molecular mechanisms by which plant cells sense sugar levels are not understood, but current models (adapted from models for sugar sensing in yeast) favour hexokinase as the primary sugar sensor . However, the hypothesis that yeast hexokinase has a signalling function has not been supported by more recent studies and the idea that hexokinase is involved in sugar sensing in plants has yet to be proven.

Trends Plant Sci, 1999 Mar, 4(3), 107 - 112
Function of the ubiquitin-proteosome pathway in auxin response; del Pozo JC et al.; Proteolysis of important regulatory proteins by the ubiquitin-proteosome pathway is a key aspect of cellular regulation in eukaryotes . Genetic studies in Arabidopsis indicate that response to auxin depends on the function of proteins in this pathway . The auxin transport inhibitor resistant 1 (TIR1) protein is part of a ubiquitin-protein-ligase complex (E3), known as SKP1 CDC53 F-boxTIR1 (SCFTIR1), that possibly directs ubiquitin-modification of protein regulators of the auxin response . In yeast, a similar E3 complex, SCFCDC4, is regulated by conjugation of the ubiquitin-related protein Rub1 to the Cdc53 protein . In Arabidopsis, the auxin-resistant1 (AXR1) gene encodes a subunit of the RUB1-activating enzyme, the first enzyme in the RUB-conjugation pathway . Loss of AXR1 results in loss of auxin response . These results suggest a model in which RUB1 modification regulates the activity of SCFTIR1, thereby directing the degradation of the repressors of the auxin response.

Trends Cell Biol, 1999 May, 9(5), 166 - 9
Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions; Harvey KF et al.; The members of an emerging family of proteins similar to Nedd4 have a unique modular structure consisting of a Ca2+/lipid-binding domain, multiple protein-protein interaction modules and a ubiquitin-protein ligase domain . Although little is known about the physiological roles of these proteins, studies in both mammals and yeast are providing evidence that members of this family might be involved in diverse cellular functions, such as regulation of membrane channels and permeases, the cell cycle and transcription . This article attempts to bring together what is currently known about these evolutionarily conserved ubiquitin-protein ligases.

Curr Opin Genet Dev, 1999 Apr, 9(2), 218 - 24
Telomeres and telomerase: broad effects on cell growth; Price CM; During the past year, major advances have been made in understanding the link between telomerase expression and cell immortality . Studies of yeast telomeres have revealed an unexpected role for the non-homologous end-joining machinery in telomere maintenance and have provided the first definitive evidence that telomeres play a critical role in meiosis . Identification of new telomere proteins has led to a better understanding of vertebrate telomere structure and function.

Mol Microbiol, 1999 May, 32(3), 557 - 68
The multiply-regulated gabA gene encoding the GABA permease of Aspergillus nidulans: a score of exons; Hutchings H et al.; We describe the cloning, sequence and expression of gabA, encoding the gamma-amino-n-butyrate (GABA) permease of the fungus Aspergillus nidulans . Sequence changes were determined for three up-promoter (gabI ) and six gabA loss-of-function mutations . The predicted protein contains 517 residues and shows 30.3% overall identity with a putative GABA permease of Arabidopsis thaliana, 29.6% identity with the yeast choline transporter and 23.4% identity with the yeast UGA4 GABA permease . Structural predictions favour 11-12 transmembrane domains . Comparison of the genomic and cDNA sequences shows the presence of 19 introns, an unusually large number of introns for, we believe, any fungal gene . In agreement with the wealth of genetic data available, transcript level analyses demonstrate that gabA is subject to carbon catabolite and nitrogen metabolite repression, omega-amino acid induction and regulation in response to ambient pH (being acid-expressed) . In agreement with this, we report consensus binding sites 5' to the coding region, six each for CreA and AREA and one for PacC, the transcription factors mediating carbon catabolite and nitrogen metabolite repression and response to ambient pH respectively.

J Biol Chem, 1999 May 14, 274(20), 14352 - 8
The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction; Kraichely DM et al.; A ligand-inducible transactivation function (AF-2) exists in the extreme carboxyl terminus of the vitamin D receptor (VDR) that is essential for 1alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-activated transcription and p160 coactivator interaction . Crystallographic data of related nuclear receptors suggest that binding of 1, 25-(OH)2D3 by VDR induces conformational changes in the ligand-binding domain (LBD), the most striking of which is a packing of the AF-2 helix onto the LBD adjacent to helices H3 and H4 . In this study, a panel of VDR helix H3 mutants was generated, and residues in helix H3 that are important for ligand-activated transcription by the full-length VDR were identified . In particular, one mutant (VDR (Y236A)) exhibited normal ligand binding and heterodimerization with the retinoid X receptor (RXR) but was transcriptionally inactive . Yeast two-hybrid studies and in vitro protein interaction assays demonstrated that VDR (Y236A) was selectively impaired in interaction with AF-2-interacting coactivator proteins such as SRC-1 and GRIP-1 . These data indicate an importance of helix H3 in the mechanism of VDR-mediated transcription, and they support the concept that helix H3 functions in concert with the AF-2 domain to form a transactivation surface for binding the p160 class of nuclear receptor coactivators.

Biochemistry, 1999 May 11, 38(19), 6093 - 103
Role of unusual amino acid residues in the proximal and distal heme regions of a plant P450, CYP73A1; Schalk M et al.; CYP73A1 is a typical plant P450 in terms of its function and primary sequence . The enzyme catalyzes the 4-hydroxylation of trans-cinnamic acid, the first oxidative step in the phenylpropanoid pathway . Its primary protein sequence exhibits some particular landmarks which are characteristic of plant P450 enzymes . The most interesting is a proline residue (Pro448), very unusual in animal P450s, just C-terminal to the invariant heme-binding cysteine . To determine the role of this proline, we substituted it with valine, isoleucine, or phenylalanine, residues found in animal P450s, using site-directed mutagenesis . Expression of the wild type and mutants in yeast indicated that replacement of Pro448 led to disruption of the heme-protein interaction, loss of catalytic activity, and either impaired expression or destabilization of the apoprotein . Pro448 is thus essential for the correct insertion of heme in the apoprotein . Another typical feature of CYP73A proteins is the presence of an alanine-alanine motif (Ala306-Ala307) on the presumed N-terminal edge of the cleft in the central part of the I helix . This cleft faces the iron on the distal side of the heme and is proposed to be essential for catalysis . Substitution of each or both Ala306 and Ala307 residues with glycines showed that they are critical for the stability of the protein and influence the positioning of the substrate in the active site . Results are discussed with reference to the structural X-ray data that are available for bacterial P450 proteins.

Ann Neurol, 1999 May, 45(5), 673 - 5
Direct evidence that mitochondrial iron accumulation occurs in Friedreich ataxia; Delatycki MB et al.; Friedreich ataxia (FRDA) is due to mutations in the FRDA gene (FRDA) . When the gene homologous to FRDA is knocked out in yeast, there is accumulation of iron in mitochondria and reduced respiratory function . So far, there is only indirect evidence to support the hypothesis that FRDA is due to accumulation of mitochondrial iron leading to increased production of free radicals . We show here that mitochondrial iron is significantly higher in fibroblasts from patients with FRDA than in control fibroblasts . This is the first direct evidence that the findings in yeast are reproducible in cells from patients with FRDA.

Cell, 1999 Apr 30, 97(3), 383 - 93
RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis; Hirayama T et al.; Ethylene is an important regulator of plant growth . We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist . Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway . RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p . Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter . Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1 . These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 351 - 9
Thermococcus barophilus sp . nov., a new barophilic and hyperthermophilic archaeon isolated under high hydrostatic pressure from a deep-sea hydrothermal vent; Marteinsson VT et al.; A novel barophilic, hyperthermophilic, anaerobic sulfur-metabolizing archaeon, strain MPT (T = type strain), was isolated from a hydrothermal vent site (Snakepit) on the Mid-Atlantic Ridge (depth, 3550 m) . Enrichments and isolation were done under 40 MPa hydrostatic pressure at 95 degrees C . Strain MPT was barophilic at 75, 80, 85, 90, 95 and 98 degrees C, and was an obligate barophile between 95 and 100 degrees C (Tmax) . For growth above 95 degrees C, a pressure of 15.0-17.5 MPa was required . The strain grew at 48-95 degrees C under atmospheric pressure . The optimal temperature for growth was 85 degrees C at both high (40 MPa) and low (0.3 MPa) pressures . The growth rate was twofold higher at 85 degrees C under in situ hydrostatic pressure compared to at low pressure . Strain MPT cells were motile, coccoid, 0.8-2.0 microns in diameter and covered by a hexagonal S-layer lattice . The optimum pH and NaCl concentration for growth at low pressure were 7.0 and 20-30 g l-1, respectively . The new isolate was an obligate heterotroph and utilized yeast extract, beef extract and peptone for growth . Growth was optimal in the presence of elemental sulfur . Rifampicin and chloramphenicol inhibited growth . The core lipids consisted of a major archaeol and a complex lipid pattern consisting of a major phospholipid . The DNA G + C content was 37.1 mol% . Sequencing of the 16S rRNA gene revealed that strain MPT belonged to the genus Thermococcus and it is proposed that this isolate should be designated as a new species, Thermococcus barophilus.

Immunol Rev, 1999 Feb, 167, 211 - 21
The mouse major histocompatibility complex: some assembly required; Amadou C et al.; We have assembled a contig of 81 yeast artificial chromosome clones that spans 8 Mb and contains the entire major histocompatibility complex (Mhc) from mouse strain C57BL/6 (H2b), and we are in the process of assembling an Mhc contig of bacterial artificial chromosome (BAC) clones from strain 129 (H2bc), which differs from C57BL/6 in the H2-Q and H2-T regions . The current BAC contig extends from Tapasin to D17Leh89 with gaps in the class II, H2-Q, and distal H2-M regions . Only four BAC clones were required to link the class I genes of the H2-Q and H2-T regions, and no new class I gene was found in the previous gap . The proximal 1 Mb of the H2-M region has been analyzed in detail and is ready for sequencing; it includes 21 class I genes or fragments, at least 14 olfactory receptor-like genes, and a number of non-class I genes that clearly establish a conserved synteny with the class I regions of the human and rat Mhc.

Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5832 - 7
poc1: an Arabidopsis mutant perturbed in phytochrome signaling because of a T DNA insertion in the promoter of PIF3, a gene encoding a phytochrome-interacting bHLH protein; Halliday KJ et al.; The phytochrome family of informational photoreceptors has a central role in regulating light-responsive gene expression, but the mechanism of intracellular signal transduction has remained elusive . In a genetic screen for T DNA-tagged Arabidopsis mutants affected in early signaling intermediates, we identified poc1 (photocurrent 1), which exhibits enhanced responsiveness to red light . This phenotype is absent in a phyB (phytochrome B) null mutant background, indicating that the poc1 mutation enhances phyB signal transduction . The T DNA insertion in poc1 was found to be located in the promoter region of PIF3, a gene encoding a basic helix-loop-helix protein . The mutant phenotype seems to result from insertion-induced overexpression of this gene in red-light-grown seedlings, consistent with PIF3 functioning as a positively acting signaling intermediate . These findings, combined with data from a separate yeast two-hybrid screen that identified PIF3 as a phytochrome-interacting factor necessary for normal signaling, provide evidence that phytochrome signal transduction may include a direct pathway to photoresponsive nuclear genes via physical interaction of the photoreceptor molecules with the potential transcriptional regulator PIF3.

Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5528 - 32
Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein; Shen WJ et al.; Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue . By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydrophobic ligands . The interaction was demonstrated in vitro by the binding of ALBP to HSL translated in vitro, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue . Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies . The HSL-ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain . These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids.

J Biol Chem, 1999 May 14, 274(20), 14422 - 8
Myb-interacting protein, ATBF1, represses transcriptional activity of Myb oncoprotein; Kaspar P et al.; Using the yeast two-hybrid system, the transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding protein . Deletion mutagenesis revealed amino acids 2484-2520 in human ATBF1 and 279-300 in v-Myb as regions required for in vitro binding of both proteins . Further experiments identified leucines Leu325 and Leu332 of the Myb leucine zipper motif as additional amino acid residues important for efficient ATBF1-Myb interaction in vitro . In co-transfection experiments, the full-length ATBF1 was found to form in vivo complexes with v-Myb and inhibit v-Myb transcriptional activity . Both ATBF1 2484-2520 and Myb 279-300 regions were required for the inhibitory effect . Finally, the chicken ATBF1 was identified, showing high degree of amino acid sequence homology with human and murine proteins . Our data reveal Myb proteins as the first ATBF1 partners detected so far and identify amino acids 279-300 in v-Myb as a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated.

J Biol Chem, 1999 May 14, 274(20), 14382 - 91
A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A; Ogris E et al.; Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells . The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned . Carboxymethylation affects PP2A activity and varies during the cell cycle . Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins . One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned . PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile . Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction . To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned . Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.

J Biol Chem, 1999 May 14, 274(20), 14331 - 6
Heterodimeric interactions between chicken ovalbumin upstream promoter-transcription factor family members ARP1 and ear2; Avram D et al.; Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription . We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1 . One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily . Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor . ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines . Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.

Plant Physiol, 1999 May, 120(1), 23 - 32
Messenger RNA-binding properties of nonpolysomal ribonucleoproteins from heat-stressed tomato cells
Stuger R, Ranostaj S, Materna T, Forreiter C.
Most cells experiencing heat stress reprogram their translational machinery to favor the synthesis of heat-stress proteins . Translation of other transcripts is almost completely repressed, but most untranslated messengers are not degraded . In contrast to yeast, Drosophila melanogaster, and HeLa cells, plant cells store repressed messengers in cytoplasmic nonpolysomal ribonucleoproteins (RNPs) . To follow the fate of untranslated transcripts, we studied protein composition, mRNA content, and RNA-binding properties of nonpolysomal RNPs from heat-stressed tomato (Lycopersicon peruvianum) cells . Contrary to the selective interaction in vivo, RNPs isolated from tomato cells bound both stress-induced and repressed messengers, suggesting that the selection mechanism resides elsewhere . This binding was independent of a cap or a poly(A) tail . The possible role of proteasomes and heat-stress granules (HSGs) in mRNA storage is a topic of debate . We found in vitro messenger-RNA-binding activity in messenger RNP fractions free of C2-subunit-containing proteasomes and HSGs . In addition, mRNAs introduced into tobacco (Nicotiana plumbaginifolia) protoplasts were found in the cytoplasm but were not associated with HSGs.

Plant Physiol, 1999 May, 120(1), 257 - 74
Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro; Sugden C et al.; We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1 . The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577 . We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits . Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543 . Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158 . The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.

Plant Physiol, 1999 May, 120(1), 53 - 64
Transformation of the collateral vascular bundles into amphivasal vascular bundles in an Arabidopsis mutant; Zhong R et al.; Arabidopsis inflorescence stems develop a vascular pattern similar to that found in most dicots . The arrangement of vascular tissues within the bundle is collateral, and vascular bundles in the stele are arranged in a ring . Although auxin has been shown to be an inducer of vascular differentiation, little is known about the molecular mechanisms controlling vascular pattern formation . By screening ethyl methanesufonate-mutagenized populations of Arabidopsis, we have isolated an avb1 (amphivasal vascular bundle) mutant with a novel vascular pattern . Unlike the collateral vascular bundles seen in the wild-type stems, the vascular bundles in the avb1 stems were similar to amphivasal bundles, i.e . the xylem completely surrounded the phloem . Furthermore, branching vascular bundles in the avb1 stems abnormally penetrated into the pith, which resulted in a disruption in the ring-like arrangement of vascular bundles in the stele . The avb1 mutation did not affect leaf venation pattern and root vascular organization . Auxin polar transport assay indicated that the avb1 mutation did not disrupt the auxin polar transport activity in inflorescence stems . The avb1 mutation also exhibited pleiotropic phenotypes, including curled stems and extra cauline branches . Genetic analysis indicated that the avb1 mutation was monogenic and partially dominant . The avb1 locus was mapped to a region between markers mi69 and ASB2, which is covered by a yeast artificial chromosome clone, CIC9E2, on chromosome 5 . Isolation of the avb1 mutant provides a novel means to study the evolutionary mechanisms controlling the arrangement of vascular tissues within the bundle, as well as the mechanisms controlling the arrangement of vascular bundles in the stele.

J Oral Pathol Med, 1999 Apr, 28(4), 178 - 82
Paediatric AIDS--related linear gingival erythema: a form of erythematous candidiasis?
Velegraki A, Nicolatou O, Theodoridou M, Mostrou G, Legakis NJ.
Three vertically HIV-infected children showed, in addition to oral candidiasis, HIV-gingivitis, which healed on antimycotic treatment . The intense linear gingival erythema of a fourth child was also clinically evaluated as a possible form of erythematous oral candidiasis . Direct microscopic examination of material from the gingival lesions of the latter disclosed yeast cells and hyphae . Subsequent culture, biochemical and serological tests identified the yeast as Candida dubliniensis . As the patient was on long-term prophylaxis with fluconazole, ketoconazole was administered and led to a good clinical response . This is the first report implicating this new Candida species as a pathogen in linear gingival erythema in a HIV-positive individual . The case reports presented provide evidence that linear gingival erythema may be of candidal origin . Further clinical and laboratory observations are required to establish whether this condition constitutes a variant of erythematous candidiasis associated with paediatric HIV infection.

Differentiation, 1999 Mar, 64(3), 161 - 71
Analysis of the pattern of QM expression during mouse development; Mills AA et al.; QM, a novel gene that was originally identified as a putative tumor suppressor gene, has since been cloned from species encompassing members of the plant, animal, and fungal kingdoms . Sequence comparison indicates that QM has been highly conserved throughout eukaryotic evolution . QM is a member of a multigene family in both mouse and man, is expressed in a broad range of tissues, and is downregulated during adipocyte differentiation . Jif-1, a chicken homolog of QM, has been reported to interact with the protooncogene c-Jun, and to inhibit transactivation of AP-1 regulated promoters in vitro . Furthermore, disruption of the yeast QM homolog is lethal . Although these studies suggest that the QM gene product plays an important role within the normal cell, the precise role of QM has remained elusive . In this study, a thorough analysis of the pattern of QM expression during mouse development was undertaken, using the techniques of whole mount in situ hybridization and whole mount immunohistochemistry, in combination with conventional immunohistochemical analysis of tissue sections . QM is expressed in numerous embryonic tissues, and is differentially expressed throughout the embryo . The cytoplasmic localization of QM is consistent with its reported association with ribosomes, and inconsistent with its previously hypothesized function as a direct modulator of the nuclear protooncogene c-Jun . QM is expressed in the developing epidermis, and is particularly strong within developing limbs . Analysis of embryos of various stages of gestation indicate that QM is downregulated in the surface ectoderm of the embryo as development proceeds . QM protein is not detectable within either nucleated or enucleated red blood cell precursors . QM is strongly expressed within chondrocytes within the transition zone of developing limb cartilage, as well as within differentiated keratinocytes of the suprabasal regions of the epidermis . Furthermore, within both cartilage and skin, there is an inverse relationship between QM expression and proliferative capacity . This pattern of QM expression suggests that this novel gene product may be involved in processes such as posttranslational protein processing which are essential for differentiation of specific tissues during embryogenesis.

Eur J Hum Genet, 1999 Apr, 7(3), 332 - 8
Refinement of the RP17 locus for autosomal dominant retinitis pigmentosa, construction of a YAC contig and investigation of the candidate gene retinal fascin; Bardien-Kruger S et al.; The RP17 locus for autosomal dominant retinitis pigmentosa has previously been mapped to chromosome 17q by linkage analysis . Two unrelated South African families are linked to this locus and the identification of key recombination events assigned the RP17 locus to a 10 cM interval on 17q22 . The work reported here refines the mapping of the locus from a 10 cM to a 1 cM interval between the microsatellite markers D17S1604 and D17S948 . A physical map of this interval was constructed using information from the Whitehead/MIT YAC contig WC 17.8 . Sequence-tagged site (STS) content mapping of seven overlapping YACs from this contig was employed in order to build the map . A BAC library was screened to cover a gap in the YAC contig and two positive BACs were identified . Intragenic polymorphisms in the retinal fascin gene provided evidence for the exclusion of this candidate as the RP17 disease gene.

Ann Transplant, 1998, 3(3), 21 - 4
Opportunistic fungal infections in patients treated with heart transplantation--own centre experiences; Konduracka E; The aim of this study was to analyse frequency, type and course of fungal infection in patients treated with heart transplantation (HT) and to estimate the efficacy of diagnostic procedures in patients before and after HT . The study was conducted on 30 patients (group I) aged 20-64 referred for HT and treated after HT in Coronary Disease Department in Cracow . Standard immunosuppressive protocol consisting of CSA-A, Aza, Prednisone was administered to all recipients after HT . As a control group 40 healthy persons aged 25-65 were examined . In all persons quantitative and mycoserological examinations were carried out according to a special protocol . In some justified cases despite serological and quantitative examinations, bronchoscopy with BAL, biopsy procedure, histological examination of tissue were performed in patients after HT . The commonest infections in patients after HT were oropharyngeal Candida infections . They mostly occurred in the first months after HT, and were observed in 67% of recipients . Besides, in two patients deep Aspergillus infections were documented . Quantitative mycological examinations were very useful in diagnosing early stages of superficial mycoses . In none of these patients before HT were permanent oropharyngeal yeast and Aspergillus colonizations observed . Diagnosis of deep Aspergillus mycoses was difficult . Even antigen detection did not allow for making definitive diagnosis, because antigen was found in some recipients without infections and in some healthy persons . Definitive diagnosis depended on histological and mycological examinations of tissue sections obtained by surgical procedure and in second case by autopsy.

Nippon Ishinkin Gakkai Zasshi, 1999, 40(2), 99 - 102
Subcutaneous phaeohyphomycosis caused by Phaeoacremonium rubrigenum in an immunosuppressed patient; Matsui T et al.; BACKGROUND: Phaeohyphomycosis refers to infection by dematiaceous fungi with pigmented hyphae or yeast-like cells in the tissue . In humans, this disease is usually considered to be an opportunistic infection . The causal agents of phaeohyphomycosis include numerous species belonging to different genera and they are increasing as a result of the development of intensive medical therapy . OBSERVATION: We report the case of a 61-year-old Japanese female under corticosteroid treatment for malignant rheumatoid arthritis . An asymptomatic subcutaneous tumor developed on the back of her left foot . Histological examination of the excised material revealed mixed cell granuloma (H&E) and the presence of branched hyphal elements (periodic acid-Schiff) . A fungus grown in pure culture was identified as Phaeoacremonium rubrigenum . CONCLUSION: The hyphomycete genus, Phaeoacremonium, was proposed in 1996 by Crous et al . Three species belonging to this genus have been isolated from clinical specimens: P . inflatipes, from a human toenail, human synovial fluid and human mycetoma of the foot, P . parasiticum, from a subcutaneous lesion on a kidney transplant patient and several other sources, and P . rubrigenum, from a human patient with pneumonia . To our knowledge, however, this is the first report of phaeohyphomycosis caused by Phaeoacremonium rubrigenum.

J Neurosci, 1999 May 15, 19(10), 3926 - 34
Postsynaptic density-93 interacts with the delta2 glutamate receptor subunit at parallel fiber synapses; Roche KW et al.; The glutamate receptor subunit delta2 has a unique distribution at the parallel fiber-Purkinje cell synapse of the cerebellum, which is developmentally regulated such that delta2 occurs at both parallel fiber synapses and climbing fiber synapses early in development but is restricted to parallel fiber synapses in adult animals . To identify proteins that might be involved in the trafficking or docking of delta2 receptors, we screened a yeast two-hybrid library with the cytosolic C terminus of delta2 and isolated a member of the postsynaptic density (PSD)-95 family of proteins, which are known to interact with the extreme C termini of NMDA receptors . We find that delta2 binds specifically to PSD-93, which is enriched in Purkinje cells . In addition, PSD-93 clusters delta2 when they are coexpressed in heterologous cells, and clustering is disrupted by point mutations of delta2 that disrupt the delta2-PSD-93 interaction . Ultrastructural localization of PSD-93 and delta2 shows they are colocalized at parallel fiber synapses; however, PSD-93 also is present at climbing fiber synapses of the adult rat, where delta2 is not found, indicating that the presence of PSD-93 alone is not sufficient for determining the synaptic expression of delta2.

J Virol, 1999 Jun, 73(6), 5018 - 25
The carboxy-terminal acidic domain of Rift Valley Fever virus NSs protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein; Yadani FZ et al.; The ambisense S segment of Rift Valley fever (RVF) virus (a phlebovirus in the Bunyaviridae family) codes for two proteins: the viral complementary-sense RNA for the N nucleoprotein and the genomic-sense RNA for the nonstructural protein NSs . Except for the fact that the NSs protein is phosphorylated and forms filamentous structures in the nuclei of infected cells (R . Swanepoel and N . K . Blackburn, J . Gen . Virol . 34:557-561, 1977), its role is poorly understood, especially since the replication cycle of all these viruses takes place in the cytoplasm . To investigate the mechanisms involved in filament formation, we expressed NSs in mammalian cells via a recombinant Semliki Forest virus and demonstrated that the protein alone was able to form structures similar to those observed in RVF virus-infected cells, indicating that the presence of other RVF virus proteins is not required for filament formation . The yeast two-hybrid system was used to show that the protein interacts with itself and to map the interacting domains . Various deletion and substitution mutants were constructed, and the mutant proteins were analyzed by immunoprecipitation, Western blotting and immunofluorescence . These experiments indicated that the 10 to 17 amino acids of the carboxy-terminal domain were involved in self-association of the protein and that deletion of this acidic carboxy-terminal domain prevents the protein from forming filaments but does not affect its nuclear localization . The role of two phosphorylation sites present in this domain was also investigated, but they were not found to have a major influence on the formation of the nuclear filament.

J Invest Dermatol, 1999 May, 112(5), 751 - 6
UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes; Peus D et al.; We have previously shown that hydrogen peroxide is an important mediator of ultraviolet B induced phosphorylation of the epidermal growth factor receptor in human keratinocytes . Here we demonstrate that physiologic doses of ultraviolet B and hydrogen peroxide stimulate activation of two related but distinct mitogen-activated protein kinase pathways: extracellular regulated kinase 1 and 2 (ERK1/2), as well as p38, the mammalian homolog of HOG1 in yeast which is a major kinase for a recently identified stress-induced signaling pathway . The time-dependent activation of ERK1/2 and p38 are distinct, and ultraviolet B-induced ERK1/2 activation is downregulated more rapidly than p38 . Using dihydrorhodamine or Amplex as specific fluorescent dye probes, we show that ultraviolet B-induced peroxides can be inhibited by ascorbic acid . Ascorbic acid strongly blocks ERK1/2 and p38 activation by ultraviolet B and hydrogen peroxide whereas pyrrolidine dithiocarbamate and butyl hydroxyanisole are less effective . Pyrrolidine dithiocarbamate was unable to inhibit ultraviolet B-induced p38 activation . Cell death was increased after ultraviolet B when ERK1/2 activation was attenuated by the specific inhibitor PD098059 . The distinct time courses and extents of activation and inhibition of ERK1/2 and p38 indicate that these pathways are separate and regulated independently in keratinocytes . Specific types of reactive oxygen species induced by ultraviolet B as well as selective activation or inhibition of specific phosphatases may mediate these responses in keratinocytes . These findings demonstrate that reactive oxygen species are important multifunctional mediators of ultraviolet B-induced ERK1/2 and p38 signaling transduction pathways and suggest that ERK1/2 may play an important part in protecting keratinocytes from cell death following oxidative stress.

Cancer Res, 1999 May 1, 59(9), 2041 - 4
Centrosomal kinase AIK1 is overexpressed in invasive ductal carcinoma of the breast; Tanaka T et al.; A centrosomal serine/threonine kinase, AIK1(3)/breast tumor amplified kinase/aurora2, which was recently identified as an oncogene, shows high amino acid identity with chromosome segregation kinases, fly Aurora, and yeast Ipl1 . Immunohistochemical analyses of invasive ductal adenocarcinomas of the breast revealed that overexpression of AIK1 was observed in 94% of the cases, irrespective of the histopathological type, whereas the protein was not detected in normal ductal and lobular cells . Benign breast lesions including fibrocystic disease and fibroadenoma (epithelial components) displayed weakly detectable AIK1 expression in part of the lesions . This is the first immunohistochemical report of AIK1 expression in primary human breast carcinomas . Although the physiological function(s) of AIK1 kinase during cell division remains to be determined, the markedly high positivity of AIK1 staining in the cancer lesions suggested a possible involvement of its overexpression in the tumorigenesis of some of breast cancer cells.

Genome, 1999 Apr, 42(2), 330 - 7
Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones; Ashikawa I et al.; To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs) . In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5 . The probe produces a unique fingerprint profile characteristic of each YAC clone . The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated . The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments . We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.

Gene, 1999 Apr 29, 231(1-2), 111 - 20
A compact gene cluster in Drosophila: the unrelated Cs gene is compressed between duplicated amd and Ddc; Tatarenkov A et al.; Cs, a gene with unknown function, and amd and Ddc, which encode decarboxylases, are among the most closely spaced genes in D . melanogaster . Untranslated 3' ends of the convergently transcribed genes Cs and Ddc are known to overlap by 88bp . A number of questions arise about the organization of this tightly-packed gene region and about the evolution and function of the Cs gene . We have now investigated this three-gene cluster in Scaptodrosophila lebanonensis (which diverged from D . melanogaster 60-65 MYA), as well as in D . melanogaster and D . simulans . Gene order and direction of transcription is the same in all three species . The Cs gene codes, in Scaptodrosophila, for a polypeptide of 544 amino acids; in D . melanogaster, it consists of 504 amino acids, which is twice as long as previously suggested, which makes the gene density even more spectacular . The Cs sequences exhibit higher number of non-synonymous substitutions between species, higher ratios of non-synonymous to synonymous substitutions, and lower codon usage bias than other genes, suggesting that Cs is less functionally constrained than the other genes . This is consistent with the failure of inducing phenotypic mutations in D . melanogaster . The function of Cs remains to be identified, but a high degree of similarity indicates that it is homologous to genes coding for a corticosteroid-binding protein in yeast and a polyamine oxidase in maize.

Environ Health Perspect, 1999 Feb, 107 Suppl 1, 89 - 108
Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals; Andersen HR et al.; The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries . Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent . Also included in the test panel were 17beta++-estradiol as a positive control and ethanol as solvent control . The test compounds were coded before distribution . Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout . 17beta-Estradiol, 17alpha-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems . Colchicine caused cytotoxicity only . Bisphenol A induced an estrogenic response in all assays . The results obtained for the remaining test compounds--tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol--varied among the assays . The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility . Also, similar methods vary in their sensitivity to estrogenic compounds . Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.

Eur J Biochem, 1999 May, 262(1), 36 - 42
Structure and splice products of the human gene encoding sds22, a putative mitotic regulator of protein phosphatase-1; Ceulemans H et al.; sds22 is a regulatory subunit of protein phosphatase-1 that is required for the completion of mitosis in yeast . It consists largely of 11 tandem leucine-rich repeats of 22 residues that are expected to mediate interactions with other polypeptides, including protein phosphatase-1 . In this paper, we report on the structure of the human gene encoding sds22, designated PPP1R7 . This gene (33 kb) comprises 11 exons, but these do not coincide with the sequences encoding the leucine-rich repeats . Up to six splice variants can be generated by exon skipping and alternative polyadenylation, as revealed by expressed sequence tag database analysis, RT-PCR and Northern blot analysis . The sds22 transcripts are expected to encode four different polypeptides . sds22alpha1 corresponds to the variant cloned previously from human brain {Renouf et al . (1995) FEBS Lett . 375, 75-78} . Sds22beta1 is truncated within the ninth repeat and has a short and different C-terminus . Both variants also exist without the sequence corresponding to exon 2, and these are termed sds22alpha2 and sds22beta2 . The 5'-flanking region of PPP1R7 contains two NF-Y-binding CCAAT boxes near the transcription start site and potential binding sites for the transcription factors c-Myb, Ik-2 and NF-1, which are conserved in the mouse gene.

DNA Res, 1999 Feb 26, 6(1), 37 - 44
Characterization of a 1200-kb genomic segment of chromosome 3p22-p21.3; Daigo Y et al.; We previously determined the nucleotide sequence and characterized the 685-kb proximal half of CEPH YAC936c1, which corresponds to a portion of human chromosome 3p21.3 . In the study reported here, we characterized the remaining 515-kb of this YAC clone corresponding to the telomeric half of its human insert . The newly sequenced region contained a total of ten genes including six reported previously: phospholipase C delta 1 (PLCD1), human activin receptor type IIB (hActR-IIB), organic cation transporter-like 1 (OCTL1), organic cation transporter-like 2 (OCTL2), oxidative stress response 1 (OSR1), and human xylulokinase-like protein (XYLB) . The remaining four genes present in the telomeric region included two known genes, MyD88 and ACAA, and two novel genes . One (designated ENGL) of the novel sequences was found to encode an amino-acid sequence homologous to the family of DNA/RNA endonucleases, especially endonuclease G . The other gene F56 revealed no significant homology to any known genes . These results disclosed complete physical and transcriptional maps of the 1200-kb region of 3p present in YAC 936c1.

Neuron, 1999 Apr, 22(4), 809 - 18
A dynamically regulated 14-3-3, Slob, and Slowpoke potassium channel complex in Drosophila presynaptic nerve terminals; Zhou Y et al.; Slob is a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium (K(Ca)) channel . A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein . Coimmunoprecipitation experiments from Drosophila heads and transfected cells confirm that 14-3-3 interacts with dSlo via Slob . All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions . Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation . 14-3-3 coexpression dramatically alters dSlo channel properties when wild-type Slob is present but not when a double serine mutant Slob that is incapable of binding 14-3-3 is present . The results provide evidence for a dSlo/Slob/14-3-3 regulatory protein complex.

Plant J, 1999 Mar, 17(6), 667 - 78
Inhibition of protoporphyrinogen oxidase expression in Arabidopsis causes a lesion-mimic phenotype that induces systemic acquired resistance; Molina A et al.; We have used an antisense expression technology in Arabidopsis based on the yeast GAL4/UAS transactivation system (Guyer et al., Genetics, 1998; 149:633-639) to reduce levels of protoporphyrinogen IX oxidase (PPO), the last common enzyme of the biosynthesis of the haem group and chlorophyll . Plants expressing the antisense PPO gene presented growth alterations and their leaves showed necrotic lesions that appeared similar to lesions characteristic of the pathogen-induced hypersensitive reaction, and seen in the so-called lesion-mimic mutants . Plants expressing the antisense gene also had high endogenous salicylic acid levels, constitutive expression of the PR-1 gene, and were resistant to Peronospora parasitica, consistent with the activation of systemic acquired resistance (SAR) . Treatment of wild-type plants with sublethal concentrations of herbicides that inhibit PPO also induced defence responses that conferred enhanced tolerance to P . parasitica . This effect was not observed in NahG and nim1 plants, which are compromised in their ability to activate SAR . These results demonstrate that genetic or chemical disruption of a metabolic pathway can lead to the induction of a set of defence responses including activation of SAR.

Bioelectrochem Bioenerg, 1999 Feb, 48(1), 3 - 16
Fundamentals of electroporative delivery of drugs and genes; Neumann E et al.; Electrooptical and conductometrical relaxation methods have given a new insight in the molecular mechanisms of the electroporative delivery of drug-like dyes and genes (DNA) to cells and tissues . Key findings are: (1) Membrane electroporation (ME) and hence the electroporative transmembrane transport of macromolecules are facilitated by a higher curvature of the membrane as well as by a gradient of the ionic strength across charged membranes, affecting the spontaneous curvature . (2) The degree of pore formation as the primary field response increases continuously without a threshold field strength, whereas secondary phenomena, such as a dramatic increase in the membrane permeability to drug-like dyes and DNA (also called electropermeabilization), indicate threshold field strength ranges . (3) The transfer of DNA by ME requires surface adsorption and surface insertion of the permeant molecule or part of it . The diffusion coefficient for the translocation of DNA (M(r) approximately 3.5 x 10(6)) through the electroporated membrane is Dm = 6.7 x 10(-13) cm2 s-1 and Dm for the drug-like dye Serva Blue G (M(r) approximately 854) is Dm = 2.0 x 10(-12) cm2 s-1 . The slow electroporative transport of both DNA and drugs across the electroporated membrane reflects highly interactive (electro-) diffusion, involving many small pores coalesced into large, but transiently occluded pores (DNA) . The data on mouse B-cells and yeast cells provide directly the flow and permeability coefficients of Serva blue G and plasmid DNA at different electroporation protocols . The physico-chemical theory of ME and electroporative transport in terms of time-dependent flow coefficients has been developed to such a degree that analytical expressions are available to handle curvature and ionic strength effects on ME and transport . The theory presents further useful tools for the optimization of the ME techniques in biotechnology and medicine, in particular in the new field of electroporative delivery of drugs (electrochemotherapy) and of DNA transfer and gene therapy.

EMBO J, 1999 May 4, 18(9), 2631 - 7
Structure and interactions of the translation initiation factor eIF1; Fletcher CM et al.; eIF1 is a universally conserved translation factor that is necessary for scanning and involved in initiation site selection . We have determined the solution structure of human eIF1 with an N-terminal His tag using NMR spectroscopy . Residues 29-113 of the native sequence form a tightly packed domain with two alpha-helices on one side of a five-stranded parallel and antiparallel beta-sheet . The fold is new but similar to that of several ribosomal proteins and RNA-binding domains . A likely binding site is indicated by yeast mutations and conserved residues located together on the surface . No interaction with recombinant eIF5 or the initiation site RNA GCCACAAUGGCA was detected by NMR, but GST pull-down experiments show that eIF1 binds specifically to the p110 subunit of eIF3 . This interaction explains how eIF1 is recruited to the 40S ribosomal subunit.

Neurochem Res, 1999 Apr, 24(4), 565 - 80
Peroxisomal disorders: clinical, biochemical, and molecular aspects; Wanders RJ; Peroxisomes are subcellular organelles catalyzing a number of indispensable functions in cellular metabolism . The importance of peroxisomes in man is stressed by the existence of an expanding group of genetic diseases in which there is an impairment in one or more peroxisomal functions . Much has been learned in recent years about these functions and many of the enzymes involved have been characterized, purified and their cDNAs cloned . This has allowed resolution of the enzymatic and molecular basis of many of the single peroxisomal enzyme deficiencies . Similarly, the molecular basis of the peroxisome biogenesis disorders is also being resolved rapidly thanks to the successful use of CHO as well as yeast mutants . In this paper we will provide an overview of the peroxisomal disorders with particular emphasis on their clinical, biochemical and molecular characteristics.

J Med Genet, 1999 Apr, 36(4), 271 - 8
Systematic characterisation of disease associated balanced chromosome rearrangements by FISH: cytogenetically and genetically anchored YACs identify microdeletions and candidate regions for mental retardation genes; Wirth J et al.; Disease associated balanced chromosome rearrangements (DBCRs) have been instrumental in the isolation of many disease genes . To facilitate the molecular cytogenetic characterisation of DBCRs, we have generated a set of >1200 non-chimeric, cytogenetically and genetically anchored CEPH YACs, on average one per 3 cM, spaced over the entire human genome . By fluorescence in situ hybridisation (FISH), we have performed a systematic search for YACs spanning translocation breakpoints . Patients with DBCRs and either syndromic or non-syndromic mental retardation (MR) were ascertained through the Mendelian Cytogenetics Network (MCN), a collaborative effort of, at present, 270 cytogenetic laboratories throughout the world . In this pilot study, we have characterised 10 different MR associated chromosome regions delineating candidate regions for MR . Five of these regions are narrowed to breakpoint spanning YACs, three of which are located on chromosomes 13q21, 13q22, and 13q32, respectively, one on chromosome 4p14, and one on 6q25 . In two out of six DBCRs, we found cytogenetically cryptic deletions of 3-5 Mb on one or both translocation chromosomes . Thus, cryptic deletions may be an important cause of disease in seemingly balanced chromosome rearrangements that are associated with a disease phenotype . Our region specific FISH probes, which are available to MCN members, can be a powerful tool in clinical cytogenetics and positional cloning.

Fundam Clin Pharmacol, 1999, 13(2), 220 - 5
Effects of tramadol on experimental inflammation; Bianchi M et al.; We examined the ability of the analgesic drug tramadol to affect the development of inflammation in rats . The acute administration of tramadol significantly reduced the edema and the hyperalgesia induced by yeast injection in the paw . Moreover, in the subcutaneous carrageenin-induced inflammation, tramadol reduced the amount of the exudate, as well as the prostaglandin (PG)E2-like bio- and immuno-activity in the exudate; on the contrary, leukotriene (LT)B4 concentrations in the exudate were not changed . However, tramadol did not affect the ability of macrophages to migrate towards the chemotactic peptide N-formyl-L-methionil-L-leucyl-L-phenylalanine (FMLP) . Our results suggest that tramadol is able to inhibit the development of different types of inflammation in the rat without affecting immune mechanisms, and contribute to explain the efficacy of this drug in the treatment of inflammatory pain.

Curr Biol, 1999 Apr 22, 9(8), 405 - 15
Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts; Mullins RD et al.; BACKGROUND: Actin filaments polymerize in vivo primarily from their fast-growing barbed ends . In cells and extracts, GTPgammaS and Rho-family GTPases, including Cdc42, stimulate barbed-end actin polymerization; however, the mechanism responsible for the initiation of polymerization is unknown . There are three formal possibilities for how free barbed ends may be generated in response to cellular signals: uncapping of existing filaments; severing of existing filaments; or de novo nucleation . The Arp2/3 complex localizes to regions of dynamic actin polymerization, including the leading edges of motile cells and motile actin patches in yeast, and in vitro it nucleates the formation of actin filaments with free barbed ends . Here, we investigated actin polymerization in soluble extracts of Acanthamoeba . RESULTS: Addition of actin filaments with free barbed ends to Acanthamoeba extracts is sufficient to induce polymerization of endogenous actin . Addition of activated Cdc42 or activation of Rho-family GTPases in these extracts by the non-hydrolyzable GTP analog GTPgammaS stimulated barbed-end polymerization, whereas immunodepletion of Arp2 or sequestration of Arp2 using solution-binding antibodies blocked Rho-family GTPase-induced actin polymerization . CONCLUSIONS: For this system, we conclude that the accessibility of free barbed ends regulates actin polymerization, that Rho-family GTPases stimulate polymerization catalytically by de novo nucleation of free barbed ends and that the primary nucleation factor in this pathway is the Arp2/3 complex.

J Cell Biol, 1999 May 3, 145(3), 425 - 35
The maize homologue of the cell cycle checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and meiotic localization patterns; Yu HG et al.; We have identified a maize homologue of yeast MAD2, an essential component in the spindle checkpoint pathway that ensures metaphase is complete before anaphase begins . Combined immunolocalization of MAD2 and a recently cloned maize CENPC homologue indicates that MAD2 localizes to an outer domain of the prometaphase kinetochore . MAD2 staining was primarily observed on mitotic kinetochores that lacked attached microtubules; i.e., at prometaphase or when the microtubules were depolymerized with oryzalin . In contrast, the loss of MAD2 staining in meiosis was not correlated with initial microtubule attachment but was correlated with a measure of tension: the distance between homologous or sister kinetochores (in meiosis I and II, respectively) . Further, the tension-sensitive 3F3/2 phosphoepitope colocalized, and was lost concomitantly, with MAD2 staining at the meiotic kinetochore . The mechanism of spindle assembly (discussed here with respect to maize mitosis and meiosis) is likely to affect the relative contributions of attachment and tension . We support the idea that MAD2 is attachment-sensitive and that tension stabilizes microtubule attachments.

Radiother Oncol, 1999 Jan, 50(1), 1 - 11
Towards prediction and modulation of treatment response; Bartelink H et al.; The purpose of this paper is to evaluate new predictive assays and their potential to modulate treatment response . Their impact is presented in the context of three EORTC clinical trials in head and neck, lung and breast cancer, showing an improvement in survival by accelerated fractionation, concomitant use of cisplatin and radiotherapy and adjuvant hormonal treatment, respectively . Assays have been developed to predict the response to treatment by measuring tumor characteristics, such as the growth potential by the labeling index after i.v . injection of IdUrd, the extent of radiation-induced stable and unstable chromosome aberrations and the induction of apoptosis . These assays could guide us in the adaptation of the individual radiation doses and fractionation schedules . The measurement of the effect of cisplatin on DNA has become feasible with the development of antibodies against DNA adducts . In a recently completed phase II dose escalation trial with concomitant radiotherapy and daily cisplatin in lung cancer, we found that patients with high DNA adduct levels measured in the buccal mucosa, had a much better survival rate than patients with a low or undetectable amount of cisplatin DNA adducts . A better understanding of the signal transduction pathways involved in radiation-induced apoptosis may help to design studies aimed at modulating the apoptotic response . We and others have recently shown that alkylphospholipids, which inhibit mitogenic signaling, induce apoptosis in a variety of tumor cell lines . In combination with ionizing radiation, these compounds cause an enhancement of apoptotic cell kill . This type of signaling-based intervention study may form the basis for new therapeutic strategies . Pretreatment levels of apoptosis may be helpful in predicting treatment outcome, although the data so far show inconsistent results . The importance of evaluating other tumor-biological parameters, including cell kinetics should be stressed . Based on assays predicting reliably the response to hormonal therapy, a more appropriate choice can be made for therapeutic intervention with hormonal therapy and for selecting the appropriate adjuvant therapy in breast cancer patients . The development of a functional estrogen receptor assay (ER-FASAY), based on a yeast growth-assay, provides a way of estimating abnormal function of the receptor in tumors with a positive estrogen receptor score as measured by a classical immuno-histochemistry assay . This yeast assay can also detect different DNA mutations of the estrogen receptor existing in an individual tumor specimen.

Genetics, 1999 May, 152(1), 191 - 9
The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development; Masloff S et al.; During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia . Previously it has been shown that this developmental process is under polygenic control . To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted . We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1(-) mutation . Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1 . A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast . However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1 . The involvement of the pro1(+) gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame . Southern hybridization experiments also indicated that pro1(+) homologues are present in other sexually propagating filamentous ascomycetes.

Appl Environ Microbiol, 1999 May, 65(5), 2179 - 83
Localized, positive charge mediates adhesion of rhodosporidium toruloides to barley leaves and polystyrene
Buck JW, Andrews JH.
The physicochemical forces that mediate attachment of yeasts to the phylloplane are unknown . Cell surface charge and hydrophobicity and adhesion to polystyrene, glass, and barley were assessed for wild-type Rhodosporidium toruloides and attachment-minus (Att-) mutants . Cells were grown under conditions promoting (excess carbon) or not promoting (excess nitrogen) capsule production . Hydrophobicity was measured by adhesion to xylenes, and surface charge characteristics were assessed by attachment to either DEAE (positive)- or carboxymethyl (CM) (negative)-Sephadex ion-exchange beads . Hydrophobicity and adhesiveness of nonencapsulated, wild-type R . toruloides decreased from mid-log to late stationary phase . Encapsulated wild-type R . toruloides cells were more hydrophobic and more adhesive than nonencapsulated cells . However, two encapsulated Att- mutants were more hydrophobic than the wild type and levels of adhesion of R . toruloides were similar on polystyrene and less hydrophobic glass surfaces . Adhesion of wild-type yeast to barley and polystyrene was correlated with attachment to CM-Sephadex beads, indicating a positive cell surface charge . Sixteen Att- mutants did not exhibit a positive cell surface charge, and wild-type yeast cells that did not attach to CM-Sephadex did not adhere to either polystyrene or barley . Wild-type R . toruloides attached to CM-Sephadex beads by the poles of the cells, indicating a localization of positive charge which was also visualized with India ink . We conclude that localized, positive charge, and not hydrophobic interactions, mediates attachment of R . toruloides to barley leaves.

J Biol Chem, 1999 May 7, 274(19), 13711 - 7
Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling; Shi X et al.; Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads . Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription . However, the DNA-binding protein(s) involved in BMP signaling has not been identified . Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor . The interaction between Smad1 and Hoxc-8 was confirmed by a "pull-down" assay and a co-immunoprecipitation experiment in COS-1 cells . Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner . Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation . Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation . When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation . Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct . Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression.

J Biol Chem, 1999 May 7, 274(19), 12975 - 8
Loss-of-function and dominant-negative mechanisms associated with hepatocyte nuclear factor-1beta mutations in familial type 2 diabetes mellitus; Tomura H et al.; Hepatocyte nuclear factor (HNF)-1beta, a homeodomain-containing transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues . Recent genetic studies have identified two HNF-1beta mutations, R177X and A263fsinsGG, in subjects with a monogenic form of type 2 diabetes . Despite the defects being in the same gene, diverse severities of disease are observed in the affected subjects . To investigate the molecular mechanism by which mutations might cause various phenotypic features, wild type and mutant proteins were transiently expressed in insulin-producing (MIN6) and hepatic (HepG2) cells . Luciferase reporter assay showed that both mutations resulted in a marked reduction of transactivation activity . Because their dimerization activity was found to be intact by the yeast two-hybrid system, it was possible that they were dominant-negative to wild type activity . When co-expressed with wild type, both of the mutants significantly decreased wild type activity in HepG2 cells . In contrast, although A263fsinsGG functioned similarly in MIN6 cells, R177X failed to affect wild type activity in this cell line . Immunohistochemical analysis of the mutants suggests that this functional divergence might be generated by the modification of nuclear localization . These results suggest that HNF-1beta mutations may impair pancreatic beta-cell function by loss-of-function and dominant-negative mechanisms.

Nippon Rinsho, 1999 Apr, 57(4), 856 - 61
{Cloning and characterization of cDNA for DRPLA interacting protein}; Okamumoho Y et al.; Dentatorubral-pallidoluysian atrophy (DRPLA) is associated with CAG repeat expansion . While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific area of the brain, predicting that the DRPLA protein interacts with other proteins, which may play a role in the pathogenesis . We isolated a DRPLA binding protein with a yeast two-hybrid system, and identified it to be a human homologue of insulin receptor substrate protein of 53 kDa (IRSp53) . The binding of the DRPLA protein with IRSp53 was ascertained by co-immunoprecipitation, and colocalization . A proline-rich region near polyglutamine of the DRPLA protein and the SH3 domain of IRSp53 were involved in the binding . Extended polyglutamine significantly reduced the binding ability in yeast cells.

Biochem Biophys Res Commun, 1999 Apr 29, 258(1), 102 - 8
Dynamics of DNA molecules in gel studied by fluorescence microscopy; Kantor RM et al.; The dynamics of individual DNA molecules in a thin gel were studied with fluorescence microscopy . Driven by an electric field, molecules hooked around isolated obstacles and became extended . By analyzing molecular images, we identified the reptation tube and primitive chain . When the field was turned off, the molecules relaxed . The relaxation time tau1 and primitive chain length <L> at equilibrium depend on N, the size of the molecule in base pairs, consistently with reptation theory . Using five yeast chromosomal DNAs ranging in size from 245 kb to 980 kb, we found that: These results constitute a way of sizing individual DNA molecules by imaging rather than by gel electrophoresis .

Exp Cell Res, 1999 May 1, 248(2), 339 - 49
Subunits and substrates of the anaphase-promoting complex; Peters JM; The initiation of anaphase and exit from mitosis depend on a ubiquitination complex called the anaphase-promoting complex (APC) or cyclosome . The APC is composed of more than 10 constitutive subunits and associates with additional regulatory factors in mitosis and during the G1 phase of the cell cycle . At the metaphase-anaphase transition the APC ubiquitinates proteins such as Pds1 in budding yeast and Cut2 in fission yeast whose subsequent degradation by the 26S proteasome is essential for the initiation of sister chromatid separation . Later in anaphase and telophase the APC promotes the inactivation of the mitotic cyclin-dependent protein kinase 1 by ubiquitinating its activating subunit cyclin B . The APC also mediates the ubiquitin-dependent proteolysis of several other mitotic regulators, including other protein kinases, APC activators, spindle-associated proteins, and inhibitors of DNA replication .

Genes Chromosomes Cancer, 1999 May, 25(1), 60 - 4
Identification of three commonly deleted regions on chromosome arm 6q in human pancreatic cancer; Abe T et al.; Pancreatic cancer has one of the poorest prognoses among malignant diseases . To understand its molecular mechanisms, we studied allelic losses on the long arm of chromosome 6 . Using 55 paired DNAs of tumors and their corresponding normal tissues and 30 microsatellite markers that spanned the entire 6q chromosome arm, we found three distinct regions of common allelic loss: region A, a less than 500-kb region bordered by D6S449 and D6S283 on 6q21 with a loss of heterozygosity (LOH) frequency of 69% (38/55); region B, a 7-cM region bordered by D6S292 and D6S308 on 6q23-q24 with a LOH frequency of 60% (33/55); and region C, a 13-cM region bordered by D6S305 and D6S264 with a LOH frequency of 51% (28/55) . We further focused on region A and constructed a physical map using yeast artificial chromosome (YAC) clones, their derived cosmid clones, and bacterial artificial chromosome (BAC) clones . Region A was completely covered by three overlapping BAC clones . Our results in the present study should shed light on the cloning and characterization of tumor suppressor genes in pancreatic carcinogenesis.

Physiol Rev, 1999 Apr, 79(2), 361 - 85
Vacuolar and plasma membrane proton-adenosinetriphosphatases; Nelson N et al.; The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature . It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes . V-ATPases have similar structure and mechanism of action with F-ATPase and several of their subunits evolved from common ancestors . In eukaryotic cells, F-ATPases are confined to the semi-autonomous organelles, chloroplasts, and mitochondria, which contain their own genes that encode some of the F-ATPase subunits . In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps . The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes . The mechanistic and structural relations between the two enzymes prompted us to suggest similar functional units in V-ATPase as was proposed to F-ATPase and to assign some of the V-ATPase subunit to one of four parts of a mechanochemical machine: a catalytic unit, a shaft, a hook, and a proton turbine . It was the yeast genetics that allowed the identification of special properties of individual subunits and the discovery of factors that are involved in the enzyme biogenesis and assembly . The V-ATPases play a major role as energizers of animal plasma membranes, especially apical plasma membranes of epithelial cells . This role was first recognized in plasma membranes of lepidopteran midgut and vertebrate kidney . The list of animals with plasma membranes that are energized by V-ATPases now includes members of most, if not all, animal phyla . This includes the classical Na+ absorption by frog skin, male fertility through acidification of the sperm acrosome and the male reproductive tract, bone resorption by mammalian osteoclasts, and regulation of eye pressure . V-ATPase may function in Na+ uptake by trout gills and energizes water secretion by contractile vacuoles in Dictyostelium . V-ATPase was first detected in organelles connected with the vacuolar system . It is the main if not the only primary energy source for numerous transport systems in these organelles . The driving force for the accumulation of neurotransmitters into synaptic vesicles is pmf generated by V-ATPase . The acidification of lysosomes, which are required for the proper function of most of their enzymes, is provided by V-ATPase . The enzyme is also vital for the proper function of endosomes and the Golgi apparatus . In contrast to yeast vacuoles that maintain an internal pH of approximately 5.5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2 . Similarly, some brown and red alga maintain internal pH as low as 0.1 in their vacuoles . One of the outstanding questions in the field is how such a conserved enzyme as the V-ATPase can fulfill such diverse functions.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 117 - 25
A mitogen-activated protein kinase (MPKA) is involved in polarized growth in the filamentous fungus, Aspergillus nidulans; Bussink HJ et al.; An Aspergillus nidulans kinase gene, which encodes a protein kinase with high similarity to mitogen-activated protein kinases involved in cell wall construction and morphogenesis in yeast species, was cloned and sequenced . Targeted deletion of the Aspergillus nidulans kinase gene indicates that this kinase is involved in germination of conidial spores and polarized growth . These defects were largely remedied on complex high osmolarity media, although abnormal swellings of hyphal tips were still observed . Glycerol (1 M) only supported the growth of compact colonies . The Aspergillus nidulans kinase gene is, thus, required for normal polarized growth at several stages of colony formation in the filamentous fungus A . nidulans.

Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 5322 - 7
Regulatory interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases; Bhalerao RP et al.; Mutation of the PRL1 gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in Arabidopsis . The yeast SNF1 protein kinase, a key regulator of glucose signaling, and Arabidopsis SNF1 homologs AKIN10 and AKIN11, which can complement the Deltasnf1 mutation, were found to interact with an N-terminal domain of the PRL1 protein in the two-hybrid system and in vitro . AKIN10 and AKIN11 suppress the yeast Deltasnf4 mutation and interact with the SNF4p-activating subunit of SNF1 . PRL1 and SNF4 bind independently to adjacent C-terminal domains of AKIN10 and AKIN11, and these protein interactions are negatively regulated by glucose in yeast . AKIN10 and AKIN11, purified in fusion with glutathione S-transferase, undergo autophosphorylation and phosphorylate a peptide of sucrose phosphate synthase in vitro . The sucrose phosphate synthase-peptide kinase activity of AKIN complexes detected by immunoprecipitation is stimulated by sucrose in light-grown Arabidopsis plants . In comparison with wild type, the activation level of AKIN immunocomplexes is higher in the prl1 mutant, suggesting that PRL1 is a negative regulator of Arabidopsis SNF1 homologs . This conclusion is supported by the observation that PRL1 is an inhibitor of AKIN10 and AKIN11 in vitro.

Chromosome Res, 1999, 7(1), 65 - 9
Characterization of an alphoid subfamily located near p-arm sequences on human chromosome 22; Eisenbarth I et al.; The centromeric heterochromatin of all human chromosomes is composed of tandemly repeated alpha satellite DNA . Here we describe another alphoid subfamily that maps to human chromosome 22 as determined by FISH . The alphoid sequences were isolated from three YAC-clones carrying DNA from the pericentromeric region of the short arm of human chromosome 22 and limited amounts of alphoid DNA . This property enabled us to map the members of the subfamily to the border of the centromeric region and the short arm of the chromosome . The new alphoid subfamily may contribute to the closure of the gap remaining between the centromeric and short-arm maps of human chromosome 22.

FEBS Lett, 1999 Mar 19, 447(1), 76 - 80
Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system; Honma Y et al.; We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages . This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system . Eos protein could interact with itself and Ikaros protein through its C-terminal portion in the yeast two hybrid assay . These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.

Trends Genet, 1999 Apr, 15(4), 141 - 5
Non-conventional infectious elements in filamentous fungi; Silar P et al.; Old data (most often in French) described phenomena involving non-conventional infectious factors in filamentous fungi . Recently, it was shown that two yeast cytoplasmic determinants are similar to known mammalian prions, in that their different states are attributed to conformational changes of normal cellular proteins . In the light of this discovery, fungal elements are now being reconsidered . This review presents four elements that affect vegetative incompatibility, conidiogenesis, morphology and cell growth . Recently, one element has been shown to be a prion analogue . The status of the others is not clear . We consider the view that non-conventional inheritance might be initiated by the appearance, in the cytoplasm, of a metabolite or a macromolecule whose production involves a positive regulatory loop.

Int J Biochem Cell Biol, 1999 Jan, 31(1), 37 - 41
Protein synthesis initiation factor 4G; Keiper BD et al.; eIF4G is a member of the class of translational initiation factors involved in mRNA recruitment to the 43S initiation complex . The proteins from yeast to mammals are present in multiple isoforms of 82-176 kDa . Mammalian eIF4G-1 is synthesized by internal initiation of translation and is specifically degraded by viral and host proteases activated by stress conditions . The role of eIF4G in protein synthesis is inferred from the presence of binding sites for other initiation factors that serve to co-localize the 5'- and 3'-termini of mRNA with RNA-helicase activity and the 40S ribosomal subunit . Growth-regulated mRNAs are preferentially translated under conditions of accentuated eIF4E-eIF4G interaction . Proteolysis of eIF4G or expression of competitor proteins interferes with its binding to either the 5'- or 3'-termini, changing the spectrum of mRNAs translated . Elevated eIF4G levels correlate with malignant cell transformation and diminished eIF4G levels, with nutritional deprivation and anoxia.

Eur J Biochem, 1999 Apr, 261(2), 379 - 91
The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase; Dopner S et al.; The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxidase were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines . Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the heme c and the hemes a and a3 . The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2 . Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket . In cytochrome c oxidase, the structural changes in both hemes refer to more subtle perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states . These changes are qualitatively different to those observed for cytochrome c oxidase upon poly-l-lysine binding . The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively . The spectroscopic studies were paralleled by steady-state kinetic measurements of the same protein combinations . The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants . The complex stability decreases in the order: wild-type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine) . This order is not exhibited by the conformational equilibria . The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cytochrome c that is defined by the lysyl residues at positions 72, 73 and 79 . On the other hand, the conformational changes in cytochrome c oxidase were found to be independent of the identity of the bound cytochrome c variant . The maximum rate constants determined from steady-state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation . Within this model, the data analysis leads to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1 . These results can be interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.

Genes Dev, 1999 Apr 15, 13(8), 954 - 65
ebi regulates epidermal growth factor receptor signaling pathways in Drosophila; Dong X et al.; ebi regulates the epidermal growth factor receptor (EGFR) signaling pathway at multiple steps in Drosophila development . Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions . However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway . ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats . The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway . Proteins with related structures regulate protein degradation . Similarly, in the developing eye, ebi promotes EGFR-dependent down-regulation of Tramtrack88, an antagonist of neuronal development.

Prog Neurobiol, 1999 Apr, 57(5), 507 - 25
Subcellular RNA compartmentalization; Mohr E; The phenomenon of mRNA sorting to defined subcellular domains is observed in diverse organisms such as yeast and man . It is now becoming increasingly clear that specific transport of mRNAs to extrasomal locations in nerve cells of the central and peripheral nervous system may play an important role in nerve cell development and synaptic plasticity . Although the majority of mRNAs that are expressed in a given neuron are confined to the cell somata, some transcript species are specifically delivered to dendrites and/or, albeit less frequently, to the axonal domain . The physiological role and the molecular mechanisms of mRNA compartmentalization is now being investigated extensively . Even though most of the fundamental aspects await to be fully characterized, a few interesting data are emerging . In particular, there are a number of different subcellular distribution patterns of different RNA species in a given neuronal cell type and RNA compartmentalization may differ depending on the electrical activity of nerve cells . Furthermore, RNA transport is different in neurons of different developmental stages . Considerable evidence is now accumulating that mRNA sorting, at least to dendrites and the initial axonal segment, enables local synthesis of key proteins that are detrimental for synaptic function, nerve cell development and the establishment and maintenance of nerve cell polarity . The molecular determinants specifying mRNA compartmentalization to defined microdomains of nerve cells are just beginning to be unravelled . Targeting appears to be determined by sequence elements residing in the mRNA molecule to which proteins bind in a manner to direct these transcripts along cytoskeletal components to their site of function where they may be anchored to await transcriptional activation upon demand.

Mol Biochem Parasitol, 1999 Mar 15, 99(1), 11 - 9
Molecular cloning and nuclear localization of a histone deacetylase homologue in Plasmodium falciparum; Joshi MB et al.; Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression and developmental events . The acetylation state of histones is controlled by a dynamic equilibrium between activities of histone acetylase and deacetylase enzymes . Histone deacetylase (HDAC) was recently suggested to be the target of a fungus-derived antiprotozoal agent exhibiting structural similarity to known HDAC inhibitors . We have initiated a study of HDAC of human malaria parasite, Plasmodium falciparum, to evaluate its potential as the target for novel antimalarials and its role in parasite development . We have isolated HDAC1 gene from the P . falciparum genomic and cDNA libraries . The nucleotide sequence contains no intervening sequence and its open reading frame (ORF) codes for a protein of 449 amino acid residues . We have named the protein, PfHDAC1, as the sequence shows significant homology to yeast, human and other eukaryotic HDACs . Northern blot analysis of the total RNA from different asexual and sexual stages of the parasite reveals the presence of single mRNA transcript, which is predominantly expressed in mature asexual blood stages and in gametocytes . Antiserum raised against a carboxyl terminal peptide immunoprecipitated an in vitro translated P . falciparum HDAC gene product and recognized an approximately 50 kDa protein in the Triton X-100 insoluble fraction of parasites . Immunoelectron microscopy analysis showed majority of the protein localized in the nucleus of P . falciparum . To our knowledge, this is the first HDAC gene isolated from the malaria parasite.

Planta, 1999 Mar, 208(1), 125 - 31
Isolation and characterization of four type-1 ribosome-inactivating proteins, with polynucleotide:adenosine glycosidase activity, from leaves of Phytolacca dioica L; Di Maro A et al.; Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L . The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29,407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I) . The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the beta-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes . Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 {designated the isoleucine (Ile) form from the N-terminal residue} and PD-L3 is identical to PD-L4 {designated the valine (Val) form from the N-terminal residue} and that there are 35 identical residues between the two forms . Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant . With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had M(r) values of 32,715 +/- 1 (PD-L1), 31,542 +/- 1 (PD-L2), 30,356 +/- 1 (PD-L3) and 29,185 +/- 1 Da (PD-L4) . The 1171 kDa difference in M(r), within the same RIP form, could be due to glycosylation . Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases . This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively . The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin 1 and even PD-S2, an RIP isolated from the seeds of the same plant . Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.

Philos Trans R Soc Lond B Biol Sci, 1999 Mar 29, 354(1383), 613 - 27
Replication of tobacco mosaic virus RNA; Buck KW; The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates . Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo . The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases . The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins . Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication . Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3 . Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication . The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase . TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies' . Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated . Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.

Diagn Microbiol Infect Dis, 1999 Apr, 33(4), 217 - 22
Trends in species distribution and susceptibility to fluconazole among blood stream isolates of Candida species in the United States; Pfaller MA et al.; National surveillance of blood stream infections (BSI) attributable to Candida spp . has been limited to date . Recent studies have suggested in increase in the proportion of BSI attributable to non-Candida albicans species and have also raised concerns regarding the emergence of antifungal resistance among Candida spp . The increased utilization of broad-spectrum antifungal agents and the recognition of Candida spp . as prominent pathogens with the potential for developing antifungal resistance, emphasize the need for ongoing surveillance of antifungal susceptibility patterns . In this investigation trends in species distribution and susceptibility to fluconazole among BSI isolates of Candida spp . referred to our laboratory by United States hospitals were evaluated over the 7-year period from 1992 to 1998 . A total of 1579 BSI isolates from more than 50 medical centers were processed . Overall, C . albicans accounted for 52% of isolates followed by C . glabrata (18%), C . parapsilosis (15%), C . tropicalis (11%), and C . krusei (2%) . The proportion of BSI isolates that were C . albicans ranged from 45% in 1992 to 60% in 1998 . Among the non-C . albicans isolates, C . glabrata succeeded C . parapsilosis as the most common species beginning in 1995 . Overall, the susceptibility of all Candida species (C . albicans plus all other species) to fluconazole remained stable (MIC90, 16 micrograms/mL) . The fluconazole MIC90 for C . albicans was 0.5-2.0 micrograms/ml for all years studied except 1995 (8.0 micrograms/mL) and was 1.0 microgram/mL overall . The present study suggests a continued prominent role of C . albicans as a cause of BSI, and a constant level of susceptibility of Candida BSI isolates to fluconazole over 7 years . These data should serve as a baseline for future surveillance efforts for anti-fungal agents tested against yeast BSI isolates.

J Biol Chem, 1999 Apr 30, 274(18), 12803 - 10
Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin . Regulation by conformational change; Huang L et al.; Point and deletion mutants of moesin were examined for F-actin binding by blot overlay and co-sedimentation, and for intra- and intermolecular interactions with N- and C-terminal domains with yeast two-hybrid and in vitro binding assays . Wild-type moesin molecules interact poorly with F-actin and each other, and bind neither C- nor N-terminal fragments . Interaction with F-actin is strongly enhanced by replacement of Thr558 with aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by deletion of the entire N-terminal membrane-binding domain of both wild type and T558D mutant molecules, and by exposure to phosphatidylinositol 4, 5-diphosphate . Activation of F-actin binding is accompanied by changes in inter- and intramolecular domain interactions . The T558D mutation renders moesin capable of binding wild type but not mutated (T558D) C-terminal or wild type N-terminal fragments . The interaction between the latter two is prevented . DelN11 truncation enables binding of wild type N and C domain fragments . These changes suggest that the T558D mutation, mimicking phosphorylation of Thr558, promotes F-actin binding by disruption of interdomain interactions between N and C domains and exposure of the high affinity F-actin binding site in the C-terminal domain . Oscillation between activated and resting state could thus provide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions.

J Cell Sci, 1999 May, 112 ( Pt 10), 1465 - 76
A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock; Weighardt F et al.; A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP) . HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region . In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs . HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene . We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins . Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells . Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1) . The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C . The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes . Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.

Biochim Biophys Acta, 1999 Apr 1, 1449(3), 211 - 6
Isolation of spermidine synthase gene (spsA) of Dictyostelium discoideum; Guo K et al.; The gene encoding spermidine synthase (spsA) was isolated from Dictyostelium discoideum using the technique of insertional mutagenesis . Northern blot analysis showed that the spsA mRNA is expressed maximally during the vegetative stage and decreases gradually during the 24 h of development . Sequencing of the genomic DNA and a full-length cDNA clone indicated the presence of one intron in a gene coding for a predicted protein (SpsA) with 284 amino acids . The sequence is highly conserved, with amino acid identities compared to spermidine synthases of humans, 59.5%, to mouse, 61.3%, and to yeast, 58.1% . A null mutant of the spsA gene is unable to grow in the absence of exogenous spermidine . Development of spsA null cells grown in the absence of spermidine produced fruiting bodies that have abnormally short stalks.

Biochim Biophys Acta, 1999 Apr 14, 1418(1), 61 - 70
Transbilayer movement and distribution of spin-labelled phospholipids in the inner mitochondrial membrane; Gallet PF et al.; The transmembrane diffusion and equilibrium distribution of spin-labelled phosphatidylethanolamine (PE*), phosphatidylcholine (PC*) and cardiolipin (CL*) were investigated in purified mitochondrial inner membranes using electron spin resonance spectroscopy . Using the back exchange technique, we found that the outside-inside movement of PE* and PC* in beef-heart inner mitochondrial membranes was rapid (t1/2 in the range 10-15 min at 30 degrees C) . The steady-state distributions in non-energised mitoplasts were approximately 30% in the inner leaflet for PC* and 39% for PE* . Within the limits of probe concentration that can possibly be used in these experiments, the initial velocity of the inward movement was not saturable with respect to the amount of analogue added to the membranes, suggesting that the spin-labelled phospholipids diffused passively between the two leaflets of the inner mitochondrial membrane . In energised mitoplasts, PC* behaviour was not affected, PE* diffused approximately two times faster toward the inner monolayer but reached the same plateau . Treatment of energised mitochondria with N-ethylmaleimide did not affect PC* diffusion, while the kinetics of PE* internalisation became identical to that of PC* . Similar results were found when PC* and PE* movements were studied in mitoplasts from beef heart, rat liver or yeast . The spin-labelled cardiolipin, which possesses four long chains, had to be introduced in the mitoplast with some ethanol . After equilibration (t1/2 of the order of 13 min at 30 degrees C), the transmembrane distribution suggested that approximately half of the cardiolipin analogue remained in the outer leaflet . These results do not allow us to determine if a specific protein (or flippase) is involved in the phospholipid transmembrane traffic within inner mitochondrial membranes, but they show that lipids can rapidly flip through the mitochondrial membrane.

Curr Biol, 1999 Apr 8, 9(7), 381 - 4
A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins; Barr FA; In recent years, a large number of coiled-coil proteins localised to the Golgi apparatus have been identified using antisera from human patients with a variety of autoimmune conditions {1} . Because of their common method of discovery and extensive regions of coiled-coil, they have been classified as a family of proteins, the golgins {1} . This family includes golgin-230/245/256, golgin-97, GM130/golgin-95, golgin-160/MEA-2/GCP170, giantin/macrogolgin and a related group of proteins - possibly splice variants - GCP372 and GCP364{2}{3}{4}{5}{6}{7}{8}{9}{10}{11} . GM130 and giantin have been shown to function in the p115-mediated docking of vesicles with Golgi cisternae {12} . In this process, p115, another coiled-coil protein, is though to bind to giantin on vesicles and to GM130 on cisternae, thus acting as a tether holding the two together {12} {13} . Apart from giantin and GM130, none of the golgins has yet been assigned a function in the Golgi apparatus . In order to obtain clues as to the functions of the golgins, the targeting to the Golgi apparatus of two members of this family, golgin-230/245/256 and golgin-97, was investigated . Each of these proteins was shown to target to the Golgi apparatus through a carboxy-terminal domain containing a conserved tyrosine residue, which was critical for targeting . The domain preferentially bound to Rab6 on protein blots, and mutations that abolished Golgi targeting resulted in a loss of this interaction . Sequence analysis revealed that a family of coiled-coil proteins from mammals, worms and yeast contain this domain at their carboxyl termini . One of these proteins, yeast Imh1p, has previously been shown to have a tight genetic interaction with Rab6 {14} . On the basis of these data, it is proposed that this family of coiled-coil proteins functions in Rab6-regulated membrane-tethering events.

Curr Biol, 1999 Apr 8, 9(7), 377 - 80
The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins; Munro S et al.; Many large coiled-coil proteins are being found associated peripherally with the cytoplasmic face of the organelles of the secretory pathway . Various roles have been proposed for these proteins, including the docking of donor vesicles or organelles to an acceptor organelle prior to fusion, and, in the case of the Golgi apparatus, the stacking of the cisternae {1} {2} {3} {4} {5} . Such critical roles require accurate recruitment to the correct organelle . For the endosomal coiled-coil protein EEA1, targeting requires a carboxy-terminal FYVE domain, which interacts with Rab5 and phosphatidylinositol 3-phosphate (PI(3)P), whereas the Golgi protein GM130 interacts with Golgi membranes via the protein GRASP65 {3} {6} {7} . In this paper, we show that two other mammalian Golgi coiled-coil proteins, golgin-245/p230 and golgin-97, have a conserved domain of about 50 amino acids at their carboxyl termini . This 'GRIP' domain is also found at the carboxyl terminus of several other large coiled-coiled proteins of unknown function, including two human proteins and proteins in the genomes of Caenorhabditis elegans and yeasts . The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were sufficient to specify Golgi targeting in mammalian cells when fused to green fluorescent protein (GFP) . This result suggests that this small domain functions to recruit specific coiled-coil proteins to the Golgi by recognising a determinant that has been well conserved in eukaryotic evolution.

Curr Biol, 1999 Apr 8, 9(7), 341 - 50
A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs; Okuhara K et al.; BACKGROUND: Alteration of chromatin structure is a key step in various aspects of DNA metabolism . DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication . For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure . At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication . RESULTS: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides . DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis . Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein . The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure . Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA . CONCLUSIONS: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

J Cell Biol, 1999 Apr 19, 145(2), 317 - 30
Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth; Kost B et al.; Pollen tube cells elongate based on actin- dependent targeted secretion at the tip . Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells . We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes . Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth . Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity . Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac . Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation . These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.

Neuroreport, 1999 Feb 25, 10(3), 563 - 8
Isolation of human delta-catenin and its binding specificity with presenilin 1; Tanahashi H et al.; We screened proteins for interaction with presenilin (PS) 1, and cloned the full-length cDNA of human delta-catenin, which encoded 1225 amino acids . Yeast two-hybrid assay, GST binding assay and immunoprecipitation demonstrated that delta-catenin interacted with a hydrophilic loop region in the endoproteolytic C-terminal fragment of PS1, but not with that of PS-2 . These results suggest that PS1 and PS2 partly differ in function . PS1 loop fragment containing the pathogenic mutation retained the binding ability . We also found another armadillo-protein, p0071, interacted with PS1.

Development, 1999 May, 126(10), 2227 - 39
The Caenorhabditis elegans gene ncc-1 encodes a cdc2-related kinase required for M phase in meiotic and mitotic cell divisions, but not for S phase; Boxem M et al.; We have identified six protein kinases that belong to the family of cdc2-related kinases in Caenorhabditis elegans . Results from RNA interference experiments indicate that at least one of these kinases is required for cell-cycle progression during meiosis and mitosis . This kinase, encoded by the ncc-1 gene, is closely related to human Cdk1/Cdc2, Cdk2 and Cdk3 and yeast CDC28/cdc2(+) . We addressed whether ncc-1 acts to promote passage through a single transition or multiple transitions in the cell cycle, analogous to Cdks in vertebrates or yeasts, respectively . We isolated five recessive ncc-1 mutations in a genetic screen for mutants that resemble larval arrested ncc-1(RNAi) animals . Our results indicate that maternal ncc-1 product is sufficient for embryogenesis, and that zygotic expression is required for cell divisions during larval development . Cells that form the postembryonic lineages in wild-type animals do not enter mitosis in ncc-1 mutants, as indicated by lack of chromosome condensation and nuclear envelope breakdown . However, progression through G1 and S phase appears unaffected, as revealed by expression of ribonucleotide reductase, incorporation of BrdU and DNA quantitation . Our results indicate that C . elegans uses multiple Cdks to regulate cell-cycle transitions and that ncc-1 is the C . elegans ortholog of Cdk1/Cdc2 in other metazoans, required for M phase in meiotic as well as mitotic cell cycles.

Mol Cell Biol, 1999 May, 19(5), 3614 - 23
The catenin p120(ctn) interacts with Kaiso, a novel BTB/POZ domain zinc finger transcription factor; Daniel JM et al.; p120(ctn) is an Armadillo repeat domain protein with structural similarity to the cell adhesion cofactors beta-catenin and plakoglobin . All three proteins interact directly with the cytoplasmic domain of the transmembrane cell adhesion molecule E-cadherin; beta-catenin and plakoglobin bind a carboxy-terminal region in a mutually exclusive manner, while p120 binds the juxtamembrane region . Unlike beta-catenin and plakoglobin, p120 does not interact with alpha-catenin, the tumor suppressor adenomatous polyposis coli (APC), or the transcription factor Lef-1, suggesting that it has unique binding partners and plays a distinct role in the cadherin-catenin complex . Using p120 as bait, we conducted a yeast two-hybrid screen and identified a novel transcription factor which we named Kaiso . Kaiso's deduced amino acid sequence revealed an amino-terminal BTB/POZ protein-protein interaction domain and three carboxy-terminal zinc fingers of the C2H2 DNA-binding type . Kaiso thus belongs to a rapidly growing family of POZ-ZF transcription factors that include the Drosophila developmental regulators Tramtrak and Bric a brac, and the human oncoproteins BCL-6 and PLZF, which are causally linked to non-Hodgkins' lymphoma and acute promyelocytic leukemia, respectively . Monoclonal antibodies to Kaiso were generated and used to immunolocalize the protein and confirm the specificity of the p120-Kaiso interaction in mammalian cells . Kaiso specifically coprecipitated with a variety of p120-specific monoclonal antibodies but not with antibodies to alpha- or beta-catenin, E-cadherin, or APC . Like other POZ-ZF proteins, Kaiso localized to the nucleus and was associated with specific nuclear dots . Yeast two-hybrid interaction assays mapped the binding domains to Arm repeats 1 to 7 of p120 and the carboxy-terminal 200 amino acids of Kaiso . In addition, Kaiso homodimerized via its POZ domain but it did not heterodimerize with BCL-6, which heterodimerizes with PLZF . The involvement of POZ-ZF proteins in development and cancer makes Kaiso an interesting candidate for a downstream effector of cadherin and/or p120 signaling.

Mol Cell Biol, 1999 May, 19(5), 3383 - 94
Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily; Dressel U et al.; Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone . Corepressors, which bind to the receptor's silencing domain, are involved in this repression . Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation . Here, we describe the characteristics of Alien, a novel corepressor . Alien interacts with TR only in the absence of hormone . Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation experiments . Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function . Immune staining shows that Alien is localized in the cell nucleus . Alien is a highly conserved protein showing 90% identity between human and Drosophila . Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function . Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96 . These properties, taken together, show that Alien has the characteristics of a corepressor . Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.

J Biol Chem, 1999 Apr 23, 274(17), 12001 - 8
Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha; Wang W et al.; A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory . To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK . One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha) . CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner . The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis . Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1 . An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner . Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites . This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance . Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner . This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers . Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

Med Mycol, 1998 Dec, 36(6), 433 - 6
Antifungal activity of a new triazole, voriconazole (UK-109,496), compared with three other antifungal agents tested against clinical isolates of filamentous fungi; Marco F et al.; Voriconazole is a new triazole antifungal agent with potent activity against yeast and moulds . We investigated the in vitro activity of voriconazole, itraconazole, amphotericin B and 5-flucytosine against 51 clinical isolates of filamentous fungi . Overall, voriconazole was active (MIC50, 0.5 mg l(-1) and MIC90, 8 mg l(-1)) against these mould isolates . Voriconazole was most active against P . boydii (MIC50, 0.12 mg l(-1)) and Aspergillus spp . (MIC90, 0.5 mg l(-1)) and least active against Fusarium spp . (MIC90, 8 mg l(-1)) and Rhizopus spp . (MIC50, 8 mg l(-1)) . Voriconazole was more active than amphotericin B against Aspergillus spp . and P . boydii . By comparison with itraconazole, voriconazole was more active against all isolates except Rhizopus spp . Based on these results, voriconazole has promising activity against commonly encountered isolates of filamentous fungi and its clinical usefulness should be established by further studies.

Plant J, 1999 Feb, 17(4), 433 - 44
Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method; Ito T et al.; For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al . Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A . thaliana chromosome 5 . We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub-libraries in which cDNAs for genes on each YAC DNA were concentrated . From each sub-library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs) . In total, 113 non-redundant groups of cDNAs were obtained . Forty-four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms . In parallel, we transposed Ds from a donor Ds-GUS-T-DNA line, Ds4391-20, already mapped to the CIC7E11/8B11 region . We obtained Ds-transposed lines and recovered their Ds-flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR) . Dot-blot analysis indicated that 20% of the lines contained transposed Ds in the CIC7E11/8B11 region, suggesting that this Ac/Ds transposon system is effective for regional insertional mutagenesis . To isolate Ds insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800 Ds-containing lines using Ds-specific and gene-specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method . We found that 49 lines contain Ds insertion mutations, and that five lines contain Ds mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region . These results indicate that combining the cDNA scanning method and the Ac/Ds transposon gives a powerful tool for regional insertional mutagenesis not only in Arabidopsis but also in other plants or crops whose genomes are not sequenced.

J Virol Methods, 1999 Mar, 78(1-2), 13 - 9
Enhanced binding to origin DNA at low pH enables easy detection of polyomavirus large T antigen by gel mobility shift assay of unfixed complexes; Peng YC et al.; Enhanced, stable binding by polyomavirus large T antigen to the viral DNA replication origin at pH 6 allowed the development of a gel mobility shift assay for the detection of large T antigen . Such assays were not possible at pH 7.6 without previous fixation, due to instability of the complexes . We demonstrated that the gel mobility shift assay at pH 6 is very sensitive, allowing the detection of as little as 5 ng large T antigen, and is highly specific for DNA containing G(A/G)GGC target sequences . This method was used to detect large T antigen in crude cell lysates from transformed yeast cell lines or nuclear extracts from infected insect cells . Large T antigen-DNA complexes remained at or near the loading well in 5% acrylamide or 1.5% agarose gels, indicating that these complexes are very large . Glycerol gradient analysis showed that protein-DNA complexes formed at pH 6 were massive, and that large T antigen also formed large complexes when incubated at low pH in the absence of DNA . These results show that pH has a major effect on binding of large T antigen to its multiple target sites in the viral origin of DNA replication, presumably by affecting protein-protein interactions that are important for the stability of large T antigen-DNA complexes.

Cell Death Differ, 1998 Oct, 5(10), 838 - 46
Identification of a new caspase homologue: caspase-14; Van de Craen M et al.; Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis . Here, we report the identification of a new murine caspase homologue, viz . caspase-14 . It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain . Murine caspase-14 shows 83% similarity to human caspase-14 . Human caspase-14 is assigned to chromosome 19p13.1 . Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos . In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8 . No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression . Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis . In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far . The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.

J Clin Microbiol, 1999 May, 37(5), 1393 - 7
Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection; Chien DY et al.; The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, E2/NS1, NS2, NS3, NS4, and NS5 regions . The U . S . Food and Drug Administration-licensed 2.0G immunoassay for the detection of anti-HCV uses proteins from the core, NS3, and NS4 regions (McHutchinson et al., Hepatology 15:19-25, 1992) . The 3.0G enzyme-linked immunosorbent assay includes the protein from the NS5 region (Uyttendaele et al., Vox Sang . 66:122-129, 1994) . The necessity of detecting antibodies to viral envelope proteins (E1 and E2) and to different genotype samples has been demonstrated previously (Chien et al., Lancet 342:933, 1993; Lok et al., Hepatology 18:497-502, 1993) . In this study we have attempted to improve the sensitivity of the anti-HCV assay by developing a single multiple-epitope fusion antigen (MEFA; MEFA-6) which incorporates all of the major immunodominant epitopes from the seven functional regions of the HCV genome . A nucleic acid sequence consisting of proteins from the viral core, E1, E2, NS3, NS4, and NS5 regions and different subtype-specific regions of the NS4 region was constructed, cloned, and expressed in yeast . The epitopes present on this antigen can be detected by epitope-specific monoclonal and polyclonal antibodies . In a competition assay, the MEFA-6 protein competed with 83 to 96% of genotype-specific antibodies from HCV genotype-specific peptides . This recombinant antigen was subsequently used to design an anti-HCV chemiluminescent immunoassay . We designed our assay using a monoclonal anti-human immunoglobulin G antibody bound to the solid phase . Because MEFA-6 is fused with human superoxide dismutase (h-SOD), we used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection . Our results indicate that MEFA-6 exposes all of the major immunogenic epitopes . Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.

Int J Radiat Biol, 1999 Mar, 75(3), 267 - 83
The effects of ionizing radiation on DNA synthesis in eukaryotic cells; Rowley R et al.; PURPOSE: To review observations of the effects of ionizing radiation on DNA synthesis in eukaryotes . CONTENT: Available information broadly falls into two categories: descriptions of the phenomenon, including dose response data and analysis; and, more recently, investigations utilizing genetic approaches . The down-regulation of DNA replication in the presence of radiation-induced DNA damage appears to be an active cellular response, termed the S-phase damage-sensing (SDS) checkpoint control (Larner et al . 1997) . Observations on a variety of eukaryotes, including man, suggest that the regulatory controls involved are highly conserved and may additionally function in G1 and G2 checkpoint controls . Budding yeast, fission yeast and human homologues are identified . CONCLUSIONS: The SDS checkpoint control appears to be comprised of a complex of checkpoint proteins that respond to the stalled replication complex . The replication complex is thought to signal down-regulation of the mitotic kinase, ensuring that the cell does not enter mitosis while S phase is delayed . Concomitantly, the checkpoint complex is believed to transmit a signal via two key checkpoint proteins (Rad3 and Cds1 in the fission yeast), in order to arrest further DNA synthesis initiation.

Annu Rev Neurosci, 1999, 22, 1 - 10
Monitoring secretory membrane with FM1-43 fluorescence; Cochilla AJ et al.; FM1-43 and similar styryl dyes have proven useful as probes for membrane trafficking because they reversibly stain membranes, are impermeable to membranes, and are more fluorescent when bound to membranes than when in solution . Because these dyes stain membranes in an activity-dependent manner, they are ideal for studies of neurotransmitter release mechanisms such as synaptic vesicle recycling, exocytosis, and endocytosis . FM dyes have been used in conjunction with other techniques such as fluorescent calcium indicator dyes and electrophysiological techniques to elucidate mechanisms of presynaptic calcium homeostasis and modulation of neurotransmitter release . Presynaptic membranes have been marked by FM dyes in studies of synaptogenesis and reinnervation . As a probe for endocytosed membranes, these dyes have been used to examine vacuole formation in yeast . These versatile membrane dyes are useful in a variety of applications.

EMBO J, 1999 Apr 1, 18(7), 1905 - 14
Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300; Ema M et al.; Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen . Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half . Their sequences are conserved between HLF and HIF1alpha . One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain) . Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed . In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism . Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD . Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP . Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.

EMBO J, 1999 Apr 1, 18(7), 1783 - 92
CLK-1 controls respiration, behavior and aging in the nematode Caenorhabditis elegans; Felkai S et al.; Mutations in the clk-1 gene of the nematode Caenorhabditis elegans result in an average slowing of a variety of developmental and physiological processes, including the cell cycle, embryogenesis, post-embryonic growth, rhythmic behaviors and aging . In yeast, a CLK-1 homologue is absolutely required for ubiquinone biosynthesis and thus respiration . Here we show that CLK-1 is fully active when fused to green fluorescent protein and is found in the mitochondria of all somatic cells . The activity of mutant mitochondria, however, is only very slightly impaired, as measured in vivo by a dye-uptake assay, and in vitro by the activity of succinate cytochrome c reductase . Overexpression of CLK-1 activity in wild-type worms can increase mitochondrial activity, accelerate behavioral rates during aging and shorten life span, indicating that clk-1 regulates and controls these processes . These observations also provide strong genetic evidence that mitochondria are causally involved in aging . Furthermore, the reduced respiration of the long-lived clk-1 mutants suggests that longevity is promoted by the age-dependent decrease in mitochondrial function that is observed in most species.

Med Mycol, 1999 Feb, 37(1), 69 - 73
Rapid extraction of fungal DNA from clinical samples for PCR amplification; Velegraki A et al.; A hexadecyltrimethylammonium bromide (CTAB) method for isolating fungal DNA from clinical samples, suitable for PCR amplification is described . Yeast and filamentous fungi DNA from clinical samples was amplified with primers complementary to the genes coding for rRNA, amplifying a 105 bp fragment and internal transcribed spacer primers amplifying fragments between 242 and 622 bp . The level of sensitivity was 10 +/- 5 yeast and 28 Aspergillus fumigatus CFU ml-1 of biological fluid.

J Hum Virol, 1999 Jan-Feb, 2(1), 33 - 7
Epstein-Barr virus-encoded nuclear protein EBNA-3 interacts with the epsilon-subunit of the T-complex protein 1 chaperonin complex; Kashuba E et al.; OBJECTIVE: To find cellular proteins that associate with EBNA-3 (also called EBNA-3A), one of the Epstein-Barr virus (EBV)-encoded growth transformation-associated nuclear proteins . METHODS: Screening human cDNA libraries in the yeast two-hybrid system and performing an analysis of interaction in vitro as well as in cell lysates . RESULTS: EBNA-3 binds to the epsilon subunit of the chaperonin containing T-complex protein 1 (epsilon-TCP-1) in the yeast two-hybrid system . The cDNA clone isolated from a human lymphocyte library was found to encode the middle and C-terminal part of epsilon-TCP-1 . The interaction was confirmed by showing that a GST fusion protein specifically precipitated EBNA-3 from CV1 cells infected with recombinant vaccinia virus expressing EBNA-3 . The interacting region was mapped to the putative apical domain of epsilon-TCP-1 . CONCLUSIONS: This study shows that large, virus-encoded transforming proteins such as EBNA-3 may receive help for their initial folding by chaperonin complexes . The recognition of the chaperonin complex likely occurs through specific interaction with one of the subunits . We suggest that nascent EBNA-3 may recognize the TCP-1 complex by interacting with the apical region of the epsilon subunit.

Cell Death Differ, 1998 Jul, 5(7), 615 - 22
Prohibitin and RACK homologues are up-regulated in trypanosomes induced to undergo apoptosis and in naturally occurring terminally differentiated forms; Welburn SC et al.; Two genes have been identified as up-regulated late during ConA-induced apoptosis in procyclic form Trypanosoma brucei rhodesiense . The first represents a homologue of prohibitin, a proto-oncogene originally described in mammals and subsequently in yeast, which is involved in cell-cycle control and senescence . The Trypanosoma prohibitin homologue appears to contain within it a putative death domain . The second gene, homologous to a family of regulatory proteins which are receptors for activated protein kinase C (RACKs), is also shown to be up-regulated in terminally differentiated bloodstream form trypanosomes . These are the first endogenous genes to be identified as up-regulated in programmed cell death (PCD) in unicellular organisms.

Cell Death Differ, 1998 Jun, 5(6), 488 - 96
Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction; Trapani JA et al.; Killer lymphocytes utilize the synergy of a membranolytic protein, perforin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined . We have previously shown that perforin specifically induces the redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has not been demonstrated . In the present study, we used confocal laser scanning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptosis . Two distinct and mutually exclusive cellular responses were observed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FITC-grB in the nucleus (maximal at 7 min; t1/2 of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cytoplasm, and escaped apoptosis . Under these conditions, DNA fragmentation was not observed for at least 13 min, indicating nuclear accumulation of grB preceded the execution phase of apoptosis . Furthermore, nuclear import of grB proceeded through an intact nuclear membrane, as the nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dextran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB . These findings indicated that perforin-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation . The cell membrane lesions formed by perforin in these experiments were not large enough to permit a 13 kDa protein (yeast cdk p13suc) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equilibrate between the cytosol and the exterior . Therefore, transmembrane pores large enough to allow passive diffusion of grB (32 kDa) into the cell are not necessary for apoptosis . Rather, a perforin-dependent signal results in a redistribution of grB from the cytoplasm to the nucleus, where it may contribute to the nuclear changes associated with apoptosis.

Cell Death Differ, 1998 Jan, 5(1), 116 - 25
Differentially expressed genes in C6.9 glioma cells during vitamin D-induced cell death program; Baudet C et al.; C6.9 rat glioma cells undergo a cell death program when exposed to 1, 25-dihydroxyvitamin D3 (1,25-D3) . As a global analytical approach, we have investigated gene expression in C6.9 engaged in this cell death program using differential screening of a rat brain cDNA library with probes derived from control and 1,25-D3-treated cells . Using this methodology we report the isolation of 61 differentially expressed cDNAs . Forty-seven cDNAs correspond to genes already characterized in rat cells or tissues . Seven cDNAs are homologous to yeast, mouse or human genes and seven are not related to known genes . Some of the characterized genes have been reported to be differentially expressed following induction of programmed cell death . These include PMP22/gas3, MGP and beta-tubulin . For the first time, we also show a cell death program induced up-regulation of the c-myc associated primary response gene CRP, and of the proteasome RN3 subunit and TCTP/mortalin genes . Another interesting feature of this 1,25-D3 induced-cell death program is the down-regulated expression of transcripts for the microtubule motor dynein heavy chain/MAP 1C and of the calcium-binding S100beta protein . Finally 15 upregulated cDNAs encode ribosomal proteins suggesting a possible involvement of the translational apparatus in this cell program . Alternatively, these ribosomal protein genes could be up-regulated in response to altered rates of cellular metabolism, as has been demonstrated for most of the other isolated genes which encode proteins involved in metabolic pathways . Thus, this study presents to our knowledge the first characterization of genes which are differentially expressed during a cell death program induced by 1, 25-D3 . Therefore, this data provides new information on the fundamental mechanisms which participate in the antineoplastic effects of 1,25-D3 and on the machinery of a cell death program in a glioma cell line.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4718 - 23
Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals; Kudla J et al.; An important effector of Ca2+ signaling in animals and yeast is the Ca2+/calmodulin-dependent protein phosphatase calcineurin . However, the biochemical identity of plant calcineurin remained elusive . Here we report the molecular characterization of AtCBL (Arabidopsis thaliana calcineurin B-like protein) from Arabidopsis . The protein is most similar to mammalian calcineurin B, the regulatory subunit of the phosphatase . AtCBL also shows significant similarity with another Ca2+-binding protein, the neuronal calcium sensor in animals . It contains typical EF-hand motifs with Ca2+-binding capability, as confirmed by in vitro Ca2+-binding assays, and it interacts in vivo with rat calcineurin A in the yeast two-hybrid system . Interaction of AtCBL1 and rat calcineurin A complemented the salt-sensitive phenotype in a yeast calcineurin B mutant . Cloning of cDNAs revealed that AtCBL proteins are encoded by a family of at least six genes in Arabidopsis . Genes for three isoforms were identified in this study . AtCBL1 mRNA was preferentially expressed in stems and roots and its mRNA levels strongly increased in response to specific stress signals such as drought, cold, and wounding . In contrast, AtCBL2 and AtCBL3 are constitutively expressed under all conditions investigated . Our data suggest that AtCBL1 may act as a regulatory subunit of a plant calcineurin-like activity mediating calcium signaling under certain stress conditions.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4313 - 8
A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2; Liu YC et al.; Recently a number of nonclass I genes were discovered in the human MHC class I region . One of these, FAT10, encodes a protein consisting of two domains with homology to ubiquitin . FAT10 mRNA is expressed constitutively in some lymphoblastoid lines and dendritic cells and in certain other cells after gamma-interferon induction . FAT10 protein expression is controlled at several levels including transcription, translation, and protein stability . Yeast two-hybrid screening of a human lymphocyte library and immunoprecipitation studies revealed that FAT10 noncovalently associated with MAD2, a protein implicated in a cell-cycle checkpoint for spindle assembly during anaphase . Thus, FAT10 may modulate cell growth during B cell or dendritic cell development and activation.

Cell Tissue Res, 1999 Apr, 296(1), 85 - 93
Formin defines a large family of morphoregulatory genes and functions in establishment of the polarising region; Zeller R et al.; Formin was originally isolated as the gene affected by the murine limb deformity (ld) mutations, which disrupt the epithelial-mesenchymal interactions regulating patterning of the vertebrate limb autopod . More recently, a rapidly growing number of genes with similarity to formin have been isolated from many different species including fungi and plants . Genetic and biochemical analysis shows that formin family members function in cellular processes regulating either cytokinesis and/or cell polarisation . Another common feature among formin family members is their requirement in morphogenetic processes such as budding and conjugation of yeast, establishment of Drosophila oocyte polarity and vertebrate limb pattern formation . Vertebrate formins are predominantly nuclear proteins which control polarising activity in limb buds through establishment of the SHH/FGF-4 feedback loop . Formin acts in the limb bud mesenchyme to induce apical ectodermal ridge (AER) differentiation and FGF-4 expression in the posterior AER compartment . Finally, disruption of the epithelial-mesenchymal interactions controlling induction of metanephric kidneys in ld mutant embryos indicates that formin might function more generally in transduction of morphogenetic signals during embryonic pattern formation.

Am J Physiol, 1999 Apr, 276(4 Pt 1), C796 - 802
Ca2+-ATPases and their expression in the mammary gland of pregnant and lactating rats; Reinhardt TA et al.; The transcellular Ca2+ fluxes required for milk production must be rigorously regulated to maintain the low cytosolic Ca2+ concentrations critical to cell function . Ca2+-ATPases play a critical role in the maintenance of this cellular Ca2+ homeostasis . Using RT-PCR and sequencing, we identified six Ca2+ pumps in lactating mammary tissue . Three plasma membrane Ca2+-ATPases (PMCAs) were found (PMCA1b, PMCA2b, and PMCA4b) . Two sarco (endo)plasmic reticulum Ca2+-ATPases (SERCAs) were identified (SERCA2 and SERCA3), and the rat homologue to the yeast Golgi Ca2+-ATPase RS-10 was also found . The pattern of mRNA expression of each of these pumps was examined in rat mammary tissue from the 7th day of pregnancy to the 21st day of lactation . Northern blots revealed increased mRNA expression for all Ca2+ pumps by the 14th day of lactation, and transcripts continued to increase through the 18th day of lactation . PMCA1b, PMCA4b, SERCA2, and SERCA3 showed the lowest levels of expression . RS-10 transcripts were more abundant than SERCA2, SERCA3, PMCA1b, and PMCA4b . RS-10 was the only pump to increase in expression before parturition . PMCA2b was the most abundant transcript found in lactating mammary tissue . At peak lactation, expression of PMCA2b approached that of actin . The high expression, high affinity for Ca2+, and high activity at low calmodulin concentrations exhibited by PMCA2b suggest that it is uniquely suited for maintenance of Ca2+ homeostasis in the lactating mammary gland . The pattern of expression and abundance of RS-10 suggest that it is a candidate for the Golgi Ca2+-ATPase shown to be important in maintaining the Golgi Ca2+ concentration required for casein synthesis and micelle formation.

RNA, 1999 Apr, 5(4), 503 - 11
The uridine in "U-turn": contributions to tRNA-ribosomal binding; Ashraf SS et al.; "U-turns" represent an important class of structural motifs in the RNA world, wherein a uridine is involved in an abrupt change in the direction of the polynucleotide backbone . In the crystal structure of yeast tRNAPhe, the invariant uridine at position 33 (U33), adjacent to the anticodon, stabilizes the exemplar U-turn with three non-Watson-Crick interactions: hydrogen bonding of the 2'-OH to N7 of A35 and the N3-H to A36-phosphate, and stacking between C32 and A35-phosphate . The functional importance of each noncanonical interaction was determined by assaying the ribosomal binding affinities of tRNAPhe anticodon stem and loop domains (ASLs) with substitutions at U33 . An unsubstituted ASL bound 30S ribosomal subunits with an affinity (Kd = 140+/-50 nM) comparable to that of native yeast tRNAPhe (Kd = 100+/-20 nM) . However, the binding affinities of ASLs with dU-33 (no 2'-OH) and C-33 (no N3-H) were significantly reduced (2,930+/-140 nM and 2,190+/-300 nM, respectively) . Surprisingly, the ASL with N3-methyluridine-33 (no N3-H) bound ribosomes with a high affinity (Kd = 220+/-20 nM) . In contrast, ASLs constructed with position 33 uridine analogs in nonstacking, nonnative, and constrained conformations, dihydrouridine (C2'-endo), 6-methyluridine (syn) and 2'O-methyluridine (C3'-endo) had almost undetectable binding . The inability of ASLs with 6-methyluridine-33 and 2'O-methyluridine-33 to bind ribosomes was not attributable to any thermal instability of the RNAs . These results demonstrate that proton donations by the N3-H and 2'OH groups of U33 are not absolutely required for ribosomal binding . Rather, the results suggest that the overall uridine conformation, including a dynamic (C3'-endo > C2'-endo) sugar pucker, anti conformation, and ability of uracil to stack between C32 and A35-phosphate, are the contributing factors to a functional U-turn.

J Cell Sci, 1999 Apr, 112 ( Pt 7), 1065 - 76
Ultrastructural distribution of a MAP kinase and transcripts in quiescent and cycling plant cells and pollen grains; Prestamo G et al.; Mitogen-activated protein kinases (MAPKs) are components of a kinase module that plays a central role in the transduction of diverse extracellular stimuli, including mitogens, specific differentiation and developmental signals and stress treatments . This shows that reversible protein phosphorylation cascades play a pivotal role in signal transduction in animal cells and yeast, particularly the entry into mitosis of arrested cells . Homologues of MAPKs have been found and cloned in various plant species, but there have been no data about their in situ localization at the subcellular level and their expression in plant cells so far . In the present paper we report the first data on the ultrastructural in situ localization of MAPK and their mRNAs in various plant cells . Proliferating and quiescent meristematic plant cells were studied to evaluate whether changes in MAPK presence, distribution and expression accompany the entry into proliferation of dormant cells . Moreover, MAPK localization was analyzed in vacuolate microspores . Polyclonal antibodies against the deduced MAPK from the tobacco Ntf6 clone were able to recognize homologue epitopes by immunocytochemical techniques in the cell types studied . The pattern of protein distribution is similar in all the cases studied: it is localized in the cytoplasm and in the nucleus, mainly in the interchromatin region . The quantitative study of the density showed that MAPK labelling is more abundant in cycling than in quiescent cells, also suggesting that, in plants, MAPK pathways might play a role in cell proliferation . RNA probes for conserved regions of the catalytic domain of plant MAPK homologue genes were used to study MAPK expression in those plant cells . In situ hybridization (ISH) showed the presence of MAPK transcripts in the three plant cell types studied, but levels were very low in quiescent cells compared to those in cycling cells . The quantification of labelling density of ISH signals strongly suggests a higher level of MAPK expression in proliferating cells, but also some basal messenger presence and/or expression in the quiescent ones . Immunogold and ISH results show the presence and distribution of MAPK proteins and mRNAs in vacuolate microspores . This represents a very dynamic stage during pollen development in which the cell nucleus is being prepared for an asymmetrical mitotic division, giving rise to both the generative and the vegetative nuclei of the bicellular pollen grain . Taken together, the data indicate a role played by MAPK in the re-entry into proliferation in plant cells.

Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 577 - 83
Molecular cloning, expression analysis, and chromosomal localization of human syntaxin 8 (STX8); Thoreau V et al.; We report the cloning of a cDNA encoding human syntaxin 8 (STX8), using the regulator (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library by the yeast two-hybrid system . This gene was found broadly transcribed and its mRNA size is about 1.3 kb . The STX8 gene maps to chromosomal band 17p12 and it encodes a 236-amino-acid protein . Syntaxin 8 contains in its C-terminal half a coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on target membrane) superfamily of proteins, which are involved in vesicular trafficking and docking . In syntaxin 8, a C-terminal hydrophobic domain may constitute a transmembrane anchor . It was recently shown that CFTR-mediated chloride currents can be regulated by syntaxin 1A, a t-SNARE family member, through direct protein-protein interaction . This raises the possibility that syntaxin 8 may also be involved in such regulations .

Genomics, 1999 Apr 15, 57(2), 209 - 18
Transcription map of Xq27: candidates for several X-linked diseases; Zucchi I et al.; Human Xq27 contains candidate regions for several disorders, yet is predicted to be a gene-poor cytogenetic band . We have developed a transcription map for the entire cytogenetic band to facilitate the identification of the relatively small number of expected candidate genes . Two approaches were taken to identify genes: (1) a group of 64 unique STSs that were generated during the physical mapping of the region were used in RT-PCR with RNA from human adult and fetal brain and (2) ESTs that have been broadly mapped to this region of the chromosome were finely mapped using a high-resolution yeast artificial chromosome contig . This combined approach identified four distinct regions of transcriptional activity within the Xq27 band . Among them is a region at the centromeric boundary that contains candidate regions for several rare developmental disorders (X-linked recessive hypoparathyroidism, thoracoabdominal syndrome, albinism-deafness syndrome, and Borjeson-Forssman-Lehman syndrome) . Two transcriptionally active regions were identified in the center of Xq27 and include candidate regions for X-linked mental retardation syndrome 6, X-linked progressive cone dystrophy, X-linked retinitis pigmentosa 24, and a prostate cancer susceptibility locus . The fourth region of transcriptional activity encompasses the FMR1 (FRAXA) and FMR2 (FRAXE) genes . The analysis thus suggests clustered transcription in Xq27 and provides candidates for several heritable disorders for which the causative genes have not yet been found .

Plant Physiol, 1999 Apr, 119(4), 1289 - 96
Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in catharanthus roseus
Menke FL, Parchmann S, Mueller MJ, Kijne JW, Memelink J.
Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells . We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract . In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid . The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes . The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression . Analysis of the expression of Str promoter/gusA fusions in transgenic C . roseus cells showed that the elicitor and MeJA act at the transcriptional level . These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

Electrophoresis, 1999 Feb, 20(2), 391 - 408
Protein kinase CK2 and its role in cellular proliferation, development and pathology; Guerra B et al.; Protein kinase CK2 is a pleiotropic, ubiquitous and constitutively active protein kinase that can use both ATP and GTP as phosphoryl donors with specificity for serine/threonine residues in the vicinity of acidic amino acids . Recent results show that the enzyme is involved in transcription, signaling, proliferation and in various steps of development . The tetrameric holoenzyme (alpha2beta2) consists of two catalytic alpha-subunits and two regulatory beta-subunits . The structure of the catalytic subunit with the fixed positioning of the activation segment in the active conformation through its own aminoterminal region suggests a regulation at the transcriptional level making a regulation by second messengers unlikely . The high conservation of the catalytic subunit from yeast to man and its role in the tetrameric complex supports this notion . The regulatory beta-subunit has been far less conserved throughout evolution . Furthermore the existence of different CK2beta-related proteins together with the observation of deregulated CK2beta levels in tumor cells and the reported association of CK2beta protein with key proteins in signal transduction, e.g . A-Raf, Mos, pg90rsk etc . are suggestive for an additional physiological role of CK2beta protein beside being the regulatory compound in the tetrameric holoenzyme.

J Mol Evol, 1999 May, 48(5), 555 - 64
Gene duplication and gene conversion in the Caenorhabditis elegans genome; Semple C et al.; A comprehensive analysis of duplication and gene conversion for 7394 Caenorhabditis elegans genes (about half the expected total for the genome) is presented . Of the genes examined, 40% are involved in duplicated gene pairs . Intrachromosomal or cis gene duplications occur approximately two times more often than expected . In general the closer the members of duplicated gene pairs are, the more likely it is that gene orientation is conserved . Gene conversion events are detectable between only 2% of the duplicated pairs . Even given the excesses of cis duplications, there is an excess of gene conversion events between cis duplicated pairs on every chromosome except the X chromosome . The relative rates of cis and trans gene conversion and the negative correlation between conversion frequency and DNA sequence divergence for unconverted regions of converted pairs are consistent with previous experimental studies in yeast . Three recent, regional duplications, each spanning three genes are described . All three have already undergone substantial deletions spanning hundreds of base pairs . The relative rates of duplication and deletion may contribute to the compactness of the C . elegans genome.

J Mol Evol, 1999 May, 48(5), 517 - 27
Diversification pattern of the HMG and SOX family members during evolution; Soullier S et al.; From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity . Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used . This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA . The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages . Particular emphasis was placed on the analysis of the SOX subfamily . For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18 . The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box . In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family . The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG . The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans.

Plant Physiol, 1999 Apr, 119(4), 1437 - 46
Arabidopsis Sec21p and Sec23p homologs . Probable coat proteins of plant COP-coated vesicles; Movafeghi A et al.; Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles . Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified . We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively) . The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat . Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell . The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins . The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins . Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes . This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes . Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems . Therefore, a further purification of the putative plant COP vesicles was undertaken.

Genes Dev, 1999 Apr 1, 13(7), 864 - 76
Specific isoforms of squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis; Norvell A et al.; Heterogeneous nuclear RNA-binding proteins, hnRNPs, have been implicated in nuclear export of mRNAs in organisms from yeast to humans . A germ-line mutation in a Drosophila hnRNP, Squid (Sqd)/hrp40, causes female sterility as a result of mislocalization of gurken (grk) mRNA during oogenesis . Alternative splicing produces three isoforms, SqdA, SqdB, and SqdS . Here we show that these isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in grk mRNA localization and protein accumulation, whereas SqdB cannot perform these functions . Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions . Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic . We show that this differential nuclear accumulation is correlated with a differential association with the nuclear import protein Transportin . Finally, we provide evidence that grk mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno . These results demonstrate the isoform-specific contributions of individual hnRNP proteins in the regulation of a specific mRNA . Moreover, these data suggest a novel role for hnRNPs in localization and translational regulation of mRNAs.

Genes Dev, 1999 Apr 1, 13(7), 841 - 50
Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing; Chua K et al.; The spliceosome catalyzes pre-mRNA splicing in two steps . After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II . Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7 . We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II . In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex . We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing . Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.

Cancer Res, 1999 Apr 1, 59(7 Suppl), 1747s - 1750s; discussion 1751s
Multiple roles for the Wilms' tumor suppressor, WT1; Davies R et al.; Wilms' tumor is a childhood kidney tumor that is a striking example of the way that cancer may arise through development gone awry . A proportion of these tumors develop as a result of the loss of function mutations in the Wilms' tumor suppressor gene, WT1 . Inherited mutations in the WT1 gene can lead to childhood kidney cancer, severe gonadal dysplasia, and life-threatening hypertension . Knockouts show that the gene is essential for the early stages of kidney and gonad formation . These tissues are completely absent in null mice . The WT1 gene encodes numerous protein isoforms, all of which share four zinc fingers . There is a large body of evidence supporting the notion that WT1 is a transcription factor, particularly a transcriptional repressor . Recently, however, we obtained evidence that WT1 colocalizes and is physically associated with splice factors . What is more, one alternative splice isoform of WT1 containing three amino acids, Lys-Thr-Ser (KTS; inserted between zinc fingers 3 and 4) is preferentially associated with splice factors, whereas the other alternative splice version, lacking these three amino acids, preferentially associates with the transcriptional apparatus . Both genetic and evolutionary considerations suggest that these two different forms of the protein have different functions . We will discuss recent evidence to further implicate WT1 in splicing . Our results raise the possibility that regulation of splicing is a crucial factor in the development of the genitourinary system, and that tumors may arise through aberrant splicing . To pursue the regulation and function of WT1 in whole animals, we have been introducing the human gene and large flanking regions cloned in yeast artificial chromosomes directly into mice . These studies have allowed us to dissect the function of WT1 at late as well as at early stages in organogenesis and to identify new sites and surprising new potential functions for the gene.

Gene, 1999 Apr 1, 230(1), 15 - 22
Characterisation of XlCdc1, a Xenopus homologue of the small (PolD2) subunit of DNA polymerase delta; identification of ten conserved regions I-X based on protein sequence comparisons across ten eukaryotic species; Reynolds N et al.; DNA polymerase delta (Pol delta), which plays keys roles in DNA replication, repair and recombination in eukaryotic cells, comprises at least two essential subunits - a large catalytic subunit (PolD1) possessing both DNA polymerase and 3'-5' exonuclease activities, and a smaller subunit (PolD2) whose function is not yet clear . Here we describe the cloning and sequencing of a Xenopus cDNA encoding a homologue of the PolD2 subunit . This protein (designated XlCdc1) is 69% identical to the human PolD2 protein and 34% identical to fission yeast Cdc1 . Alignment of PolD2 protein sequences across ten eukaryotic species identifies 36 invariant amino-acid positions . These 36 residues are located within ten conserved regions (designated I-X) likely to have key functional roles . Consistent with this, the mutations in six previously identified yeast mutant PolD2 proteins map within conserved regions III, VI, VII and VIII . Several of the invariant amino acids are also conserved across the archaeal DNA polymerase II DP1 protein family.

J Biol Chem, 1999 Apr 16, 274(16), 11369 - 75
A TATA element is required for tRNA promoter activity and confers TATA-binding protein responsiveness in Drosophila Schneider-2 cells; Trivedi A et al.; In contrast to yeast and mammalian systems, which depend principally on internal promoter elements for tRNA gene transcription, insect systems require additional upstream sequences . To understand the function of the upstream sequences, we have asked whether the Bombyx mori tRNACAla and tRNASGAla genes, which are absolutely dependent on these sequences in vitro, also require them for transcription in vivo . We introduced wild-type and mutant versions of the Bombyx tRNAAla genes into Drosophila Schneider-2 cells and found that the tRNACAla gene is efficiently transcribed and that its transcription depends strongly on the distal segment of its upstream promoter . In contrast, the tRNASGAla gene is inefficiently transcribed, and this inefficiency results from lack of a specific sequence within the distal tRNACAla upstream promoter . This sequence, 5'-TTTATAT-3', is sufficient to increase the activity of the tRNASGAla promoter to that of the tRNACAla promoter . Moreover, promoters containing the 5'-TTTATAT-3' element are stimulated by increased levels of cellular TATA-binding protein . Together these results indicate that, in insect cells, a TATA-like element is specifically required to form functional TATA-binding protein-containing complexes that promote efficient transcription of tRNA genes.

J Biol Chem, 1999 Apr 16, 274(16), 11334 - 8
Binding of CtIP to the BRCT repeats of BRCA1 involved in the transcription regulation of p21 is disrupted upon DNA damage; Li S et al.; Mutations in BRCA1 are responsible for nearly all of the hereditary ovarian and breast cancers, and about half of those in breast cancer-only kindreds . The ability of BRCA1 to transactivate the p21 promoter can be inactivated by mutation of the conserved BRCA1 C-terminal (BRCT) repeats . To explore the mechanisms of this BRCA1 function, the BRCT repeats were used as bait in a yeast two-hybrid screen . A known protein, CtIP, a co-repressor with CtBP, was found . CtIP interacts specifically with the BRCT repeats of BRCA1, both in vitro and in vivo, and tumor-derived mutations in this region abolished these interactions . The association of BRCA1 with CtIP was also abrogated in cells treated with DNA-damaging agents including UV, gamma-irradiation, and adriamycin, a response correlated with BRCA1 phosphorylation . The transactivation of the p21 promoter by BRCA1 was diminished by expression of exogenous CtIP and CtBP . These results suggest that the binding of the BRCT repeats of BRCA1 to CtIP/CtBP is critical in mediating transcriptional regulation of p21 in response to DNA damage.

J Biol Chem, 1999 Apr 16, 274(16), 11186 - 93
Identification of the APS protein as a novel insulin receptor substrate; Moodie SA et al.; In order to identify novel substrates involved in insulin receptor signaling, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the cytoplasmic domain of the human insulin receptor as bait . Here we describe the isolation and characterization of an interacting protein, APS, which contains pleckstrin homology and Src homology 2 domains and several potential tyrosine phosphorylation sites . APS mRNA and protein are expressed primarily in skeletal muscle, heart, and adipose tissue, and in differentiated 3T3-L1 adipocytes . We show that APS associates with phosphotyrosines situated within the activation loop of the insulin receptor via the APS Src homology 2 domain . Insulin stimulation of 3T3-L1 adipocytes resulted in rapid tyrosine phosphorylation of endogenous APS on tyrosine 618, whereas platelet-derived growth factor treatment resulted in no APS phosphorylation . In summary, we have identified a new insulin receptor substrate that is primarily expressed in insulin-responsive tissues and in 3T3-L1 adipocytes whose phosphorylation shows insulin receptor specificity . These findings suggest a potential role for APS in insulin-regulated metabolic signaling pathways.

J Biol Chem, 1999 Apr 16, 274(16), 10951 - 62
Alternative splicing determines the intracellular localization of the novel nuclear protein Nop30 and its interaction with the splicing factor SRp30c; Stoss O et al.; We report on the molecular cloning of a novel human cDNA by its interaction with the splicing factor SRp30c in a yeast two-hybrid screen . This cDNA is predominantly expressed in muscle and encodes a protein that is present in the nucleoplasm and concentrated in nucleoli . It was therefore termed Nop30 (nucleolar protein of 30 kDa) . We have also identified a related cDNA with a different carboxyl terminus . Sequencing of the NOP gene demonstrated that both cDNAs are generated by alternative 5' splice site usage from a single gene that consists of four exons, spans at least 1800 nucleotides, and is located on chromosome 16q21-q23 . The alternative 5' splice site usage introduces a frameshift creating two different carboxyl termini . The carboxyl terminus of Nop30 is rich in serines and arginines and has been found to target the protein into the nucleus, whereas its isoform is characterized by proline/glutamic acid dipeptides in its carboxyl terminus and is predominantly found in the cytosol . Interaction studies in yeast, in vitro protein interaction assays, and co-immunoprecipitations demonstrated that Nop30 multimerizes and binds to the RS domain of SRp30c but not to other splicing factors tested . Overexpression of Nop30 changes alternative exon usage in preprotachykinin and SRp20 reporter genes, suggesting that Nop30 influences alternative splice site selection in vivo.

J Biol Chem, 1999 Apr 16, 274(16), 10685 - 8
Interaction of heterotrimeric G protein Galphao with Purkinje cell protein-2 . Evidence for a novel nucleotide exchange factor; Luo Y et al.; The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined . To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system . Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system . Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed . Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice (Nordquist, D . T., Kozak, C . A., and Orr, H . T . (1988) J . Neurosci . 8, 4780-4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A . R., Feddersen, R . M., Nguyen, M . S., and Koller, B . H . (1997) Mol . Cell . Neurosci . 9, 63-76) . Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated {35S}methionine-labeled Galphao . In addition, when Galphao and Pcp2 were cotransfected into COS cells, Galphao was detected in immunoprecipitates of Pcp2 . To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2 . Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat . These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function.

J Biol Chem, 1999 Apr 16, 274(16), 10677 - 80
Identification of NSF as a beta-arrestin1-binding protein . Implications for beta2-adrenergic receptor regulation; McDonald PH et al.; Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane . Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions . We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells . beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF . In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding . Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization . Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization . The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.

Methods, 1999 Apr, 17(4), 275 - 86
Database search strategies used to isolate cell death proteins; Gururajan R et al.; The identification of proteins involved in the early phases of cell death has relied primarily on the modular organization of shared sequences and structural motifs of previously identified proteins in the apoptotic machinery . This property has facilitated the isolation of proteins that interact with each other through structural domains using yeast two-hybrid cloning . Likewise, the conservation in primary sequence of the various shared domains has promoted the use of polymerase chain reaction and database search strategies to isolate additional family members . Here, we discuss the use of database search strategies in the isolation of novel death proteins, as well as how similar strategies may be extended to discover additional, novel cell death proteins .

Mol Cell Endocrinol, 1999 Jan 25, 147(1-2), 103 - 12
CBP-dependent and independent enhancing activity of steroid receptor coactivator-1 in thyroid hormone receptor-mediated transactivation; Ikeda M et al.; Full-length of steroid receptor coactivator-1 (F-SRC-1) has been shown to interact with thyroid hormone receptors (TRs) in a ligand-dependent manner and to stimulate receptor-dependent transcription . To identify functional domains of F-SRC-1, several internal deletion mutants of F-SRC-1 were constructed . Although in vitro pull down assay with TR showed interaction of all of these mutants with TR, lack of mid legion (amino acids 398-1172) lost enhancing activity of TR-mediated transcription in a transient transfection assay . However, F-SRC-1 mutant lacking CBP-interacting domain still preserved enhancing activity . Surprisingly, F-SRC-1 mutants also increased basal level of viral promoter activity depending upon their deleted region . Yeast activation function assay revealed that these F-SRC-1 mutants had intrinsic activation function when bound to DNA . Analyses of small fragments of F-SRC-1 identified three separable activation domains . In vitro binding assay showed that TBP and TFIIB bound to C-terminal half of F-SRC-1 . These results suggest that F-SRC-1 can function via both CBP-dependent and independent manners using various sets of activation domains and that direct interactions between F-SRC-1 and TBP or TFIIB may not be important for CBP-independent transcription.

Mol Endocrinol, 1999 Apr, 13(4), 644 - 57
Expression cloning and characterization of PREB (prolactin regulatory element binding), a novel WD motif DNA-binding protein with a capacity to regulate prolactin promoter activity; Fliss MS et al.; Previous studies have implied that a transcription factor(s) other than Pit-1 is involved in homeostatic regulation of PRL promoter activity via Pit-1-binding elements . One such element, 1P, was employed to clone from a rat pituitary cDNA expression library a novel 417-amino acid WD protein, designated PREB (PRL regulatory element binding) protein . PREB contains two PQ-rich potential transactivation domains, but no apparent DNA-binding motif, and exhibits sequence-specific binding to site 1P, to a site nonidentical to that for Pit-1 . The PREB gene (or a related gene) is conserved, as an apparently single copy, in rat, human, fly, and yeast . A single approximately 1.9-kb PREB transcript accumulates in GH3 rat pituitary cells, to levels similar to Pit-1 mRNA . PREB transcripts were detected in all human tissues examined, but the observation of tissue-specific multiple transcript patterns suggests the possibility of tissue-specific alternative splicing . RT-PCR analysis of human brain tumor RNA samples suggested region-specific expression of PREB transcripts in brain . Western and immunocytochemical analysis implied that PREB accumulates specifically in GH3 cell nuclei . Transient transfection employing PREB-negative C6 rat glial cells showed that PREB is as active as, and additive with, Pit-1 in transactivation of a PRL promoter construct, and that PREB, but not Pit-1, can mediate transcriptional activation by protein kinase A (PKA) . Expression in GH3 cells of a GAL4-PREB fusion protein both strongly transactivated a 5XGAL indicator construct and yielded a further stimulation of expression of this construct by coexpressed PKA, implying that PREB can mediate both basal and PKA-stimulated transcriptional responses in pituitary cells . These observations imply that PREB will prove to play a significant transcriptional regulatory role, both in the pituitary and in other organs in which transcripts of its gene are expressed.

J Cell Sci, 1999 May, 112 ( Pt 9), 1303 - 11
Inhibition of clathrin-coated pit assembly by an Eps15 mutant; Benmerah A et al.; Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis . However, its precise function remains unknown . Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast . Results presented here show that EH domains are required for correct coated pit targeting of Eps15 . Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits . This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited . Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane . This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.

Biochemistry, 1999 Mar 30, 38(13), 4227 - 34
Effects of ion gradients on H+ transport mediated by human MDR 1 protein; Santai CT et al.; In the previous paper we presented a variety of data consistent with significant perturbations in 9.3 yeast plasma membrane ion transport upon overexpression of the hu MDR 1 protein . Thus, in this paper, we compare formation of DeltapH for inside-out yeast plasma membrane vesicles (ISOV) prepared from control 9.3/pVT versus 9.3/hu MDR 1 yeast . Since MDR 1 ATPase activity has a broader, more alkaline pH profile relative to endogenous yeast H+ ATPase activity, we analyzed H+ pumping at pH >/= 8.0 in detail in order to selectively amplify hu MDR 1 contributions to H+ movement over those of the endogenous yeast H+ ATPase . We observed: (1) imposition of a Cl- gradient oriented outside to in enhances acidification for 9.3/pVT ISOV (as expected), but decreases acidification for 9.3/hu MDR 1 ISOV; (2) imposition of a Cl- gradient oriented inside to out decreases acidification for 9.3/pVT ISOV (as expected) but enhances acidification for 9.3/hu MDR 1 ISOV; (3) a Na+ gradient oriented in the same direction as the Cl- gradient amplifies the effects due to hu MDR 1 when both gradients are oriented inside to out, but not outside to in . The data are most easily explained by interesting Na+, Cl-, and ATP-dependent H+ transport mediated by hu MDR 1 protein as previously suggested {Hoffman and Roepe (1997) Biochemistry 36, 11153-11168} . These data may help to resolve a variety of conflicting reports in the literature regarding ion transport mediated by hu MDR 1 and have implications for the physiology of a number of polarized epithelia in which hu MDR 1 is endogenously expressed.

Leuk Lymphoma, 1999 Mar, 33(1-2), 181 - 6
The mechanisms of death of an erythroleukemic cell line by p53: involvement of the microtubule and mitochondria; Kato MV; A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established . These cells showed typical characteristics of apoptosis, when they were cultured at 32 degrees C . In this process, p53 recovered the wild-type p53 function and the expression of the p21 (waf1/cip1/sdi1), cyclin G1 and gadd45 genes was increased . However, no significant changes were detected in the expression of the mdm2, bcl-2, bax, fas and fasl genes, suggesting the existence of other genes associated with apoptosis . Genes up-regulated by p53 were screened by the mRNA differential display method . One of the up-regulated genes was identified as the elongation factor 1 alpha (EF-1 alpha) gene . EF-1 alpha is also a microtubule-severing protein . Upon the temperature-shift, the cells developed the morphology and the localization of alpha-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules . The microtubule-severing associated with up-regulation of EF-1 alpha by p53 may be a cause of the cell death . On the other hand, the function of cyclin G1 is not so clear despite the fact that 1-2-3 cells showed a significant increase of the cyclin G1 gene during the early stage of apoptosis . The yeast two-hybrid system was used to identify cyclin G1-associated proteins . One is a cytochrome c (Cyt c) oxidase subunit II (COXII) . Cyclin G1 and COXII were co-immunoprecipitated from an extract of human osteosarcoma cell line that expressed high levels of cyclin G1 . COX activity was also increased by temperature-shift in this cell line . The pattern of changes in COX activity was closely reflected by the expression of the cyclin G1 gene . Cyclin G1 and COXII associate physically with each other in vivo and that activation of COXII by binding to cyclin G1 upregulated by p53 may be associated with apoptosis . These two new pathways, p53-EF-1 alpha-microtubule-severing (-distortion of cytoskeleton) and p53-cyclin G1-COXII (-CytC, ATP-caspase-3 activation), may cooperate to induce apoptosis in this cell line.

Leuk Lymphoma, 1999 Mar, 33(1-2), 119 - 26
CRKL binding to BCR-ABL and BCR-ABL transformation; Kolibaba KS et al.; The SH2-SH3 domain-containing adaptor protein CRKL is the predominant tyrosine phosphorylated protein in chronic myelogenous leukemia (CML) neutrophils and BCR-ABL-expressing cell lines . The amino terminal CRKL SH3 domain binds directly to a proline-rich region in the C-terminus of BCR-ABL . BCR-ABL mutants with deletions of this region were constructed to assess biologic effects of eliminating the CRKL binding site . Yeast two-hybrid analysis and gel overlay assays show eradication of the direct interaction of CRKL with BCR-ABL in the proline deletion mutants . However, these BCR-ABL mutants transform myeloid cells to growth factor independence, and in these cells CRKL is tyrosine phosphorylated and associates with BCR-ABL . These findings suggest both direct and indirect interactions of CRKL with BCR-ABL . Thus, disruption of the direct interaction with BCR-ABL has not excluded a role for CRKL in BCR-ABL-mediated transformation.

Nature, 1999 Mar 25, 398(6725), 344 - 8
Effects of altered gene order or orientation of the locus control region on human beta-globin gene expression in mice; Tanimoto K et al.; The five human beta-type-globin genes, epsilon, Ggamma, Agamma, delta and beta, are close together and are regulated by a locus control region (LCR) located at the 5' end of the locus . Here we investigate the functional consequences of this organization with respect to temporal regulation of the individual genes, by using recombination techniques to invert the order of either the genes or the LCR in vivo . Our analysis of transgenic mice bearing either normal or mutant transgenes leads to two new observations . First, the position of the epsilon-globin gene next to the LCR is mandatory for its expression during the yolk-sac stage of erythropoiesis . Second, LCR activity is orientation dependent, and so the LCR does not act as a simple enhancer to stimulate transcription of the globin genes . Thus, in the absence of any change in transgene integration position, transgene copy number, trans-acting factors or other resident genetic information, simple inversion of the human genes or the LCR fundamentally alters the transcription of beta-type globin genes.

Br J Haematol, 1999 Mar, 104(4), 665 - 71
Molecular characterization of deletion at 11q22.1-23.3 in mantle cell lymphoma; Monni O et al.; Chromosomal deletions at 11q21-23 have recently been reported to be common aberrations in mantle cell lymphoma (MCL) . To characterize the structure of the deletion, we studied 41 cases of MCL by fluorescence in situ hybridization using a YAC contig, which spans the region at 11q22.1-23.3 . 17 MCLs were studied using a set of 20 yeast artificial chromosomes (YACs) in a contig, and nine of these cases showed deletion of 11q22-23 . The deletion spanned several megabases in all but one case, where only YAC 755b11 at 11q23.1, covering approximately a 1.6 Mb of DNA, was deleted . Analysis of additional 24 MCLs with YAC 755b11 revealed the deletion in 49% of all cases (20/41) . The deleted region at 11q22.1-23.3 was discontinuous in five lymphomas and in the majority of the cases the distal breakpoint occurred between YACs 785e12 and 911f2 at 11q23.3 . We conclude that the deletion of 11q22-23 and particularly the deletion of YAC 755b11 are very common in MCL and may be important in the genesis or progression of the disease.

Virology, 1999 Apr 10, 256(2), 270 - 9
Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif; Liu L et al.; It has previously been reported that complementary-sense gene products of wheat dwarf virus (WDV), a geminivirus of the genus Mastrevirus that infects monocotyledonous plants, bind to human and maize retinoblastoma (Rb) protein . Rb proteins control cell-cycle progression by sequestering transcription factors required for entry into S-phase, suggesting that the virus modifies the cellular environment to produce conditions suitable for viral DNA replication . Using a yeast two-hybrid assay, we have investigated whether the complementary-sense gene products of bean yellow dwarf virus, a mastrevirus that is adapted to dicotyledonous plants, also bind maize Rb protein . We demonstrate that whereas RepA binds to Rb protein, Rep does not, suggesting that RepA alone regulates host gene expression and progression of cells to S-phase . RepA mutants containing L --> I, C --> S, C --> G, and E --> Q mutations within the consensus Rb protein binding motif LXCXE retained the ability to bind to Rb, but with reduced efficiency . Most notably, the E --> Q mutation reduced binding by approximately 95% . Nonetheless, all LXCXE mutants were able to replicate in tobacco protoplasts and to systemically infect Nicotiana benthamiana and bean, in which they produced wild-type symptoms .

Mol Genet Metab, 1999 Apr, 66(4), 373 - 5
Progress toward positional cloning of ovine neuronal ceroid lipofuscinosis, a model of the human late-infantile variant CLN6; Broom MF et al.; The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases with severe neurodegenerative pathology . An ovine model (OCL) has well-defined parallels with the human disease at the biochemical and pathological levels . The gene for OCL is located in the chromosomal region OAR7q13-15 . This region is syntenic with HAS15q21-23, suggesting that OCL and CLN6 represent mutations in orthologous genes . New microsatellite markers have been developed enabling further refinement of the OCL critical region .

Mol Genet Metab, 1999 Apr, 66(4), 367 - 72
Progress in neuropathology of the neuronal ceroid lipofuscinoses; Goebel HH et al.; Since the last, 6th, International Congress on Neuronal Ceroid-Lipofuscinoses, neuropathological advances in neuronal ceroid lipofuscinoses (NCL) have been made in several areas: (1) In adult NCL (ANCL) lipopigments have now been repeatedly confirmed to contain subunit c of mitochondrial ATP synthase and even sphingolipid activators (saposins) . ANCL lipopigments have also been confirmed in extracerebral tissues including skin, skeletal muscle, and spleen, but not yet lymphocytes (2) . Among circulating blood cells not only B cells and subclasses of T lymphocytes, i.e., CD4(+), CD8(+), and CD56 cells, but also monocytes have been found to contain NCL lipopigments, indicating that this precursor cell in the digesting macrophage system also has an impaired metabolic catabolism for lipopigments (3) . Immunohistochemical studies indicate that microglial reaction in NCL brain is limited to resident microglia without contribution by circulating monocytes (4) . The granular osmiophilic deposit (GROD) type of NCL has now been established not only in infantile, but also in late-infantile, juvenile, and protracted-juvenile NCL (5) . A European Tissue Registry established within the framework of a European Concerted Action on Neuronal Ceroid-Lipofuscinosis may form the basis for additional collaborative studies on NCL, including both biopsy and autopsy tissues .

Mol Genet Metab, 1999 Apr, 66(4), 339 - 43
Genetic heterogeneity of neuronal ceroid lipofuscinosis in The Netherlands; Taschner PE et al.; An overview of patients in the Netherlands who are known to us with neuronal ceroid lipofuscinosis (NCL) is presented . Several CLN genes involved in NCL have been isolated or mapped . We have analyzed families with different types of NCL with polymorphic markers linked to CLN loci to investigate the genetic heterogeneity of NCL in the Netherlands . Haplotype analysis suggests that in addition to the CLN2 and CLN6 genes another gene is involved in at least one family with late infantile NCL in the Netherlands . The CLN2 and CLN6 loci have also been excluded in a family with protracted juvenile NCL .

Mol Genet Metab, 1999 Apr, 66(4), 332 - 6
Progress toward the cloning of CLN6, the gene underlying a variant LINCL; Auger KJ et al.; Marked clinical heterogeneity is seen in the late-infantile subtype of NCL (LINCL), complicating genetic analysis . In addition to the classical subtype, encoded by CLN2 on chromosome 11p15.5, several variant subtypes have also been described . In this paper, we report our progress in cloning a variant LINCL gene mapped in a small group of Costa Rican families . Clinically, these patients appear similar to classical LINCL patients, except onset of the disease is delayed and the course is milder . Extended haplotype analysis confirms the localization of this gene to chromosome 15q21-22, where CLN6 has also been mapped . Using now-standard positional cloning techniques, we have developed a physical map of our candidate region . These clones have been used to order genetic markers, STSs, and ESTs in this region and will be used for the identification of the disease gene transcript .

Genomics, 1999 Apr 1, 57(1), 156 - 9
Identification of a zeta-crystallin (quinone reductase)-like 1 gene (CRYZL1) mapped to human chromosome 21q22.1; Kim MY et al.; To identify a new gene(s) located on the yeast artificial chromosome (YAC) clone D142H8 that was mapped to human chromosome 21q22.1, purified YAC DNA from the clone was utilized directly as a probe to screen a human brain cDNA library after the suppression of human repetitive DNA . One cDNA clone hybridizing specifically to the YAC D142H8 DNA was identified . The clone has an insert of 1341 bp and the longest open reading frame of 349 amino acids . A search of GenBank revealed that the clone has a high degree of homology to zeta-crystallin (quinone reductase) at the amino acid level, and its nucleotide sequence represents the expressed sequence from the 50-kb segment of the human chromosome 21q11.1 . Thus a new gene was named CRYZL1 (zeta-crystalline-like 1) . Genomic Southern blot with total human and yeast DNAs suggests that CRYZL1 might be a single-copy gene . The fluorescence in situ hybridization procedure was applied, and the results showed that the gene mapped to the human chromosome 21q22.1 subband . The CRYZL1 mRNA was expressed in heart, brain, skeletal muscle, kidney, pancreas, liver, and lungs but at different levels in different tissues .

Genomics, 1999 Apr 1, 57(1), 84 - 93
Genomic organization of the human galpha14 and Galphaq genes and mutation analysis in chorea-acanthocytosis (CHAC); Rubio JP et al.; Chorea-acanthocytosis (CHAC) (OMIM 200150) is a rare neurological syndrome characterized by neurodegeneration in combination with morphologically abnormal red cells (acanthocytosis) . A partial yeast artificial chromosome contig of the CHAC critical region on chromosome 9q21 has been constructed, and 21 expressed sequence tags have been mapped . We have subsequently cloned Galpha14, a member of the G-protein alpha-subunit multigene family, and have identified Galphaq in the contig . The genomic structure of both genes has been established after construction of a bacterial artificial chromosome contig that showed Galphaq and Galpha14 to be in a head-to-tail arrangement (Cen-Galphaq-Galpha14-qter) . Northern analysis found Galphaq to be ubiquitously expressed and Galpha14 to display a more restricted pattern of expression . Mutation analysis of the coding regions and splice sites for Galphaq and Galpha14 in 10 affected individuals from different families identified no changes likely to cause disease; however, two distinct single nucleotide polymorphisms in the coding region of Galpha14 have been identified . This study has excluded two plausible candidate genes from involvement in CHAC and has provided a solid platform for a positional cloning initiative .

Genomics, 1999 Apr 1, 57(1), 70 - 8
Closing in on the BPES gene on 3q23: mapping of a de Novo reciprocal translocation t(3;4)(q23;p15.2) breakpoint within a 45-kb cosmid and mapping of three candidate genes, RBP1, RBP2, and beta'-COP, distal to the breakpoint; De Baere E et al.; BPES is a genetic disorder presenting with blepharophimosis, ptosis of the eyelids, epicanthus inversus, and telecanthus . BPES type I is associated with female infertility, whereas type II presents without additional symptoms . Hitherto, it remains unknown whether BPES type I results from a defect in a single gene or from a contiguous gene syndrome . Previous cytogenetic and linkage analyses have assigned a BPES locus to 3q23, in a 5-cM interval between D3S1615 and D3S1316 . In this report, we describe the molecular and physical characterization of the 3q23 breakpoint in a BPES patient with a t(3;4)(q23;p15.2) translocation . Eight YACs located around and within the D3S1615-D3S1316 interval were mapped relative to the 3q23 breakpoint; 5 YACs spanning the 3q23 breakpoint were identified . Thirteen STSs and ESTs were localized on the YAC map . Subsequent hybridization of 2 YACs spanning the breakpoint to the Human RPCI1 PAC Library and the Human Chromosome 3 LLNL Cosmid Library resulted in the identification of 12 PACs and 50 cosmids respectively, allowing the construction of a detailed PAC and cosmid physical map . A refined position-telomeric to the breakpoint-of 3 candidate genes, cellular retinol-binding proteins 1 and 2 (RBP1, RBP2) and the coatomer beta' subunit (beta'-COP), was obtained on this physical map . Furthermore, a PAC and cosmid contig encompassing the breakpoint was constructed . PAC 169-C 10 and cosmid 11-L 10 crossing the breakpoint have sizes of 110 and 45 kb, respectively . The isolation of coding sequences in these clones and in the rest of the contig will greatly facilitate further efforts toward positional cloning of the gene(s) involved in BPES .

Genomics, 1999 Apr 1, 57(1), 24 - 35
A radiation hybrid breakpoint map of the acute myeloid leukemia (AML) and limb-girdle muscular dystrophy 1A (LGMD1A) regions of chromosome 5q31 localizing 122 expressed sequences; Horrigan SK et al.; We have constructed a high-resolution map of a 6-Mb interval of human chromosome 5, band q31, incorporating 175 sequence tagged sites, of which 33 are genetic polymorphisms and 122 are nonredundant expressed sequences . The map was assembled initially as a YAC contig, incorporating data from radiation hybrid maps . To improve resolution and to identify errors in the databases, a radiation hybrid breakpoint map was developed for the interval, which included hybrids from both Stanford G3 and GeneBridge 4 panels . This novel approach facilitated the integration of one RH panel with another and enabled the identification and localization of new, previously unmapped ESTs from the radiation hybrid databases . ESTs were assembled into overlapping transcription units and ordered with respect to polymorphic markers in the region, resulting in a comprehensive map that incorporates markers from multiple different types of maps . This map of 5q31 will facilitate gene discovery efforts for several disorders, including limb-girdle muscular dystrophy type 1A and the genes deleted in acute myeloid leukemias and myelodysplasia . The study demonstrates the utility of a radiation hybrid breakpoint panel for correction of map errors and for the efficient identification of new transcript units in a large genomic interval .

Plant Cell Physiol, 1999 Jan, 40(1), 119 - 23
Mapping of 25 drought-inducible genes, RD and ERD, in Arabidopsis thaliana; Taji T et al.; We mapped 25 Arabidopsis thaliana drought-inducible genes . Responsive to Dehydration (RD) and Early Responsive to Dehydration (ERD), to the Arabidopsis genome and compared map positions with those of mutants that show environmental stress response . We hybridized CIC yeast artificial chromosome (YAC) library filters with the cDNAs and determined the map positions of 18 corresponding genes . We screened the P1 library with 7 other clones and analyzed segregation of their restriction fragment length polymorphisms (RFLP) in recombinant inbred (RI) lines . One cDNA could be mapped because it had been sequenced by the Arabidopsis genome sequencing program.

J Biol Chem, 1999 Apr 9, 274(15), 10382 - 7
Repression of human fibroblast growth factor 2 by a novel transcription factor; Ueba T et al.; Here we describe the cloning of the regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) using a yeast one-hybrid screening with a defined motif in FGF-2 promoter as a target sequence . Overexpression of human RFT (RFT-A) reduces FGF-2 RNA and protein levels in both normal and tumor cell lines . Its splice variants, RFT-A' and RFT-B, have deletions in the putative DNA binding domain and fail to bind FGF-2 promoter and repress FGF-2 gene expression . The ratios of RFT isoforms differ between normal and tumor cells, with the splice variants dominating in tumor cells . Overexpression of RFT-A induces glioma cell death . Our data suggest that regulation of FGF-2 by RFT is important for cellular functions and may be impaired in certain tumors.

J Biol Chem, 1999 Apr 9, 274(15), 10277 - 86
Multiple distinct coiled-coils are involved in dynamin self-assembly; Okamoto PM et al.; Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes . Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission . In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers . In this study we sought to define self-assembly regions in dynamin . Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S . Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site . Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer . Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants . Self-association was also observed between different dynamin isoforms . Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition . In addition, our data strongly suggests a parallel model for dynamin subunit self-association.

J Biol Chem, 1999 Apr 9, 274(15), 10259 - 67
Spi-C, a novel Ets protein that is temporally regulated during B lymphocyte development; Bemark M et al.; A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa promoter kappaY element as a bait . The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C . However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain . Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C . Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappaY element reporter gene upon co-transfection of HeLa cells . Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages . Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.

J Biol Chem, 1999 Apr 9, 274(15), 10145 - 53
The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis; Liou ML et al.; The tumor necrosis factor receptor, p60 (TNF-R1), transduces death signals via the association of its cytoplasmic domain with several intracellular proteins . By screening a mammalian cDNA library using the yeast two-hybrid cloning technique, we isolated a ubiquitin-homology protein, DAP-1, which specifically interacts with the cytoplasmic death domain of TNF-R1 . Sequence analysis reveals that DAP-1 shares striking sequence homology with the yeast SMT3 protein that is essential for the maintenance of chromosome integrity during mitosis (Meluh, P . B., and Koshland, D . (1995) Mol . Biol . Cell 6, 793-807) . DAP-1 is nearly identical to PIC1, a protein that interacts with the PML tumor suppressor implicated in acute promyelocytic leukemia (Boddy, M . N., Howe, K., Etkin, L . D., Solomon, E., and Freemont, P . S . (1996) Oncogene 13, 971-982), and the sentrin protein, which associates with the Fas death receptor (Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C . F., Chang, H . M., and Yeh, E . T . (1996) J . Immunol . 157, 4277-4281) . The in vivo interaction between DAP-1 and TNF-R1 was further confirmed in mammalian cells . In transient transfection assays, overexpression of DAP-1 suppresses NF-kappaB/Rel activity in 293T cells, a human kidney embryonic carcinoma cell line . Overexpression of either DAP-1 or sentrin causes apoptosis of TNF-sensitive L929 fibroblast cell line, as well as TNF-resistant osteosarcoma cell line, U2OS . Furthermore, the dominant negative Fas-associated death domain protein (FADD) protein blocks the cell death induced by either DAP-1 or FADD . Collectively, these observations highly suggest a role for DAP-1 in mediating TNF-induced cell death signaling pathways, presumably through the recruitment of FADD death effector.

Cell, 1999 Mar 19, 96(6), 879 - 91
Transmembrane structure of an inwardly rectifying potassium channel; Minor DL Jr et al.; Inwardly rectifying potassium channels (K(ir)), comprising four subunits each with two transmembrane domains, M1 and M2, regulate many important physiological processes . We employed a yeast genetic screen to identify functional channels from libraries of K(ir) 2.1 containing mutagenized M1 or M2 domains . Patterns in the allowed sequences indicate that M1 and M2 are helices . Protein-lipid and protein-water interaction surfaces identified by the patterns were verified by sequence minimization experiments . Second-site suppressor analyses of helix packing indicate that the M2 pore-lining inner helices are surrounded by the M1 lipid-facing outer helices, arranged such that the M1 helices participate in subunit-subunit interactions . This arrangement is distinctly different from the structure of a bacterial potassium channel with the same topology and identifies helix-packing residues as hallmark sequences common to all K(ir) superfamily members.

Antimicrob Agents Chemother, 1999 Apr, 43(4), 836 - 45
High-frequency, in vitro reversible switching of Candida lusitaniae clinical isolates from amphotericin B susceptibility to resistance; Yoon SA et al.; Recent studies have revealed an increase in the incidence of serious infections caused by non-albicans Candida species . Candida lusitaniae is of special interest because of its sporadic resistance to amphotericin B (AmB) . The present in vitro study demonstrated that, unlike other Candida species, C . lusitaniae isolates frequently generated AmB-resistant lineages form previously susceptible colonies . Cells switching from a resistant colony to a susceptible phenotype were also detected after treatment with either UV light, heat shock, or exposure to whole blood, all of which increased the frequency of switching . In some C . lusitaniae lineages, after a cell switched to a resistant phenotype, the resistant phenotype was stable; in other lineages, colonies were composed primarily of AmB-susceptible cells . Although resistant and susceptible lineages were identical in many aspects, their cellular morphologies were dramatically different . Switching mechanisms that involve exposure to antifungals may have an impact on antifungal therapeutic strategies as well as on standardized susceptibility testing of clinical yeast specimens.

Scand J Immunol, 1999 Mar, 49(3), 293 - 301
Peripheral blood T-cell receptor beta-chain V-repertoire in atopic dermatitis patients after in vitro exposure to Pityrosporum orbiculare extract; Johansson C et al.; The yeast Pityrosporum orbiculare belongs to the normal cutaneous flora but is also considered to be one of the factors that may contribute to atopic dermatitis (AD) . In the present study we investigated the possibility that P . orbiculare can act with superantigen activity in AD . P . orbiculare-reactive T-cell lines (TCLs) were obtained after stimulation of peripheral blood mononuclear cells (PBMC) with P . orbiculare extract . T-cell receptor beta-chain V-segment (TCRBV) usage was investigated using monoclonal antibodies and flow cytometry . We could not find any difference in TCRBV usage between AD patients (n = 10) and healthy controls (n = 5), either in fresh PBMC or in P . orbiculare-reactive TCLs . Compared with their original PBMCs the P . orbiculare-reactive TCLs showed a decreased usage of several TCRBVs, although increased usage of certain TCRBVs could be seen in some of the individuals . Further analysis of the CDR3-length polymorphism exhibited a shift in CDR3-length distribution, indicating oligoclonal expansion of T cells specific to different antigens in the P . orbiculare extract . In conclusion we have not found any evidence for superantigen activity in P . orbiculare extract, but our data support the importance of classical major histocompatibility complex (MHC)-restricted allergens in P . orbiculare.

Mol Cells, 1999 Feb 28, 9(1), 84 - 90
Characterization of two new channel protein genes in Arabidopsis; Pih KT et al.; Aquaporins, small channel proteins, found in a variety of organisms are members of the major intrinsic protein (MIP) superfamily and have been shown to facilitate water transport when expressed in Xenopus oocytes . We isolated two Arabidopsis cDNAs, SIMIP and SITIP, that encode protein homologues of the MIP superfamily . SIMIP exhibits a high degree of sequence homology to PIP3 and MIP1, and thus may belong to the plasmamembrane intrinsic protein (PIP) subfamily, whereas salt-stress inducible tonoplast intrinsic protein (SITIP) is highly homologous to VM23 and gamma-TIP, and therefore may belong to the TIP subfamily . Expression studies revealed that the two genes showed a different expression pattern . The SIMIP gene was expressed in a tissue-specific manner, for example, its highest transcript level is found in flowers, relatively low levels in siliques, and very low level in leaves and roots . In contrast, SITIP was expressed in nearly equal amounts in all the tissues we examined . Also, the expression of SIMIP and SITIP showed a temporal regulation pattern . For example, the highest expression level was at 1 week after germination . In addition, the transcript levels of SIMIP and SMTIP were increased upon NaCl and ABA treatments . The biological function of the 2 genes were investigated using two NaCl stress-sensitive yeast mutant strains . The mutant yeast cells expressing these 2 genes were more resistant to high NaCl conditions . The results suggest that the proteins encoded by these genes may be involved in the osmoregulation in plants under high osmotic stress such as under a high salt condition.

Genetics, 1999 Apr, 151(4), 1517 - 29
Trans-acting factors required for inclusion of regulated exons in the Ultrabithorax mRNAs of Drosophila melanogaster; Burnette JM et al.; Alternatively spliced Ultrabithorax mRNAs differ by the presence of internal exons mI and mII . Two approaches were used to identify trans-acting factors required for inclusion of these cassette exons . First, mutations in a set of genes implicated in the control of other alternative splicing decisions were tested for dominant effects on the Ubx alternative splicing pattern . To identify additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested first for dominant enhancement of the haploinsufficient Ubx haltere phenotype and second for effects on the splicing pattern . Inclusion of the cassette exons in Ubx mRNAs was reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing . Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck . The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p . Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII . Possible roles of identified factors are discussed in the context of the resplicing strategy for generation of alternative Ubx mRNAs.

Development, 1999 May, 126(9), 1845 - 57
YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis; Moore AW et al.; The Wilms' Tumour gene WT1 has important functions during development . Knock-out mice were shown to have defects in the urogenital system and to die at embryonic day E13.5, probably due to heart failure . Using a lacZ reporter gene inserted into a YAC construct, we demonstrate that WT1 is expressed in the early proepicardium, the epicardium and the subepicardial mesenchymal cells (SEMC) . Lack of WT1 leads to severe defects in the epicardial layer and a concomitant absence of SEMCs, which explains the pericardial bleeding and subsequent embryonic death observed in Wt1 null embryos . We further show that a human-derived WT1 YAC construct is able to completely rescue heart defects, but only partially rescues defects in the urogenital system . Analysis of the observed hypoplastic kidneys demonstrate a continuous requirement for WT1 during nephrogenesis, in particular, in the formation of mature glomeruli . Finally, we show that the development of adrenal glands is also severely affected in partially rescued embryos . These data demonstrate a variety of new functions for WT1 and suggest a general requirement for this protein in the formation of organs derived from the intermediate mesoderm.

Biotechnol Bioeng, 1999 Feb 5, 62(3), 324 - 35
Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium; Nyberg GB et al.; Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function . Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured . Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates . These digests typically contain large amounts of peptides, which may be utilized as amino acids . This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium . A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements . Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical "consistency index." The consistency index tests whether redundancies are satisfied within expected experimental error . For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account .

Biotechnol Bioeng, 1998 Nov 5, 60(3), 277 - 82
A pH-controlled fed-batch process can overcome inhibition by formate in NADH-dependent enzymatic reductions using formate dehydrogenase-catalyzed coenzyme regeneration; Neuhauser W et al.; The NAD-dependent, formate dehydrogenase-catalyzed oxidation of formate anion into CO2 is known as the method for the regeneration of NADH in reductive enzymatic syntheses . Inhibition by formate and inactivation by alkaline pH-shift that occurs when oxidation of formate is carried out at pH approximately 7.0 may, however, hamper the efficient application of this NADH recycling reaction . Here, we have devised a fed-batch process using pH-controlled feeding of formic acid that can overcome enzyme inhibition and inactivation . The reaction pH is thus kept constant by addition of acid, and formate dehydrogenase is supplied continuously with substrate as required, but the concentration of formate is maintained at a constant, non- or weakly inhibitory level throughout the enzymatic conversion, thus enabling a particular NADH-dependent dehydrogenase to operate stably and at high reaction rates . For xylitol production from xylose using yeast xylose reductase (Ki,Formate 182 mM), a fed-batch conversion of 0.5M xylose yielded productivities of 2.8 g (L h)-1 that are three-fold improved when contrasted to a conventional batch reaction that employed equal initial concentrations of xylose and formate .

Biotechnol Bioeng, 1998 Sep 5, 59(5), 647 - 50
Nonflocculent versus total biomass ratio as a criterion for starting the biomass separation process
Podgornik A, Koloini T, Raspor P.
We introduce the ratio of nonflocculent versus total biomass as a criterion for starting cell separation from the medium . This criterion can be applied for the automation of the process regardless of the process dynamics . Its minimum indicates the optimum period of time for the start of the separation process with regard not only to nonflocculent cell concentration, but also medium attributes . In contrast to the concentration of nonflocculent cells, which has two minima, first at the beginning of the process and another broader one in the period during which maximum flocculation is present, the ratio has a single minimum and can therefore be implemented as a criterion for cell separation . To calculate the ratio value, in addition to an on-line method for nonflocculent biomass measurement described elsewhere, an on-line method for the total biomass of flocculent yeast is proposed . It is based on the absorbency measurement of the cell biomass, previously deflocculated by EDTA . Therefore, it can be applied in bioprocesses with transparent media and yeast that can be deflocculated by EDTA .

Biotechnol Bioeng, 1998 Sep 5, 59(5), 595 - 604
Secondary metabolite scale-up to minimize homolog impurity levels
Junker B, Reddy J, Olewinski R, Gailliot P, Byrne K, Gbewonyo K.
A mutant strain of Streptomyces hygroscopicus was found to produce up to 9.0 units/L of an immunoregulant precursor, immunomycin, with up to 3.5% of a lower homolog impurity under either dual fed-batch or batch conditions . Glycerol and valine were key nutrients influencing productivity and impurity levels . Soybean oil was successfully substituted for glycerol as a carbon source to minimize shot additions to batch culture . The remainder of the production medium was composed largely of defined components with the exception of yeast extract . Valine limitation increased lower homolog formation while decreasing higher homolog formation; excess valine decreased lower homolog formation below 2-3% while increasing higher homolog formation . Higher homolog formation in the presence of valine seemed to be slower than lower homolog formation in the absence of valine . Valine was believed to be the major butyrate precursor; consequently its availability influenced the impurity profile . A preliminary cost analysis suggests that elimination of added valine from the cultivation and replacement of glycerol with soybean oil can result in a 6.6-fold reduction in media costs relative to the original fed-batch process .

FEBS Lett, 1999 Mar 5, 446(1), 189 - 93
The BAH (bromo-adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation; Callebaut I et al.; Using sensitive methods of sequence analysis including hydrophobic cluster analysis, we report here a hitherto undescribed family of modules, the BAH (bromo-adjacent homology) family, which includes proteins such as eukaryotic DNA (cytosine-5) methyltransferases, the origin recognition complex 1 (Orc1) proteins, as well as several proteins involved in transcriptional regulation . The BAH domain appears to act as a protein-protein interaction module specialized in gene silencing, as suggested for example by its interaction within yeast Orc1p with the silent information regulator Sir1p . The BAH module might therefore play an important role by linking DNA methylation, replication and transcriptional regulation.

FEBS Lett, 1999 Mar 5, 446(1), 35 - 9
Interaction of tissue transglutaminase with nuclear transport protein importin-alpha3; Peng X et al.; Tissue transglutaminase is a multifunctional enzyme which has been involved in the regulation of cell growth, differentiation, and apoptosis . Recently, nuclear localization of tTG has been reported indicating the potential of active nuclear transport . In this study we use the yeast two-hybrid assay and co-immunoprecipitation to show that tTG interacts with the nuclear transport protein importin-alpha3 . Using electron microscopy we demonstrate that nuclear expression of tTG in a non-small cell lung cancer cell line is induced by retinoic acid (RA) . These data suggest that importin-alpha3 could mediate active nuclear transport of tTG which may be important for the regulation of critical cellular processes.

Chem Biol, 1999 Apr, 6(4), 221 - 35
The identification of myriocin-binding proteins; Chen JK et al.; BACKGROUND: Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2) and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis . Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography . RESULTS: Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays . Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates . Analyses of these polypeptides establish conclusively that they are murine LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis . CONCLUSION: The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides.

Trends Genet, 1999 Feb, 15(2), 51 - 8
How the worm was won . The C . elegans genome sequencing project; Wilson RK; The genome sequence of the free-living nematode Caenorhabditis elegans is nearly complete, with resolution of the final difficult regions expected over the next few months . This will represent the first genome of a multicellular organism to be sequenced to completion . The genome is approximately 97 Mb in total, and encodes more than 19,099 proteins, considerably more than expected before sequencing began . The sequencing project--a collaboration between the Genome Sequencing Center in St Louis and the Sanger Centre in Hinxton--has lasted eight years, with the majority of the sequence generated in the past four years . Analysis of the genome sequence is just beginning and represents an effort that will undoubtedly last more than another decade . However, some interesting findings are already apparent, indicating that the scope of the project, the approach taken, and the usefulness of having the genetic blueprint for this small organism have been well worth the effort.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3969 - 74
MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1; Bellacosa A et al.; The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity . To identify novel human genes that may function in MMR, we employed the yeast interaction trap . Using the MMR protein MLH1 as bait, we cloned MED1 . The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity . Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI) . These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH . In addition, these results suggest that cytosine methylation may play a role in human DNA repair.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3836 - 41
Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain; Lee JT et al.; Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic) . In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing . It remains unclear what elements make up the Xic . We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation . To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning . We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes . Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding . The autosomes became late-replicating and hypoacetylated on histone H4 . A deletion of the Xist 5' sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells . Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing . Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

Zentralbl Bakteriol, 1999 Feb, 289(1), 63 - 77
Inflammatory and immunopharmacological activities of meta-periodate oxidized zymosan; Ohno N et al.; Zymosan (ZYM), a strong complement-activating yeast cell preparation composed mainly of mannan and beta-glucan moieties, is a potent inflammatory substance with immunopharmacological activity . We previously analyzed the metabolism of ZYM in mice and found that it was deposited in liver and spleen for at least several months and then gradually oxidatively degraded . In this paper, we prepared oxidized ZYM by sodium metaperiodate oxidation (NaIO4) and borohydride reduction (I/B-ZYM) and/or limited hydrolysis of oxidized moieties (I/B/H-ZYM) . Activities of the resulting products were assessed by (i) vascular permeability in mice, (ii) H2O2 synthesis by macrophages, (iii) TNF-alpha synthesis by macrophages, and (iv) reactivity to anti-ZYM sera . As a general trend, NaIO4, oxidation products exhibited reduced, but still significant, activity . Interestingly, the H2O2 production induced by I/B/H-ZYM was significantly reduced after extensive sonication . Antagonist(s) for H2O2 synthesis were concomitantly solubilized by sonication of I/B/H-ZYM . On the contrary, TNF-alpha production induced by I/B/H-ZYM was comparable with that of ZYM . These facts strongly suggest that highly branched 1,3-beta- and 1,6-beta-glucosidic linkages resistant to NaIO4 oxidation are important for biological activity of ZYM . Further, the minimal structure in ZYM necessary for biological activity may depend on the activity tested.

Gene, 1999 Mar 18, 229(1-2), 215 - 21
Interaction of the p23/p198 protein with ErbB-3; Yoo JY et al.; The processes by which the kinase inactive receptor ErbB-3 transmits the signals of its ligand, heregulin (HRG), are incompletely understood . We used a yeast two-hybrid system to identify ErbB-3 interacting proteins that may participate in HRG signal transduction . We found that the protein p23, the human homolog of the mouse transplantation antigen P198, interacted with the cytoplasmic domain of ErbB-3 in the yeast two-hybrid system . P23 bound the 26-amino-acid juxtamembrane domain of ErbB-3 in vitro . The N-terminal end of p23 contained the ErbB-3 interacting region . P23 also bound to ErbB-3 in a human breast cell line . Two p23 mRNA transcripts were detected in normal human epithelial tissues including those of the heart, placenta, lung, brain, kidney, pancreas, skeletal muscle, and liver . These same transcripts were also detected in ErbB-3 overexpressing human tumor cell lines derived from breast and lung carcinomas, and a sarcoma . Transfection of p23 resulted in suppression of colony formation of the ErbB-3 overexpressing human breast cancer cell line, AU565, a decreased rate of cell growth, and induction of differentiation . The interaction of ErbB3 and p23 may play a role in regulation of proliferation of ErbB-3 expressing cells.

Gene, 1999 Mar 18, 229(1-2), 193 - 201
YAC fragmentation with repetitive and single-copy sequences: detailed physical mapping of the presenilin 1 gene on chromosome 14; Del-Favero J et al.; We constructed new LYS2 fragmentation vectors that allow direct acentric and centric fragmentation of yeast artificial chromosomes (YACs) and selection of fragmented YACs in yeast strain AB1380 . The fragmentation vectors were used efficiently with repetitive (e.g., Alu), low-copy (e.g., CA-repeats) and single-copy (e.g., exons) sequences . High recombination efficiencies were obtained in fragmenting two different CEPH YACs with the Alu consensus sequence as target sequences for homologous recombination . Analysis of the acentric Alu fragmentation panel of 788H12, containing the presenilin 1 (PSEN1) gene for familial Alzheimer's disease (AD), indicated that high-resolution YAC fragmentation panels covering the entire parent YAC are obtained . Also, marker content analysis of the fragmentation panel indicated that fragmented YACs were propagated stably without rearrangements . The same fragmentation vectors were used efficiently for fragmentation of 788H12 with unique sequences, i.e., exons 3 and 12 of PSEN1 and D14S77, a polymorphic CA repeat, as target sequences . Together, our YAC fragmentation data of 788H12 provided a size estimate for the coding region of PSEN1 of 60kb and a more precise localization of D14S77 at 25kb upstream of PSEN1.

Gene, 1999 Mar 18, 229(1-2), 109 - 16
Zimp encodes a homologue of mouse Miz1 and PIAS3 and is an essential gene in Drosophila melanogaster; Mohr SE et al.; The related mouse proteins Miz1 and PIAS3, which have predicted zinc finger domains, interact with the transcription factors Msx2 and STAT3, modulating the ability of Msx2 and STAT3 to regulate transcription . Here, we describe a Drosophila gene, zimp, that encodes a protein with similarity to Miz1 and PIAS3 . The zimp gene appears to be post-transcriptionally regulated, as three alternatively spliced forms are detected in a cDNA library screen and on an RNA blot . In addition, all three zimp transcripts are detected in embryonic mRNA, but only two of the transcripts are detected in adult mRNA . The three transcripts have the ability to encode two proteins, of 554 and 522 amino acids . The two Zimp amino acid sequences share an amino-terminal 515-amino-acid region and differ in their carboxy-termini . These proteins and related proteins in other organisms, including mammals, C . elegans, yeast, and plants, share a highly conserved region predicted to form a zinc finger . Deletion of the zimp gene or P-element insertion in zimp is lethal; thus, zimp is an essential gene in Drosophila . These data underscore the potential importance of Zimp-related proteins cross-species, and conservation of the putative zinc finger domain suggests that it is functionally important.

Anal Biochem, 1999 Apr 10, 269(1), 10 - 6
Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein; Hosfield T et al.; We have studied the cleavage efficiency of the protease enterokinase (EK) using the novel vector pESP4 . pESP4 is a yeast expression vector equipped with ligation-independent cloning sites, a GST purification tag, and a FLAG epitope tag . EK is used to cleave the FLAG and GST tags leaving the protein of interest without any extraneously added amino acids . We have found that EK is relatively permissive of the amino acid residue downstream of the recognition sequence (the P'1 position) . This makes EK an ideal choice to use as a protease to cleave any protein of interest cloned within the pESP4 yeast expression vector .

FEBS Lett, 1999 Feb 26, 445(2-3), 366 - 70
Modelling of a voltage-dependent Ca2+ channel beta subunit as a basis for understanding its functional properties; Hanlon MR et al.; Structure prediction methods have been used to establish a domain structure for the voltage-dependent calcium channel beta subunit, beta1b . One domain was identified from homology searches as an SH3 domain, whilst another was shown, using threading algorithms, to be similar to yeast guanylate kinase . This domain structure suggested relatedness to the membrane-associated guanylate kinase protein family, and that the N-terminal domain of the beta subunit might be similar to a PDZ domain . Three-dimensional model structures have been constructed for these three domains . The extents of the domains are consistent with functional properties and mutational assays of the subunit, and provide a basis for understanding its modulatory function.

FEBS Lett, 1999 Feb 26, 445(2-3), 351 - 5
Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains; Wixler V et al.; The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms . Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1 . In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit . In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified . Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits . The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins . They, therefore, may be involved in the regulation of general functions of integrins.

Mol Cell Biochem, 1999 Jan, 191(1-2), 129 - 34
Protein kinase CK2alpha may induce gene expression but unlikely acts directly as a DNA-binding transcription-activating factor; Ackermann K et al.; The gene-inducing property ofCK2alpha, a Ser/Thr protein kinase that appears normally to be complexed to a CK2beta protein controlling activity and substrate selectivity, has been unclear . We show here that CK2alpha induces in human JEG-3 cells the expression of Aromatase, an estrogen-synthesis key enzyme, which is regulated at transcriptional level . Electrophoretic mobility shift assays indicate that CK2alpha binds to the Aromatase gene promoter . To test for CK2alpha's transactivating ability as a DNA-binding protein, a CK2alpha binding site was cloned in front of indicator genes . The constructs were used to transform a yeast-based one-hybrid system . Overexpression of activation-domain fused CK2alpha in this system, i.e., CK2alpha in its native configuration, failed to activate the transcription machinery . The data indicate CK2alpha to affect gene expression at the level of transcription via an indirect as yet unknown mechanism rather than directly as a DNA-binding transcription-activating protein.

Eur J Hum Genet, 1999 Jan, 7(1), 77 - 87
Lamellar ichthyosis: further narrowing, physical and expression mapping of the chromosome 2 candidate locus; Parmentier L et al.; Lamellar ichthyosis (LI) is an autosomal recessive genodermatosis which has been shown to be both clinically and genetically heterogeneous . Keratinocyte transglutaminase (or transglutaminase 1: TGM1) has been demonstrated to be the disease-causing gene in some families, whilst in others, a second unidentified LI gene was mapped to chromosome 2q33-35 (ICR2B locus) . In this study, we present a physical map that encompasses the ICR2B locus, including the mapping of new microsatellite markers . Based on this new map, genotyping additional families highly suggests a reduction in size of the candidate interval . The final interval is covered by a single yeast artificial chromosome (937-H-3) which is 2.2Mb in length . Fine mapping of potential candidate transcripts was also focused on this region.

Eur J Hum Genet, 1999 Jan, 7(1), 68 - 76
Molecular cytogenetic detection of 9q34 breakpoints associated with nail patella syndrome; Silahtaroglu A et al.; The nail patella syndrome (NPS1) is an autosomal dominant disorder characterised by dysplasia of the finger nails and skeletal abnormalities . NPS1 has been mapped to 9q34, to a 1 cM interval between D9S315 and the adenylate kinase gene (AK1) . We have mapped the breakpoints within the candidate NPS1 region in two unrelated patients with balanced translocations . One patient {46,XY,t(1;9)(q32.1;q34)} was detected during a systematic survey of old cytogenetic files in Denmark and southern Sweden . The other patient {46,XY,t(9;17)(q34.1;q25)} was reported previously . D9S315 and AK1 were used to isolate YACs, from which endclones were used to isolate PACs . Two overlapping PAC clones span the 9q34 breakpoints in both patients, suggesting that NPS1 is caused by haploinsufficiency due to truncation or otherwise inactivation of a gene at or in the vicinity of the breakpoints.

J Mol Evol, 1999 Mar, 48(3), 291 - 302
Novel predicted RNA-binding domains associated with the translation machinery; Aravind L et al.; Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis . A small domain consisting of 60-65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation . Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases . Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors . Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes . We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes . The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions.

Eur J Biochem, 1999 Feb, 259(3), 762 - 9
Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin; Knight A et al.; Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system . The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin . The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo . We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine {poly(K)} to enable binding of DNA . We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line) . Transfection was dependent on HC and was neuronal cell type specific . HC may prove a useful targeting ligand for future neuronal gene therapy.

J Biol Chem, 1999 Apr 2, 274(14), 9847 - 53
Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain; Nishiya N et al.; The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains . Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts . We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5 . These two proteins are associated in mammalian cells . From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3 . As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays . Paxillin also binds to the Pro-2 domain of PTP-PEST . In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.

J Biol Chem, 1999 Apr 2, 274(14), 9744 - 51
The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D; Schurmann A et al.; ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF) . In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200) . ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay . Similarly, full-length {35S}mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity . Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy . Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes . Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes . These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.

J Biol Chem, 1999 Apr 2, 274(14), 9378 - 85
HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus; Lambert H et al.; Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively . Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers . Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers . Ser90 phosphorylation was sufficient and necessary for causing this change in structure . Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15 . Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions . One domain was insensitive to phosphorylation and corresponded to the C-terminal alpha-crystallin domain . The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein . Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation . A deletion within this domain of residues Arg5-Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers . We propose that HSP27 forms stable dimers through the alpha-crystallin domain . These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain.

J Biol Chem, 1999 Apr 2, 274(14), 9141 - 8
Direct interaction of Alzheimer's disease-related presenilin 1 with armadillo protein p0071; Stahl B et al.; Alzheimer's disease-related presenilins are thought to be involved in Notch signaling during embryonic development and/or cellular differentiation . Proteins mediating the cellular functions of the presenilins are still unknown . We utilized the yeast two-hybrid system to identify an interacting armadillo protein, termed p0071, that binds specifically to the hydrophilic loop of presenilin 1 . In vivo, the presenilins constitutively undergo proteolytic processing, forming two stable fragments . Here, we show that the C-terminal fragment of presenilin 1 directly binds to p0071 . Nine out of 10 armadillo repeats in p0071 are essential for mediating this interaction . Since armadillo proteins, like beta-catenin and APC, are known to participate in cellular signaling, p0071 may function as a mediator of presenilin 1 in signaling events.

Genes Chromosomes Cancer, 1999 Apr, 24(4), 315 - 21
Molecular cytogenetic delineation of the breakpoint at 18q21.1 in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue; Akagi T et al.; Extranodal malignant non-Hodgkin lymphoma of mucosa-associated lymphoid tissue type (MALT lymphoma) represents a subtype of B-cell lymphoid malignancies with distinct clinicopathological features and is often associated with a favorable prognosis . Recent cytogenetic studies have revealed that t(11;18)(q21;q21) is a characteristic chromosomal aberration in low-grade B-cell MALT-type lymphoma . In the present study, we employed florescence in situ hybridization analysis using contiguous YAC clones mapped to the 18q21.1 region to identify a YAC clone, y789F3, encompassing the breakpoint of t(11;18)(q21;q21) in a MALT lymphoma . PI artificial chromosome (PAC) contigs constructed on this YAC clone were used to analyze the breakpoint region . PAC clone 264m4 was observed on normal chromosome 18 and on der(18), and PAC clone 879n 10 on normal chromosome 18 and on der(II), confirming that the breakpoint is located between these two PAC clones . We also found that a region of approximately 500 kb between the two PAC clones was deleted . These results indicate that the locus between PAC clones 264m4 and 879n 10 at 18q21.1 involved in t(11;18) translocation or associated deletion plays an important role in the development of MALT lymphoma.

Eur J Biochem, 1999 Feb, 260(1), 127 - 36
Molecular characterization of the B' regulatory subunit gene family of Arabidopsis protein phosphatase 2A; Haynes JG et al.; Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a diverse array of reversible protein phosphorylation events in plants . We have identified a novel Arabidopsis gene (AtB' delta) which encodes a 55-kDa B' type regulatory subunit of PP2A . The protein encoded by this gene is 57-63% identical and 69-74% similar to the previously identified AtB' genes . The AtB' delta gene appears to be expressed in all Arabidopsis organs indicating its protein product has a basic housekeeping function in plant cells . Unlike certain mRNAs derived from the AtB' gamma gene, AtB' delta mRNAs do not fluctuate significantly in response to heat stress . Further analysis of cDNA sequences derived from the AtB' genes identified an alternatively spliced cDNA derived from AtB' gamma . This cDNA differs from the previously identified AtB' gamma cDNA by the absence of a 133-bp region in its 5' untranslated region . The missing 133-bp region appears to constitute an unspliced intron and its presence in the AtB' gamma gene was confirmed by PCR using Arabidopsis genomic DNA as a template . AtB' gamma mRNA containing the 133-bp intron accumulate in all Arabidopsis organs and their levels fluctuate differentially in response to heat stress . The 133-bp insert contains two short open reading frames and hence might serve as a translational control mechanism affecting AtB' gamma protein synthesis . Finally we show, using both the yeast two hybrid system and in vitro binding assays, that the B' subunit of Arabidopsis PP2A is able to associate with other PP2A subunits, supporting the notion that the B' protein serves as a regulator of PP2A activity in plants.

Am J Hum Genet, 1999 Apr, 64(4), 1119 - 26
Delineation of the critical deletion region for congenital heart defects, on chromosome 8p23.1; Devriendt K et al.; Deletions in the distal region of chromosome 8p (del8p) are associated with congenital heart malformations . Other major manifestations include microcephaly, intrauterine growth retardation, mental retardation, and a characteristic hyperactive, impulsive behavior . We studied genotype-phenotype correlations in nine unrelated patients with a de novo del8p, by using the combination of classic cytogenetics, FISH, and the analysis of polymorphic DNA markers . With the exception of one large terminal deletion, all deletions were interstitial . In five patients, a commonly deleted region of approximately 6 Mb was present, with breakpoints clustering in the same regions . One patient without a heart defect or microcephaly but with mild mental retardation and characteristic behavior had a smaller deletion within this commonly deleted region . Two patients without a heart defect had a more proximal interstitial deletion that did not overlap with the commonly deleted region . Taken together, these data allowed us to define the critical deletion regions for the major features of a del8p.

Science, 1999 Mar 26, 283(5410), 2089 - 91
Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A; Seeling JM et al.; Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies . The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin . A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system . Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants . The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors . B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.

Eur J Biochem, 1999 Feb, 260(1), 208 - 16
Delta 9-fatty acid desaturase from arachidonic acid-producing fungus . Unique gene sequence and its heterologous expression in a fungus, Aspergillus; Sakuradani E et al.; Based on the sequence information for delta 9-desaturase genes (from rat, mouse and yeast), which are involved in the desaturation of palmitic acid and stearic acid to palmitoleic acid and oleic acid, respectively, the corresponding cDNA and genomic gene were cloned from the fungal strain, Mortierella alpina 1S-4, which industrially produces arachidonic acid . There was a cytochrome b5-like domain linked to the carboxyl terminus of this Mortierella desaturase, as also seen in the yeast delta 9-desaturase . The Mortierella delta 9-desaturase genomic gene had only one intron, in which a novel phenomenon was observed: there was a GC-end at the 5'-terminus instead of a GT-end that is, in general, found in introns of eukaryotic genes . The full-length cDNA clone was expressed under the control of an amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in drastic changes in the fatty acid composition in the transformant cells; the contents of palmitoleic acid (16:1) and oleic acid (18:1) increased significantly, with accompanying decreases in palmitic acid (16:0) and stearic acid (18:0) . These changes were controlled by the addition of maltose as a carbon source to the medium . Also, the expression of the gene caused a significant change in the lipid composition in the Aspergillus transformant . Genomic Southern blot analysis of the transformant with the Mortierella delta 9-desaturase gene as a probe confirmed the integration of this gene into the genome of A . oryzae.

Genes Dev, 1999 Mar 15, 13(6), 675 - 85
Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage; Chen L et al.; The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage . We have found that the 14-3-3 proteins Rad24 and Rad25 physically interact with Chk1 in fission yeast . Association of Chk1 with 14-3-3 proteins is stimulated in response to DNA damage . DNA damage results in phosphorylation of Chk1 and the 14-3-3 proteins bind preferentially to the phosphorylated form . Genetic analysis has independently implicated both Rad24 and Rad25 in the DNA-damage checkpoint pathway . We suggest that DNA damage-dependent association of phosphorylated Chk1 with 14-3-3 proteins mediates an important step along the DNA-damage checkpoint pathway, perhaps by directing Chk1 to a particular substrate or to a particular location within the cell . An additional role for 14-3-3 proteins in the DNA-damage checkpoint has been suggested based on the observation that human Chk1 can phosphorylate Cdc25C in vitro creating a 14-3-3 binding site . Our results suggest that in fission yeast the interaction between the 14-3-3 proteins and Cdc25 does not require Chk1 function and is unaffected by DNA damage, in sharp contrast to the interaction between the 14-3-3 proteins and Chk1.

Trends Biochem Sci, 1999 Jan, 24(1), 22 - 5
Dealing with energy demand: the AMP-activated protein kinase; Kemp BE et al.; The AMP-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that is found in all eukaryotes . AMPK activity is regulated by vigorous exercise, nutrient starvation and ischemia/hypoxia, and modulates many aspects of mammalian cell metabolism . The AMPK yeast homolog, Snf1p, plays a major role in adaption to glucose deprivation . In mammals, AMPK also has diverse roles that extend from energy metabolism through to transcriptional control.

J Cell Biol, 1999 Mar 22, 144(6), 1123 - 33
minifly, a Drosophila gene required for ribosome biogenesis; Giordano E et al.; We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene . Genetic analysis shows that mfl is essential for Drosophila viability and fertility . While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility . Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis . We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease . mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes . In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.

Genomics, 1999 Mar 15, 56(3), 344 - 9
Radiation hybrid mapping of chromosomal region 2p15-p16: integration of expressed and polymorphic sequences maps at the Carney complex (CNC) and Doyne honeycomb retinal dystrophy (DHRD) loci; Taymans SE et al.; Chromosomal region 2p15-p16, which corresponds to the genetic interval flanked by polymorphic markers D2S119 and D2S378 and covers a genetic distance of approximately 16 cM, is underrepresented in the existing maps of chromosome 2 . This is primarily due to two large gaps of unknown physical distance within the known yeast and bacterial artificial chromosome (YAC and BAC, respectively) maps . In constructing a YAC/BAC contig covering 2p15-p16, a total of 55 sequence-tagged sites (25 of which are polymorphic), including new sequences derived from chromosomal walking, and 38 expressed sequence tags were screened by a commercially available RH panel (Stanford G3) . A total of 45 of these sequences were placed; 32 of them were assigned at unique sites . The high-resolution TNG3 RH panel was then used to define further the chromosomal order of markers contained in the region flanked by D2S391 and D2S2153 . This region harbors the genes for two autosomal dominant disorders, Carney complex (CNC), a multiple neoplasia syndrome, and Doyne honeycomb retinal dystrophy (DHRD), a disease leading to blindness at a young age . This is the first attempt to order cloned sequences in chromosomal region 2p15-p16, an area apparently resistant to YAC cloning . Construction of the 2p15-p16 RH map is critical for identifying the genes responsible for CNC and DHRD, as well as for the molecular elucidation of a chromosomal region that is frequently rearranged in tumors .

Genomics, 1999 Mar 15, 56(3), 303 - 9
The genomic organization of type I keratin genes in mice; Sato H et al.; We isolated two new keratin cDNAs by screening a cDNA library constructed from poly(A)+ RNA of the dorsal and abdominal skin of C57BL/10J mice with a probe of human KRT14 . Due to its high sequence homology to human keratin 17 cDNA, one full-length cDNA is most likely to be mouse keratin 17 (Krt1-17) cDNA . The other is the putative full-length cDNA of a novel type I keratin gene, designated Krt1-c29 . These two keratin genes were mapped to the distal portion of Chromosome 11, where the mouse keratin gene complex-1 (Krt1) is localized . To elucidate the genomic organization of Krt1 in mice, we carried out genetic and physical analyses of Krt1 . A large-scale linkage analysis using intersubspecific backcrosses suggested that there are two major clusters in Krt1, one containing Krt1-c29, Krt1-10, and Krt1-12 and the other containing Krt1-14, -15, -17, and -19 . Truncation experiments with two yeast artificial chromosome clones containing the two clusters above have revealed that the gene order of Krt1 is centromere-Krt1-c29-Krt1-10-Krt1-12-Krt1-13-K rt1-15-Krt1-19-Krt1-14-K rt1-17-telomere . Finally, we analyzed sequence divergence between the genes belonging to the Krt1 complex . The results clearly indicated that genes are classified into two major groups with respect to phylogenetic relationship . Each group consists of the respective gene cluster demonstrated by genetic and physical analyses in this study, suggesting that the physical organization of the Krt1 complex reflects the evolutionary process of gene duplication of this complex .

Genomics, 1999 Mar 15, 56(3), 296 - 302
Genomic organization and biological characterization of the novel human CC chemokine DC-CK-1/PARC/MIP-4/SCYA18; Guan P et al.; The chemokines are a group of chemotactic molecules that appear to regulate the directed movement of white blood cells in vitro and in vivo and may therefore play important roles in inflammation and immunity . The genes encoding the chemokines are clustered in close physical proximity to each other . A large cluster of human CC chemokine genes resides on chromosome 17 . We have used this information in a positional cloning approach to identify novel chemokine genes within this cluster . We constructed a YAC contig encompassing the MIP-1alpha (HGMW-approved symbol SCYA3) gene region and used exon trapping and sequence analysis to isolate novel chemokine genes . Using this approach, a gene encoding a chemokine named MIP-4, based on its homology with MIP-1alpha (49.5% identity at the nucleotide level and 59.6% at the predicted amino acid level), was found . The MIP-4 gene (HGMW-approved symbol SCYA18) consists of three exons spread over 7.1 kb and is separated from the MIP-1alpha gene by 16 kb . The MIP-4 gene encodes a 750-bp mRNA that is expressed in lung and macrophages but not in brain or muscle . The mRNA encodes an 89-amino-acid protein and includes a predicted signal peptide of 21 amino acids . Recombinant or synthetic MIP-4 induced calcium mobilization in naive and activated T lymphocyte subpopulations in vitro . Injection of synthetic MIP-4 into the peritoneal cavity of mice led to the accumulation of both CD4(+) and CD8(+) T lymphocytes, but not monocytes or granulocytes . These observations provide new information concerning the arrangement of the CC chemokine gene cluster on human chromosome 17 and indicate that the MIP-4 gene product is chemotactic in vivo for both CD4(+) and CD8(+) T lymphocytes and may therefore be implicated in both humoral and cell-mediated immunity .

Genomics, 1999 Mar 15, 56(3), 274 - 87
Genetic and physical analyses of the centromeric and pericentromeric regions of human chromosome 5: recombination across 5cen; Puechberty J et al.; Human centromeres are poorly understood at both the genetic and the physical level . In this paper, we have been able to distinguish the alphoid centromeric sequences of chromosome 5 from those of chromosome 19 . This result was obtained by pulsed-field gel electrophoresis after cutting genomic DNA with restriction endonucleases NcoI (chromosome 5) and BamHI (chromosome 19) . We could thus define a highly polymorphic marker, representing length variations of the D5Z1 domain located at the q arm boundary of the chromosome 5 centromere . The centromeric region of chromosome 5 was then analyzed in full detail . We established an approximately 4.6-Mb physical map of the whole region with five rare-cutting enzymes by using nonchimeric YACs, two of which were shown to contain the very ends of 5cen on both sides . The p-arm side of 5cen was shown to contain an alphoid subset (D5Z12) different from those described thus far . Two genes and several putative cDNAs could be precisely located close to the centromere . Several L1 elements were shown to be present within alpha satellites at the boundary between alphoid and nonalphoid sequences on both sides of 5cen . They were used to define STSs that could serve as physical anchor points at the junction of 5cen with the p and q arms . Some STSs were placed on a radiation hybrid map . One was polymorphic and could therefore be used as a second centromeric genetic marker at the p arm boundary of 5cen . We could thus estimate recombination rates within and around the centromeric region of chromosome 5 . Recombination is highly reduced within 5cen, with zero recombinants in 58 meioses being detected between the two markers located at the two extremities of the centromere . In its immediate vicinity, 5cen indeed exerts a direct negative effect on meiotic recombination within the proximal chromosomal DNA . This effect is, however, less important than expected and is polarized, as different rates are observed on both arms if one compares the 0 cM/Mb of the p proximal first 5.5 Mb and the 0.64 cM/Mb of the q proximal first 5 Mb to the sex-average 1.02 cM/Mb found throughout the entire chromosome 5 . Rates then become close to the average when one goes further within the arms . Finally, most recombinants (21/22), irrespective of the arm, are of female origin, thus showing that recombination around 5cen is essentially occurring in the female lineage .

Nature, 1999 Mar 11, 398(6723), 165 - 9
CBP-independent activation of CREM and CREB by the LIM-only protein ACT; Fimia GM et al.; Transcriptional activation by CREB and CREM requires phosphorylation of a serine residue within the activation domain (Ser 133 in CREB; Ser 117 in CREM) which as a result interacts with the coactivator CBP . The activator CREM is highly expressed in male germ cells and is required for post-meiotic gene expression . Using a two-hybrid screen, we have isolated a testis-derived complementary DNA encoding a protein that we term ACT (for activator of CREM in testis), a LIM-only protein which specifically associates with CREM . ACT is expressed coordinately with CREM in a tissue- and developmentally regulated manner . It strongly stimulates CREM transcriptional activity in yeast and mammalian cells and contains an intrinsic activation function . As ACT bypasses the classical requirements for activation, namely phosphorylation of Ser 117 and interaction with CBP, it represents a new route for transcriptional activation by CREM and CREB . ACT may define a previously undiscovered class of tissue-specific coactivators whose function could be specific for distinct cellular differentiation programmes.

Leukemia, 1999 Mar, 13(3), 369 - 75
Identification of a novel molecular partner of the E2A gene in childhood leukemia; Brambillasca F et al.; The 'promiscuous' E2A gene, at 19p13.3, is fused with two different molecular partners, PBX1 and HLF, following two chromosome translocations recurrent in childhood pre-B ALL . We have identified a novel gene, FB1, by virtue of its fusion with E2A and by a combination of molecular techniques . FB1 was localized on 19q13.4, suggesting that the novel chimera originated by a cryptic rearrangement of chromosome 19 . Two FB1 transcripts, of 1.2 kb and 1.1 kb, are differentially expressed at low level in a variety of human tissues, including hemopoietic cell lines from different lineages . Accordingly, FB1 cDNA displays high homology with a number of cDNA clones from different human tissues . High homology was found also with cDNA clones from mouse and rat, suggesting that the sequence might be conserved at least among mammals . The function of the putative FB1 protein, however, is currently unknown as database sequence comparisons have failed to reveal strong homology with known proteins . The E2A/FB1 fusion appears to be a recurrent feature of pre-B ALLs, suggesting that it might have a role in the development and/or progression of leukemogenesis.

Leukemia, 1999 Mar, 13(3), 348 - 57
MDS1/EVI1 enhances TGF-beta1 signaling and strengthens its growth-inhibitory effect but the leukemia-associated fusion protein AML1/MDS1/EVI1, product of the t(3;21), abrogates growth-inhibition in response to TGF-beta1; Sood R et al.; MDS1/EVI1, located on chromosome 3 band q26, encodes a zinc-finger DNA-binding transcription activator not detected in normal hematopoietic cells but expressed in several normal tissues . MDS1/EVI1 is inappropriately activated in myeloid leukemias following chromosomal rearrangements involving band 3q26 . The rearrangements lead either to gene truncation, and to expression of the transcription repressor EVI1, as seen in the t(3;3)(q21;q26) and inv(3)(q21q26), or to gene fusion, as seen in the t(3;21)(q26;q22) which results in the fusion protein AML1/MDS1/EVI1 . This fusion protein contains the DNA-binding domain of the transcription factor AML1 fused in-frame to the entire MDS1/EVI1 with the exclusion of its first 12 amino acids . In this report, we have analyzed the response of the hematopoietic precursor cell line 32Dcl3, expressing either the normal protein MDS1/EVI1 or the fusion protein AML1/MDS1/EVI1, to factors that control cell differentiation or cell replication . The 32Dcl3 cells are IL-3-dependent for growth and they differentiate into granulocytes when exposed to G-CSF . They are growth-inhibited by TGF-beta1 . We show that whereas the expression of MDS1/EVI1 has no effect on granulocytic differentiation induced by G-CSF, expression of AML1/MDS1/EVI1 blocks differentiation resulting in cell death . This effect is similar to that previously described by others for 32Dcl3 cells that express transgenic Evil . Furthermore, we show that whereas the expression of the fusion protein AML1/MDS1/EVI1 completely abrogates the growth-inhibitory effect of TGF-beta1 and allows 32Dcl3 cells to proliferate, expression of the normal protein MDS1/EVI1 has the opposite effect, and it strengthens the response of cells to the growth-inhibitory effect of TGF-beta1 . By using the yeast two-hybrid system, we also show that EVI1 (contained in its entirety in MDS1/EVI1 and AML1/MDS1/EVI1) physically interacts with SMAD3, which is an intracellular mediator of TGF-beta1 signaling . Finally, we have correlated the response of the cells to G-CSF or TGF-beta1 with the ability of the normal and fusion proteins to activate or repress promoters which they can directly regulate by binding to the promoter site . We propose that mutations of MDS1/EVI1 either by gene truncation resulting in the transcription repressor EVI1 or by gene fusion to AML1 lead to an altered cellular response to growth and differentiation factors that could result in leukemic transformation . The different response of myeloid cells ectopically expressing the normal or the fusion protein to G-CSF and TGF-beta1 could depend on the different transactivation properties of these proteins resulting in divergent expression of downstream genes regulated by the two proteins.

Ann Thorac Surg, 1999 Jan, 67(1), 231 - 3
Surgical management of necrotizing Candida esophagitis; Gaissert HA et al.; Invasive esophageal candidiasis produced transmural necrosis leading to perforation in 2 patients aged 10 and 27 years . Both patients survived after esophageal resection and complete diversion . One patient with acute leukemia and neutropenia experienced systemic candidiasis, which resolved after esophagectomy . Esophagectomy and diversion for yeast-induced necrosis may lead to complete recovery and resolution of disseminated candidiasis when combined with systemic antifungal therapy.

Oncogene, 1999 Mar 11, 18(10), 1867 - 79
Cloning and characterization of mPAL, a novel Shc SH2 domain-binding protein expressed in proliferating cells; Schmandt R et al.; Shc adaptor proteins play a role in linking activated cell surface receptors to the Ras signaling pathway in response to receptor mediated tyrosine kinase activation . While the function of Shc in the activation of the Ras pathway via binding to Grb2 has been well characterized, it is becoming increasingly apparent that Shc participates in additional signaling pathways through interactions with other cytoplasmic proteins . Using the yeast two-hybrid system, we have identified a unique Shc binding protein designated PAL (Protein expressed in Activated Lymphocytes) with no similarity to other known proteins . mPAL binds specifically to the Shc SH2 domain and unlike previously described Shc SH2 domain-protein interactions, the association of mPAL and Shc is phosphotyrosine-independent . Both mPAL RNA and protein expression are restricted to tissues containing actively dividing cells and proliferating cells in culture . mPAL expression is induced upon growth factor stimulation and is down-regulated upon growth inhibition . This pattern, and timing of mPAL expression and its association with the Shc adaptor molecule suggests a role for this protein in signaling pathways governing cell cycle progression.

Biochem Pharmacol, 1999 Apr 15, 57(8), 877 - 80
Selectivity of the molecular chaperone-specific immunosuppressive agent 15-deoxyspergualin: modulation of Hsc70 ATPase activity without compromising DnaJ chaperone interactions; Brodsky JL; The immunosuppressive and cytostatic agent 15-deoxyspergualin (DSG) binds to the Hsc70 class of molecular chaperones with a K(D) = 4 microM . Because Hsc70s represent a diverse group of cellular effectors and because Hsc70 function frequently requires a DnaJ molecular chaperone, the specificity of DSG for different Hsc70s and the ability of DSG to block the productive interaction between an Hsc70 and its DnaJ partner were examined . DSG stimulated the ATPase activity of a mammalian and yeast cytosolic Hsc70 from 20 to 40%, but was unable to elicit such a response in a homologous Hsc70, Binding Protein (BiP), that resides in the lumen of the endoplasmic reticulum . In addition, the DnaJ-stimulated Hsc70 ATPase activity and the DnaJ-mediated release of an unfolded polypeptide from an Hsc70 were unaffected by DSG . These results indicate that Hsc70s exhibit substrate selectivity for DSG and that DSG does not compromise Hsc70 functions that require DnaJs . Thus, the immunosuppressive and cytostatic effects of DSG may be specific for a subset of cellular Hsc70s and confined to DnaJ-independent Hsc70-mediated activities.

J Biol Chem, 1999 Mar 26, 274(13), 9083 - 91
Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor; Bachand F et al.; The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1) . As Vpr is not part of the p55 Gag polyprotein precursor (Pr55(gag)), its incorporation requires an anchor to associate with the assembling viral particles . Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55(gag) corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein . In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55(gag) using the yeast two-hybrid system and the maltose-binding protein pull-down assay . The present study provides genetic and biochemical evidence indicating that the Pr55(gag) can physically interact with the Vpr protein . Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6(gag) completely abolish the interaction between Vpr and the Pr55(gag) and, as a consequence, prevent Vpr virion incorporation . In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55(gag) . Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal alpha-helical structure is essential for the Vpr-Pr55(gag) interaction . Vpr and the Pr55(gag) demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction . Finally, the interaction is efficiently competed using anti-Vpr sera . Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6(gag) . The development of Pr55(gag)-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.

J Biol Chem, 1999 Mar 26, 274(13), 8737 - 45
Identification and characterization of potential effector molecules of the Ras-related GTPase Rap2; Nancy V et al.; In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDS-like factor (Rlf) . These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras . Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro . When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2, i.e . the endoplasmic reticulum . However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo . Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed . Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase.

J Biol Chem, 1999 Mar 26, 274(13), 8570 - 6
Cloning and characterization of human prostate coactivator ARA54, a novel protein that associates with the androgen receptor; Kang HY et al.; Androgen receptor (AR) is a member of the steroid receptor superfamily that may require coactivators for proper or maximal transactivation . Using a yeast two-hybrid screening followed by mammalian cell analyses, we identified a novel ligand-dependent AR-associated protein, ARA54, which consists of 474 amino acids with a molecular mass of 54 kDa . We demonstrated that ARA54 might function as a preferential coactivator for AR-mediated transactivation in human prostate cancer DU145 cells . Interestingly, our data also showed that ARA54 could significantly enhance the transcriptional activity of LNCaP mutant AR (ARt877a) but not wild type AR or another mutant AR (ARe708k) in the presence of 10 nM 17beta-estradiol or 1 microM hydroxyflutamide . These results imply that both ARA54 and the positions of the AR mutation (877 versus 708) might contribute to the specificity of AR-mediated transactivation . Our findings further demonstrated that the C-terminal domain of ARA54 can serve as a dominant negative inhibitor and exogenous full-length ARA54 can reverse this squelching effect on AR transcriptional activity . Co-expression of ARA54 with other AR coactivators, such as ARA70 or SRC-1, showed additive stimulation of AR-mediated transactivation, which indicates that these cofactors may function individually as AR coactivators to induce AR target gene expression . Through our findings, we have identified and characterized a novel AR coactivator, ARA54, which may play an important role in the AR signaling pathway in human prostate.

Naturwissenschaften, 1999 Feb, 86(2), 51 - 61
Peroxin puzzles and folded freight: peroxisomal protein import in review; Crookes WJ et al.; Peroxisomes are organelles that perform a variety of functions, including the metabolism of hydrogen peroxide and the oxidation of fatty acids . Peroxisomes do not possess organellar DNA; all peroxisomal matrix proteins are post-translationally translocated into the organelle . The mechanism of peroxisomal protein translocation has been the subject of vigorous research in the past decade . Many of the proteins (peroxins, abbreviated Pex) that play critical roles in peroxisome biogenesis have been identified through functional complementation of yeast strains and of Chinese hamster ovary cell lines that are defective in peroxisome biogenesis . Researchers are now turning towards biochemical and genetic analyses of these peroxins to define their roles in peroxisome biogenesis and to discover interacting protein partners . Evidence suggests that some of the interacting partners include molecular chaperones . Several current models for peroxisomal protein import are presented.

Biochim Biophys Acta, 1999 Feb 10, 1430(1), 103 - 10
Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin; Sorrentino S et al.; A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform . Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma . Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate . It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects . Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk . Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin . Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein . Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin . When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.

Mol Cell Biol, 1999 Apr, 19(4), 3145 - 55
SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents; Duan H et al.; SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer . SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa . SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences . In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis . SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24 . Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator . When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions) . Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation . Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.

Mol Cell Biol, 1999 Apr, 19(4), 3062 - 72
Hypersensitive site 2 specifies a unique function within the human beta-globin locus control region to stimulate globin gene transcription; Bungert J et al.; The human beta-globin locus control region (LCR) harbors both strong chromatin opening and enhancer activity when assayed in transgenic mice . To understand the contribution of individual DNase I hypersensitive sites (HS) to the function of the human beta-globin LCR, we have mutated the core elements within the context of a yeast artificial chromosome (YAC) carrying the entire locus and then analyzed the effect of these mutations on the formation of LCR HS elements and expression of the genes in transgenic mice . In the present study, we examined the consequences of two different HS2 mutations . We first generated seven YAC transgenic lines bearing a deletion of the 375-bp core enhancer of HS2 . Single-copy HS2 deletion mutants exhibited severely depressed HS site formation and expression of all of the human beta-globin genes at every developmental stage, confirming that HS2 is a vital, integral component of the LCR . We also analyzed four transgenic lines in which the core element of HS2 was replaced by that of HS3 and found that while HS3 is able to restore the chromatin-opening activity of the LCR, it is not able to functionally replace HS2 in mediating high-level globin gene transcription . These results continue to support the hypothesis that HS2, HS3, and HS4 act as a single, integral unit to regulate human globin gene transcription as a holocomplex, but they can also be interpreted to say that formation of a DNase I hypersensitive holocomplex alone is not sufficient for mediating high-level globin gene transcription . We therefore propose that the core elements must productively interact with one another to generate a unique subdomain within the nucleoprotein holocomplex that interacts in a stage-specific manner with individual globin gene promoters.

Mol Cell Biol, 1999 Apr, 19(4), 2782 - 90
Conserved loop I of U5 small nuclear RNA is dispensable for both catalytic steps of pre-mRNA splicing in HeLa nuclear extracts; Segault V et al.; The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts . Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing . However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation . Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity . Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5' and 3' halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing . Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts . This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP . Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

Mol Cell Biol, 1999 Apr, 19(4), 2724 - 33
Human SWI-SNF component BRG1 represses transcription of the c-fos gene; Murphy DJ et al.; Yeast and mammalian SWI-SNF complexes regulate transcription through active modification of chromatin structure . Human SW-13 adenocarcinoma cells lack BRG1 protein, a component of SWI-SNF that has a DNA-dependent ATPase activity essential for SWI-SNF function . Expression of BRG1 in SW-13 cells potentiated transcriptional activation by the glucocorticoid receptor, which is known to require SWI-SNF function . BRG1 also specifically repressed transcription from a transfected c-fos promoter and correspondingly blocked transcriptional activation of the endogenous c-fos gene . Mutation of lysine residue 798 in the DNA-dependent ATPase domain of BRG1 significantly reduced its ability to repress c-fos transcription . Repression by BRG1 required the cyclic AMP response element of the c-fos promoter but not nearby binding sites for Sp1, YY1, or TFII-I . Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb protein, transcriptional repression by BRG1 was weak unless wild-type Rb was also supplied . Interestingly, Rb-dependent repression by BRG1 was found to take place through a pathway that is independent of transcription factor E2F.

Eur J Cell Biol, 1999 Jan, 78(1), 21 - 32
Subunits of the eukaryotic cytosolic chaperonin CCT do not always behave as components of a uniform hetero-oligomeric particle; Roobol A et al.; The chaperonin CCT is an hetero-oligomeric molecular chaperone complex . Studies in yeast suggest each of its eight gene products are required for its major identified functions in producing native tubulins and actins . However, it is unclear whether these eight components always form a single particle, covering all functions, or else can also exist as heterogeneous mixtures and/or free subunits in cells . Using mouse P19 embryonal carcinoma cells, which divide rapidly, yet in retinoic acid adopt a neuronal phenotype, admixed with occasional (approximately 10%) fibroblast-like cells, together with a panel of peptide-specific antibodies raised to 7 of the 8 CCT subunits we show that; (1) adoption of a post mitotic phenotype is accompanied by reduced CCT protein expression, significantly more so for CCTbeta, CCTdelta, CCTepsilon, and CCTtheta than for CCTalpha (TCP-1), CCTgamma and CCTzeta; (2) CCTalpha is detected preferentially over other subunits in neurites of P19 neurons; (3) small amounts of CCTalpha and gamma are localised in nuclei (i.e . are not exclusively cytoplasmic), selectively so compared with other subunits; (4) numerous cytosolic foci exist in the cytoplasm which, when detected by double immunofluorescence can contain only one of the subunits probed for; (5) while a "core" chaperonin particle can be immunoprecipitated under native conditions, epitope access is modified both by nucleotides and by non-CCT co-precipitating proteins . Collectively, these findings indicate that CCT subunits are not only components of the hetero-oligomeric chaperonin particle but exist as significant populations of free subunits or smaller oligomers in cells.

Nat Genet, 1999 Mar, 21(3), 305 - 8
High-resolution mapping of quantitative trait loci in outbred mice; Talbot CJ et al.; Screening the whole genome of a cross between two inbred animal strains has proved to be a powerful method for detecting genetic loci underlying quantitative behavioural traits, but the level of resolution offered by quantitative trait loci (QTL) mapping is still too coarse to permit molecular cloning of the genetic determinants . To achieve high-resolution mapping, we used an outbred stock of mice for which the entire genealogy is known . The heterogeneous stock (HS) was established 30 years ago from an eight-way cross of C57BL/6, BALB/c, RIII, AKR, DBA/2, I, A/J and C3H inbred mouse strains . At the time of the experiment reported here, the HS mice were at generation 58, theoretically offering at least a 30-fold increase in resolution for QTL mapping compared with a backcross or an F2 intercross . Using the HS mice we have mapped a QTL influencing a psychological trait in mice to a 0.8-cM interval on chromosome 1 . This method allows simultaneous fine mapping of multiple QTLs, as shown by our report of a second QTL on chromosome 12 . The high resolution possible with this approach makes QTLs accessible to positional cloning.

Nat Genet, 1999 Mar, 21(3), 297 - 301
SLC7A7, encoding a putative permease-related protein, is mutated in patients with lysinuric protein intolerance; Borsani G et al.; Lysinuric protein intolerance (LPI, MIM 222700) is an autosomal recessive multisystem disorder found mainly in Finland and Italy . On a normal diet, LPI patients present poor feeding, vomiting, diarrhoea, episodes of hyperammoniaemic coma and failure to thrive . Hepatosplenomegaly, osteoporosis and a life-threatening pulmonary involvement (alveolar proteinosis) are also seen . LPI is caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in kidney and intestine . Metabolic derangement is characterized by increased renal excretion of CAA, reduced CAA absorption from intestine and orotic aciduria . The gene causing LPI was assigned using linkage analysis to chromosome 14q11.2 near the T-cell receptor alpha/delta chains locus, and a critical region has been defined . We have identified two new transcripts (SLC7A8 and SLC7A7) homologous to amino acid transporters, highly expressed in kidney and mapping in the LPI critical region . Mutational analysis of both transcripts revealed that SLC7A7 (for solute carrier family 7, member 7) is mutated in LPI . In five Italian patients, we found either an insertion or deletion in the coding sequence, which provides evidence of a causative role of SLC7A7 in LPI . Furthermore, we detected a splice acceptor change resulting in a frameshift and premature translation termination in four unrelated Finnish patients . This mutation may represent the founder LPI allele in Finland.

Plant Mol Biol, 1999 Jan, 39(1), 171 - 6
Genetic and physical characterization of a region of Arabidopsis chromosome 1 containing the CLAVATA1 gene; Williams RW et al.; With the advance of Arabidopsis as a model system for understanding plant genetics, development and biochemistry, a detailed description of the genome is necessary . As such, focused projects are underway to map and sequence the Arabidopsis nuclear genome . We have characterized a region of chromosome 1, surrounding the CLAVATA1 (CLV1) locus . Three (RFLP) clones were mapped relative to clv1-1, and were used to construct an ca . 700 kb yeast artificial chromosome (YAC) contig . Three cosmids spanning the CLV1 locus were analyzed and ca . 24 kb of genomic DNA was sequenced, including a continuous stretch of 18 kb . In addition to generating clones in this region of chromosome 1, we have analyzed the size, spacing and organization of several contiguous genes.

Plant Mol Biol, 1999 Jan, 39(1), 45 - 61
Molecular characterization of four beta-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica; Yamamoto E et al.; Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization . As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations . We have previously reported that alpha-tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297-308, 1998) . In order to ascertain associations of other tubulins with dinitroaniline resistance, four beta-tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes . Sequence analysis of the four beta-tubulin cDNA classes identified no missense mutations . Identified nucleotide substitutions did not result in amino acid replacements . These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast . Expression of the four beta-tubulins was highest in inflorescences . This is in contrast to alpha-tubulin TUA1 that is expressed predominantly in roots . Collectively, these results imply that beta-tubulin genes are not associated with dinitroaniline resistance in goosegrass . Phylogenetic analysis of the four beta-tubulins, together with three alpha-tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.

Plant Mol Biol, 1999 Jan, 39(2), 325 - 33
Expression of a proteasome alpha-type subunit gene during tobacco development and senescence; Bahrami AR et al.; Proteasomes degrade specific proteins that have been targeted for proteolysis by ubiquitination . In animals and yeast nuclear-localised proteasomes play a role in regulating the cell cycle, and other developmental processes, via control of the levels of regulatory nuclear proteins such as cyclins and transcription factors . A cDNA, NtPSA1, isolated from tobacco styles was found to have high similarity to human and yeast genes, PRCI_human and PRCI_yeast with 63.4% and 51.6% overall identity respectively . These genes are believed to encode non-catalytic alpha-type subunits of 26S proteasomes and like NtPSA1 have putative nuclear localisation signals . NtPSA1 RNA was found to accumulate to varying levels in different parts of the plant and at different developmental stages . In particular, the level of NtPSA1 RNA was high in young dividing and expanding tissues, and declined during the senescence of both leaves and flowers . These results suggest that a role of proteasomes in plant nuclei may be to regulate developmental events by controlling the levels of regulatory proteins in proliferating and developing tissues, rather than to degrade and recycle proteins during senescence.

J Leukoc Biol, 1999 Mar, 65(3), 341 - 4
Sphingosine 1-phosphate: a prototype of a new class of second messengers; Spiegel S; Sphingosine 1-phosphate (SPP) is an important sphingolipid-derived second messenger in mammalian cells that acts to promote proliferation and to inhibit apoptosis . Various growth factors increase the intracellular concentration of SPP by activating sphingosine kinase, the molecular cloning of which has revealed that it defines a new type of lipid kinase . Cell fate is influenced by the balance between the intracellular concentration of SPP and that of ceramide, a pro-apoptotic sphingolipid metabolite . The observation that a similar "rheostat" is a determinant of cell survival in yeast cells exposed to heat shock indicates that it is an evolutionarily conserved mechanism of stress regulation . SPP also acts extracellularly to inhibit cell motility and to influence cell morphology, effects that appear to be mediated by the G protein-coupled receptor EDG1 . These observations indicate that SPP is the prototype of a new class of lipid mediators that exert both intracellular and extracellular actions.

Development, 1999 Apr, 126(8), 1729 - 37
Spatially regulated SpEts4 transcription factor activity along the sea urchin embryo animal-vegetal axis; Wei Z et al.; Because the transcription of the SpHE gene is regulated cell-autonomously and asymmetrically along the maternally determined animal-vegetal axis of the very early sea urchin embryo, its regulators provide an excellent entry point for investigating the mechanism(s) that establishes this initial polarity . Previous studies support a model in which spatial regulation of SpHE transcription relies on multiple nonvegetal positive transcription factor activities (Wei, Z., Angerer, L . M . and Angerer, R . C . (1997) Dev . Biol . 187, 71-78) and a yeast one-hybrid screen has identified one, SpEts4, which binds with high specificity to a cis element in the SpHE regulatory region and confers positive activation of SpHE promoter transgenes (Wei, Z., Angerer, R . C . and Angerer, L . M . (1999) Mol . Cell . Biol . 19, 1271-1278) . Here we demonstrate that SpEts4 can bind to the regulatory region of the endogenous SpHE gene because a dominant repressor, created by fusing SpEts4 DNA binding and Drosophila engrailed repression domains, suppresses its transcription . The pattern of expression of the SpEts4 gene is consistent with a role in regulating SpHE transcription in the nonvegetal region of the embryo during late cleavage/early blastula stages . Although maternal transcripts are uniformly distributed in the egg and early cleaving embryo, they rapidly turn over and are replaced by zygotic transcripts that accumulate in a pattern congruent with SpHE transcription . In addition, in vivo functional tests show that the SpEts4 cis element confers nonvegetal transcription of a beta-galactosidase reporter gene containing the SpHE basal promoter, and provide strong evidence that the activity of this transcription factor is an integral component of the nonvegetal transcriptional regulatory apparatus, which is proximal to, or part of, the mechanism that establishes the animal-vegetal axis of the sea urchin embryo.

J Mol Recognit, 1998 Winter, 11(1-6), 270 - 2
Direct measurement of intraparticle fluid velocity in superporous agarose beads; Larsson PO et al.; Superporous agarose beads contain both normal diffusion pores and special, very wide superpores through which part of the chromatographic flow is transported, a situation that may greatly improve the chromatographic performance . For the first time such pore flow was measured directly by following the movement of microparticles (dyed yeast cells) through superporous beads packed in a chromatographic bed . The passage of the microparticles through the superpores and through the interstitial pores was recorded by a microscope/video camera . The video recordings were subsequently used to determine flow paths as well as the convective fluid velocities in both the superpores and the interstitial pores . The superpore fluid velocity was found to be proportional to the ratio between the squares of the respective pore diameters, which is in agreement with the Kozeny-Carman equation . Values for two-dimensional and three-dimensional tortuosity of the flow paths were measured and calculated respectively.

Mol Cell, 1999 Feb, 3(2), 247 - 53
Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits; Phelan ML et al.; Protein complexes of the SWI/SNF family remodel nucleosome structure in an ATP-dependent manner . Each complex contains between 8 and 15 subunits, several of which are highly conserved between yeast, Drosophila, and humans . We have reconstituted an ATP-dependent chromatin remodeling complex using a subset of conserved subunits . Unexpectedly, both BRG1 and hBRM, the ATPase subunits of human SWI/SNF complexes, are capable of remodeling mono-nucleosomes and nucleosomal arrays as purified proteins . The addition of INI1, BAF155, and BAF170 to BRG1 increases remodeling activity to a level comparable to that of the whole hSWI/SNF complex . These data define the functional core of the hSWI/SNF complex.

FEMS Microbiol Rev, 1999 Jan, 23(1), 39 - 68
Nuclear movement in filamentous fungi; Fischer R; One of the most striking features of eukaryotic cells is the organization of specific functions into organelles such as nuclei, mitochondria, chloroplasts, the endoplasmic reticulum, vacuoles, peroxisomes or the Golgi apparatus . These membrane-surrounded compartments are not synthesized de novo but are bequeathed to daughter cells during cell division . The successful transmittance of organelles to daughter cells requires the growth, division and separation of these compartments and involves a complex machinery consisting of cytoskeletal components, mechanochemical motor proteins and regulatory factors . Organelles such as nuclei, which are present in most cells in a single copy, must be precisely positioned prior to cytokinesis . In many eukaryotic cells the cleavage plane for cell division is defined by the location of the nucleus prior to mitosis . Nuclear positioning is thus absolutely crucial in the unequal cell divisions that occur during development and embryogenesis . Yeast and filamentous fungi are excellent organisms for the molecular analysis of nuclear migration because of their amenability to a broad variety of powerful analytical methods unavailable in higher eukaryotes . Filamentous fungi are especially attractive models because the longitudinally elongated cells grow by apical tip extension and the organelles are often required to migrate long distances . This review describes nuclear migration in filamentous fungi, the approaches used for and the results of its molecular analysis and the projection of the results to other organisms.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 3298 - 3302
Channel-mediated high-affinity K+ uptake into guard cells from Arabidopsis; Bruggemann L et al.; Potassium uptake by higher plants is the result of high- or low-affinity transport accomplished by different sets of transporters . Although K+ channels were thought to mediate low-affinity uptake only, the molecular mechanism of the high-affinity, proton-dependent K+ uptake system is still scant . Taking advantage of the high-current resolution of the patch-clamp technique when applied to the small Arabidopsis thaliana guard cells densely packed with voltage-dependent K+ channels, we could directly record channels working in the concentration range of high-affinity K+ uptake systems . Here we show that the K+ channel KAT1 expressed in Arabidopsis guard cells and yeast is capable of mediating potassium uptake from media containing as little as 10 microM of external K+ . Upon reduction of the external K+ content to the micromolar level the voltage dependence of the channel remained unaffected, indicating that this channel type represents a voltage sensor rather than a K+-sensing valve . This behavior results in K+ release through K+ uptake channels whenever the Nernst potential is negative to the activation threshold of the channel . In contrast to the H+-coupled K+ symport shown to account for high-affinity K+ uptake in roots, pH-dependent K+ uptake into guard cells is a result of a shift in the voltage dependence of the K+ channel . We conclude that plant K+ channels activated by acid pH may play an essential role in K+ uptake even from dilute solutions.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2674 - 7
Transcriptional activation by artificial recruitment in mammalian cells; Nevado J et al.; We show that the typical "nonclassical" activator, which comprises a fusion protein bearing a component of the transcriptional machinery fused to a DNA-binding domain, activates transcription in mammalian cells only weakly when tested with an array of promoters . However, as found in analogous "artificial recruitment" experiments performed in yeast, these activators work synergistically with "classical" activators . The effect of the classical activator in such experiments requires that it be tethered to DNA, a requirement that cannot be overcome by expression of that classical activator at high levels . The effect of the one nonclassical activator that does elicit significant levels of transcription when working alone (i.e., that bearing TATA box-binding protein) is strongly influenced by promoter architecture . The results, consistent with those of analogous experiments in yeast {see the accompanying paper: Gaudreau, L., Keaveney, M., Nevado, J., Zaman, Z., Bryant, G . O., Struhl, K . & Ptashne, M . (1999) Proc . Natl . Acad . Sci . USA 96, 2668-2673}, suggest that classical activators, presumably by virtue of their abilities to interact with multiple targets, have a functional flexibility that nonclassical activators lack.

Vet Immunol Immunopathol, 1999 Feb 1, 67(2), 141 - 52
Granulocyte function in dogs experimentally infected with a Swedish granulocytic Ehrlichia species; Lilliehook I et al.; Granulocyte function was studied in six dogs inoculated with a Swedish granulocytic Ehrlichia species and in four control dogs . Whole blood chemiluminescence (CL) was enhanced in the dogs with granulocytic ehrlichiosis . Both CL after stimulation with zymosan and spontaneous CL was significantly increased at peak of infection compared with pre-infection levels . Ingestion of FITC-labelled serum-opsonized yeast cells was high and stable in both groups . The ingestion was lower when the yeast cells were opsonized with anti-yeast IgG . However, there was no difference between groups . The labelling intensity of anti-human CD11b, CD18 and CD32 mAb on the granulocytes in dogs with ehrlichiosis was similar to that in control dogs . The opsonic activity in serum collected at the peak of infection was not different from serum drawn prior to inoculation . Opsonic activity was investigated both by yeast cell ingestion and by chemiluminescence after stimulation with zymosan . The serum from infected dogs enhanced the respiratory burst without stimulation with zymosan of leukocytes from healthy dogs . This suggests that serum at the peak of infection contains granulocyte activators . In this study we found normal phagocytosis together with evidence of enhanced oxidative metabolism in the granulocytes from dogs with granulocytic ehrlichiosis.

Nucleic Acids Res, 1999 Apr 1, 27(7), 1762 - 5
An improved method for routine preparation of intact artificial chromosome DNA (340-1000 kb) for transfection into human cells; Compton ST et al.; The transfer of high molecular weight (HMW) DNA into mammalian cells is an important strategy for assessing human gene expression and chromosome structure and function . However, using current methods, it is difficult to dependably prepare intact HMW DNA because of the susceptibility of the DNA to degradation and physical shearing . Here we describe a strategy whereby intact artificial chromosome DNA (as large as 1 Mb) can be routinely prepared from yeast . Strict adherence to this protocol has resulted in: (i) >90% of liquid DNA preparations containing largely intact DNA; (ii) transfection efficiencies for the development of stable human clonal cell lines ranging from 5 x 10(-7) to 8.8 x 10(-5); and (iii) the presence of markers from both YAC arms in 30-42% of the human fibrosarcoma cell HT1080 clones and 100% of the CF lung epithelial cell lines IB3-1 and CFT1 clones, suggesting that the HMW DNA is potentially intact in a substantial proportion of clones . Using this protocol for DNA preparation, successful transfection of functional 1 Mb human artificial chromosome DNA into human cells has also been achieved . This methodology should prove useful to those interested in using HMW human DNA for gene expression and functional analysis or for linear artificial chromosome construction, since integrity is absolutely critical for the success of these studies.

Methods, 1999 Jan, 17(1), 52 - 9
Assays for analyzing exonucleases in vitro; Ross J; Ribonucleases play essential roles in cell growth, differentiation, and the response to stress . This article deals with exoribonucleases, enzymes that degrade RNAs beginning at either the 5' or 3' end and proceed down the length of the RNA . The preparation of a crude extract of a mammalian 3'-to-5' exonuclease is described . Assay conditions for both 5'-to-3' and 3'-to-5' exonucleases are given . One of these is a yeast enzyme that is known to be involved in mRNA decay . Others are vertebrate exonucleases that are presumed to have a role in mRNA stability but have not yet been proven to do so .

J Biol Chem, 1999 Mar 19, 274(12), 8316 - 21
Cloning and characterization of androgen receptor coactivator, ARA55, in human prostate; Fujimoto N et al.; Androgen receptor (AR) is a hormone-activated transcriptional factor that can bind to androgen response elements and that regulates the transcription of target genes via a mechanism that presumably involves cofactors . We report here the cloning of a novel AR coactivator ARA55 using a yeast two-hybrid system . ARA55 consists of 444 amino acids with the predicted molecular mass of 55 kDa and its sequence shows very high homology to mouse hic5, a TGF-beta1-inducible gene . Yeast and mammalian two-hybrid systems and co-immunoprecipitation assays all prove ARA55 can bind to AR in a ligand-dependent manner . Transient transfection assay in prostate cancer DU145 cells further demonstrates that ARA55 can enhance AR transcriptional activity in the presence of 1 nM dihydrotestosterone or its antagonists such as 100 nM 17beta-estradiol or 1 microM hydroxyflutamide . Our data also suggest the C-terminal half of ARA55, which includes three LIM motifs, is sufficient to interact with AR . Northern blot and polymerase chain reaction quantitation showed ARA55 can be expressed differently in normal prostate and prostate tumor cells . Together, our data suggests that ARA55 may play very important roles in the progression of prostate cancer by the modulation of AR transactivation.

J Biol Chem, 1999 Mar 19, 274(12), 7689 - 94
Interactions between neurogranin and calmodulin in vivo; Prichard L et al.; Neurogranin is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) within its IQ domain at serine 36 . Since CaM binds to neurogranin through the IQ domain, PKC phosphorylation and CaM binding are mutually exclusive . Consequently, we hypothesize that neurogranin may function to concentrate CaM at specific sites in neurons and release free CaM in response to increased Ca2+ and PKC activation . However, it has not been established that neurogranin interacts with CaM in vivo . In this study, we examined this question using yeast two-hybrid methodology . We also searched for additional proteins that might interact with neurogranin by screening brain cDNA libraries . Our data illustrate that CaM binds to neurogranin in vivo and that CaM is the only neurogranin-interacting protein isolated from brain cDNA libraries . Single amino acid mutagenesis indicated that residues within the IQ domain are important for CaM binding to neurogranin in vivo . The Ile-33 --> Gln point mutant completely inhibited and Arg-38 --> Gln and Ser-36 --> Asp point mutants reduced neurogranin/CaM interactions . These data demonstrate that CaM is the major protein that interacts with neurogranin in vivo and support the hypothesis that phosphorylation of neurogranin at Ser-36 regulates its binding to CaM.

J Clin Invest, 1999 Mar, 103(5), 723 - 9
Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts; Stagg AR et al.; We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness . Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum . Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5-14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days . TRMA fibroblasts were rescued from death with 10-30 nM thiamine (in the range of normal plasma thiamine concentrations) . Positive terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis . We assessed cellular uptake of {3H}thiamine at submicromolar concentrations . Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400-550 nM; Vmax 11 pmol/min/10(6) cells) in addition to a low-affinity unsaturable component . Mutant cells lacked detectable high-affinity uptake . At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype . Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine . We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death.

J Virol, 1999 Apr, 73(4), 2841 - 53
Hepatitis C virus core protein interacts with cellular putative RNA helicase; You LR et al.; The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters . To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein . One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained . This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth . In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein . Additionally, the protein's interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains . Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus . Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein . Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity . All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf . These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism . This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.

J Virol, 1999 Apr, 73(4), 2587 - 95
EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance; Shire K et al.; The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1) . To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1 . We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1 . EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins . EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions . We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions . Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP . An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient replication of oriP plasmids at wild-type levels . Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.

DNA Cell Biol, 1999 Feb, 18(2), 141 - 5
Characterization of the sequence and expression of a Ykt6 prenylated SNARE from rat; Catchpoole DR et al.; The Ykt6 protein represents a novel soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE), as it is the only one known without a hydrophobic transmembrane region at the carboxy terminus . For this SNARE, however, membrane interaction is thought to be mediated through a cysteine/aliphatic/aliphatic/methionine or histidine (CAAX) C-terminal motif, a consensus sequence involved in prenylated membrane anchoring . To date, two full-length Ykt6 cDNAs have been reported, these being in yeast and human, with a further protein predicted from a Caenorhabditis elegans cosmid . Using a mouse EST clone identified as having 65% homology with the human Ykt6, we isolated a cDNA clone encoding the rat Ykt6 homolog (rYkt6) . Sequence analysis of rYkt6 demonstrated that a high level of species conservation exists between the rat and human prenylated SNAREs, as both the nucleotide and amino acid sequences share >90% homology . Mammalian Ykt6 is shown here for the first time to be constitutively expressed in a variety of tissues . The species conservation and ubiquitous expression of prenylated SNAREs hence may be indicative of an important and central role for these proteins in cellular protein trafficking.

Plant Cell, 1999 Mar, 11(3), 377 - 92
LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen; Schwacke R et al.; During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes . Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth . Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed . Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids . Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline . To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters . LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization . Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity . Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Arch Med Res, 1999 Jan-Feb, 30(1), 69 - 73
Anti-inflammatory activity of Debaryomyces hansenii Cu,Zn-SOD; Garcia-Gonzalez A et al.; BACKGROUND: Cu,Zn-superoxide-dismutase, Cu,Zn-SOD, can be obtained from different sources with different anti-inflammatory activities . In this study we compared the anti-inflammatory capacity of the marine yeast Debaryomyces hanseii Cu,Zn-SOD (Dh-SOD) with that of bovine erythrocytes (Be-SOD) in a preventive and a therapeutic fashion . METHODS: Edema was induced by carrageenan injection into the rat hind paw and was evaluated using a mercury plethysmograph . Development of the inflammatory process was followed by volume displacement at time 0 (carrageenan injection), 1, 2, 3, 4, 5, 6, 9, 12, and 24 h thereafter . Three different SOD doses were used in preliminary experiments to prevent edema: 10, 100, and 1,000 U/kg . RESULTS: The results indicate that, at the lowest dose (10 U/kg), both SOD samples are effective in reducing inflammation in both the prostaglandin and amplification phases (-24.8% and -17.5% in the case of Be-SOD, and 11.8% and -18.7% in the case of Dh-SOD, respectively) (p < 0.05) . At 100 U/kg, Be-SOD also shows good anti-inflammatory activity in all edema phase (-27.1% in the serotonin phase; -19.4% in the prostaglandin phase; and -20% in the amplification phase) (p < 0.05), but Dh-SOD was less effective (-10.9%, -9.1%, and -5.7%) . At the highest dose tested (1000 U/kg), Dh-SOD was, again, more effective than Be-SOD in all three edema phases (-33.1% and -1.5%; -17.9% and -2.6%; and -13.8% and 6.7%, respectively) (p < 0.05) . When evaluated as a therapeutic alternative, single doses of Dh-SOD at 1,000 U/kg, and Be-SOD at 100 U/kg, both showed good anti-inflammatory activities (-31.7% and -23.5%, respectively) (p < 0.05) . CONCLUSION: For therapy purposes alone, Dh-SOD appears to be a better anti-inflammatory agent than Be-SOD in carrageenan-induced edema.

Bioessays, 1999 Jan, 21(1), 76 - 83
Construction of mammalian artificial chromosomes: prospects for defining an optimal centromere; Schindelhauer D; Two reports have shown that mammalian artificial chromosomes (MAC) can be constructed from cloned human centromere DNA and telomere repeats, proving the principle that chromosomes can form from naked DNA molecules transfected into human cells . The MACs were mitotically stable, low copy number and bound antibodies associated with active centromeres . As a step toward second-generation MACs, yeast and bacterial cloning systems will have to be adapted to achieve large MAC constructs having a centromere, two telomeres, and genomic copies of mammalian genes . Available construction techniques are discussed along with a new P1 artificial chromosome (PAC)-derived telomere vector (pTAT) that can be joined to other PACs in vitro, avoiding a cloning step during which large repetitive arrays often rearrange . The PAC system can be used as a route to further define the optimal DNA elements required for efficient MAC formation, to investigate the expression of genes on MACs, and possibly to develop efficient MAC-delivery protocols.

Bioessays, 1999 Jan, 21(1), 5 - 16
Replication origins in metazoan chromosomes: fact or fiction?
DePamphilis ML.
The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin . The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases . Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication . Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial . In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone . Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification . Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.

Mol Gen Genet, 1999 Feb, 261(1), 50 - 7
Genetic and physical analysis of a YAC contig spanning the fungal disease resistance locus Asc of tomato (Lycopersicon esculentum); Mesbah LA et al.; The Alternaria stem canker disease of tomato is caused by the necrotrophic fungal pathogen Alternaria alternata f . sp . lycopersici (AAL) . The fungus produces AAL toxins that kill the plant tissue . Resistance to the fungus segregates as a single locus, called Asc, and has been genetically mapped on chromosome 3 of tomato . We describe here the establishment of a 1383-kb YAC contig covering the Asc locus and a series of plants selected for recombination events around the Asc locus . It was shown that the YAC contig corresponds to a genetic distance of at least 11.2 cM . Thus, the recombination rate in the Asc region is six times higher (123 kb/cM) than the average for the tomato genome . Furthermore, the Asc locus could be localised to a 91-kb fragment, thus paving the way for the cloning and identification of the Asc gene(s) by complementation.

Hum Genet, 1999 Jan, 104(1), 77 - 82
Refined genetic and physical positioning of the gene for Doyne honeycomb retinal dystrophy (DHRD); Kermani S et al.; Doyne honeycomb retinal dystrophy (DHRD) is a late-onset autosomal dominant disorder that causes degeneration of the retina and can lead to blindness . We have previously assigned DHRD to a 5-cM region of chromosome 2p16 between marker loci D2S2739 and D2S378 . Using sequence-tagged sites (STSs), expressed sequence tags (ESTs) and polymorphic markers within the DHRD region, we have identified 18 yeast artificial chromosomes (YACs) encompassing the DHRD locus, spanning approximately 3 Mb . The YAC contig was constructed by STS content mapping of these YACs and incorporates 13 STSs, including four genes and six polymorphic marker loci . We also report the genetic mapping of two families with a dominant drusen phenotype to the DHRD locus, and genetic refinement of the disease locus to a critical interval flanked by microsatellite marker loci D2S2352 and D2S2251, a distance of approximately 700 kb . These studies exclude a number of candidate genes and provide a resource for construction of a transcriptional map of the region, as a prerequisite to identification of the DHRD disease-causing gene and genes for other diseases mapping in the region, such as Malattia leventinese and Carney complex.

Hum Genet, 1999 Jan, 104(1), 56 - 63
Fibroblast growth factor homologous factor 2 (FHF2): gene structure, expression and mapping to the Börjeson-Forssman-Lehmann syndrome region in Xq26 delineated by a duplication breakpoint in a BFLS-like patient; Gecz J et al.; Borjeson-Forssman-Lehmann syndrome (BFLS) is a syndromal X-linked mental retardation, which maps by linkage to the q26 region of the human X chromosome . We have identified a male patient with BFLS-like features and a duplication, 46,Y,dup(X)(q26q28), inherited from his phenotypically normal mother . Fluorescence in situ hybridisation using yeast artificial chromosome clones from Xq26 localised the duplication breakpoint to an approximately 400-kb interval in the Xq26.3 region between DXS155 and DXS294/DXS730 . Database searches and analysis of available genomic DNA sequence from the region revealed the presence of the fibroblast growth factor homologous factor gene, FHF2, within the duplication breakpoint interval . The gene structure of FHF2 was determined and two new exons were identified, including a new 5' end exon, 1B . FHF2 is a large gene extending over approximately 200 kb in Xq26.3 and is composed of at least seven exons . It shows tissue-specific alternative splicing and alternative transcription starts . Northern blot hybridisation showed highest expression in brain and skeletal muscle . The FHF2 gene localisation and tissue-specific expression pattern suggest it to be a candidate gene for familial cases of the BFLS syndrome and other syndromal and non-specific forms of X-linked mental retardation mapping to the region.

Plant J, 1999 Jan, 17(1), 19 - 30
A 100 kDa polypeptide associates with the V0 membrane sector but not with the active oat vacuolar H(+)-ATPase, suggesting a role in assembly; Li X et al.; The vacuolar H(+)-ATPase (V-ATPase) is responsible for acidifying endomembrane compartments in eukaryotic cells . Although a 100 kDa subunit is common to many V-ATPases, it is not detected in a purified and active pump from oat (Ward J.M . and Sze H . (1992) Plant Physiol . 99, 925-931) . A 100 kDa subunit of the yeast V-ATPase is encoded by VPH1 . Immunostaining revealed a Vph1p-related polypeptide in oat membranes, thus the role of this polypeptide was investigated . Membrane proteins were detergent-solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining . A 100 kDa polypeptide was not associated with the fully assembled ATPase; however, it was part of an approximately 250 kDa V0 complex including subunits of 36 and 16 kDa . Immunostaining with an affinity-purified antibody against the oat 100 kDa protein confirmed that the polypeptide was part of a 250 kDa complex and that it had not degraded in the approximately 670 kDa holoenzyme . Co-immunoprecipitation with a monoclonal antibody against A subunit indicated that peripheral subunits exist as assembled V1 subcomplexes in the cytosol . The free V1 subcomplex became attached to the detergent-solubilized V0 sector after mixing, as subunits of both sectors were co-precipitated by an antibody against subunit A . The absence of this polypeptide from the active enzyme suggests that, unlike the yeast Vph1p, the 100 kDa polypeptide in oat is not required for activity . Its association with the free Vo subcomplex would support a role of this protein in V-ATPase assembly and perhaps in sorting.

J Dairy Sci, 1999 Feb, 82(2), 429 - 37
Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows; Knowles SO et al.; Organic selenized yeast enriched with selenoamino acids or inorganic sodium selenate (Na2SeO4) was administered per os three times weekly as a drench for 133 d to previously unsupplemented cows that were grazing low Se pastures . Treatment groups received the equivalent of 2 or 4 mg of Se/d of either supplement form . Control cows did not receive a drench . Samples of blood and milk were collected regularly throughout the trial . Selenium concentrations in blood, milk, casein, and liver and glutathione peroxidase activity in blood and liver are reported as responses per milligram of Se intake . Mean blood Se concentrations in treated cows increased steadily and, by d 133, were 4.7 to 8.8 times that in controls . Selenized yeast was 2 to 3 times more effective than was Na2SeO4, and low Se intakes were 27% more efficient per milligram of Se administered than were high Se intakes at increasing milk Se concentration . Casein Se content mirrored that of milk; among all treated and control cows and throughout the trial, the molar ratio of Se in casein as a percentage of the Se in whole milk was constant at 71 +/- 1.2% . The Se concentration in liver biopsies taken on d 133 was indicative of total Se intake during the trial and ranged from 920 to 3920 nmol of Se/kg of fresh weight . These results demonstrate the differing efficacy of organic and inorganic Se dietary supplements to increase dairy cow Se status and to enhance Se content of milk and casein.

Development, 1999 Apr, 126(7), 1467 - 82
derrière: a TGF-beta family member required for posterior development in Xenopus; Sun BI et al.; TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm . Using a yeast-based selection scheme, we isolated derriere, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo . Unlike Vg1, derriere is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posterior mesoderm by mid-gastrula . The derriere expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF) . In turn, derriere activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes . derriere is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion . When misexpressed ventrally, derriere induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone . When misexpressed dorsally, derriere suppresses head formation . derriere can also posteriorize neurectoderm, but appears to do so indirectly . Together, these data suggest that derriere expression is compatible only with posterior fates . In order to assess the in vivo function of derriere, we constructed a dominant interfering Derriere protein (Cm-Derriere), which preferentially blocks Derriere activity relative to that of other TGFbeta family members . Cm-derriere expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure . Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated . This phenotype can be rescued by wild-type derriere and by VegT . Our findings indicate that derriere plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.

J Biol Chem, 1999 Mar 12, 274(11), 7292 - 301
Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions; Strumberg D et al.; A Ser740 --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16) . We characterized the cleavage complexes induced by the top2(S740W) in the human c-myc gene . In addition to resistance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone . Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference . C at position -2 and G at position +6 were observed for the top2(S740W) in addition to the previously reported C-1 and G+5 for the wild-type top2 . The VP-16-induced top2(S740W) cleavage complexes were also more stable . The most stable sites had strong preference for C-1, whereas the most reversible sites showed no base preference at positions -1 or -2 . Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium . These results indicate that the Ser740 --> Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors . Thus, residue 740 of top2 appears critical for both DNA and drug interactions.

J Biol Chem, 1999 Mar 12, 274(11), 6839 - 47
Genetic selection of mutations in the high affinity K+ transporter HKT1 that define functions of a loop site for reduced Na+ permeability and increased Na+ tolerance; Rubio F et al.; Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations . The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+ . A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection . Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance . By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated . Several independent mutations were isolated at the Asn-365 site . N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants . Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast . In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations . The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants.

Curr Opin Plant Biol, 1998 Dec, 1(6), 511 - 9
Cellular differentiation in the shoot epidermis; Martin C et al.; The recent advances in defining genes involved in shoot epidermal cell differentiation are impressive, especially the characterisation of genes involved in cellular patterning . The additional influences of environment and hormones on cellular patterning have recently been emphasised, and important connections have been made to changes in vegetative and reproductive growth phases . Despite these advances the cellular basis for differentiation remains less well defined, but now genetic and cell biological analysis from yeast may provide important models on which to develop further understanding.

Curr Opin Plant Biol, 1998 Jun, 1(3), 230 - 4
Sugar regulation of gene expression in plants; Smeekens S; The molecular details of sugar sensing and sugar-mediated signal transduction pathways are unclear but recent results suggest that hexokinase functions as an important plant sugar sensor in a way that is similar to that found in yeast . The use of mutants in Arabidopsis defective in specific signaling steps is of particular importance because these give access to the genes encoding components in the signaling pathways . In addition, the physiological analysis of such mutants may reveal the interaction of sugar-induced signaling pathways and those induced by other stimuli such as environmental or biotic stress.

Curr Opin Plant Biol, 1998 Apr, 1(2), 116 - 22
To pair or not to pair: chromosome pairing and evolution; Moore G; Chromosome pairing in wild-type wheat closely resembles the process in both yeast and Drosophila . The recent characterisation of a mutant Ph1 wheat and the observation that chromosome pairing in the absence of Ph1 more closely resembles that of mammals and maize has shed light on the evolution of chromosome pairing in the cereals.

Biochem Biophys Res Commun, 1999 Mar 5, 256(1), 249 - 54
Cloning of a GADD34-like gene that interacts with the zinc-finger transcription factor which binds to the p21(WAF) promoter; Hasegawa T et al.; A histone deacetylase inhibitor has been shown to induce differentiation of many cancer cells and senescence-like state of human fibroblasts . Previously, our data suggested that the region responsive to trichostatin A (TSA), a specific inhibitor of histone deacetylase, treatment in the p21(WAF1) promoter is located -100 bp upstream from transcription initiation site and contains a GC-box where both Sp1 and Sp3 are responsible . Here we show that another zinc-finger transcription factor, BFCOL1, which binds to the proximal proalpha2(I) collagen promoter, could also bind to this GC-box of the p21 promoter . In addition, we cloned a gene whose product interacts with this factor by yeast two-hybrid method . The cloned gene was a variant of GADD34 and lacking one PEST region . We found that this cDNA product decreased the DNA binding activity of BFCOL1 to the GC-rich region of p21 minimal promoter .

Biochem Biophys Res Commun, 1999 Mar 5, 256(1), 162 - 9
Spatiotemporal, allelic, and enforced expression of Ximpact, the Xenopus homolog of mouse imprinted gene impact; Yamada Y et al.; Mouse Impact is an imprinted gene encoding an evolutionarily conserved protein of unknown function . We isolated cDNA for the Xenopus homolog of Impact (Ximpact), since the clawed frog not only provides an excellent model for the study of gene function in early development but also allows the generation of interspecific F1 hybrids required for the examination of allelic expression status . The predicted product of Ximpact shows an extreme sequence similarity to those of mouse Impact and its homologs in nematoda, fission yeast, and budding yeast . The transcript of Ximpact is present in oocytes as well as in early embryos, and its spatial distribution is ubiquitous in both embryonic and adult stages . An RT-PCR-RFLP assay using the reciprocal interspecific F1 hybrids and a DNA polymorphism between X . laevis and X . borealis showed that Ximpact is expressed biallelically when analyzed as a whole embryo . Overexpression of Ximpact by RNA microinjection resulted in a higher than normal rate of gastrulation defects, suggesting the need for tight control of its dosage in early development .

Biochem Biophys Res Commun, 1999 Mar 5, 256(1), 52 - 6
Inactivation of mitogen-activated protein kinases by a mammalian tyrosine-specific phosphatase, PTPBR7; Ogata M et al.; Mitogen-activated protein kinase (MAPK) is inactivated through dephosphorylation of tyrosyl and threonyl regulatory sites . In yeast, both dual-specificity and tyrosine-specific phosphatases are involved in dephosphorylation . In mammals, however, no tyrosine-specific phosphatase has been identified molecularly to dephosphorylate MAPK in vivo . Recently, we and others have cloned a murine tyrosine-specific phosphatase, PTPBR7/PTP-SL, which is expressed predominantly in the brain . Here we report inactivation of the extracellular signal-regulated kinase (ERK) family MAPK by PTPBR7 . PTPBR7 made complexes with ERK1/ERK2 in vivo and dephosphorylated ERK1 in vitro . When overexpressed in mammalian cells, wild-type PTPBR7 suppressed the phosphorylation and activation of ERK by epidermal growth factor (EGF), nerve growth factor (NGF), and constitutively active MEK1, a mutant MAPK kinase . In contrast, catalytically inactive and ERK-binding-deficient mutants revealed little inhibition on the ERK cascade . These results indicate that PTPBR7 suppresses MAPK directly in vivo .

Mutat Res, 1999 Mar 8, 424(1-2), 207 - 19
Repair of DNA lesions: mechanisms and relative repair efficiencies; Braithwaite E et al.; DNA is frequently damaged by endogenous agents inside the cells . Some exogenous agents such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and may thus contribute to the 'background' DNA damage in humans . DNA lesions are normally removed by various repair mechanisms . The major repair mechanisms for various DNA lesions are summarized . In contrast to the extensively studied repair mechanisms, much less is known about the relative repair efficiencies of various DNA lesions . Since DNA repair is a crucial defense against carcinogenesis, it may constitute an important factor affecting the carcinogenicity of DNA damaging agents . We have adopted a human cell-free system for measuring relative DNA repair efficiencies based on the concept of repair competition between acetylaminofluorene adducts and other DNA lesions of interest . Using this in vitro system, we determined the relative repair efficiencies of PAH adducts induced by: anti-(+/-)-benzo{a}pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), anti-(+/-)-benz{a}anthracene-trans-3,4-dihydrodiol-1,2-epoxide (BADE-I), anti-(+/-)-benz{a}anthracene-trans-8,9-dihydrodiol-10, 11-epoxide (BADE-II), anti-(+/-)-benzo{b}fluoranthene-trans-9, 10-dihydrodiol-11,12-epoxide (BFDE), anti-(+/-)-chrysene-trans-1, 2-dihydrodiol-3,4-epoxide (CDE), and anti-(+/-)-dibenzo{a, l}pyrene-trans-11,12-dihydrodiol-13,14-epoxide (DBPDE) . While damage by BPDE, DBPDE, CDE, and BFDE were repaired by nucleotide excision repair as efficiently as AAF adducts, the repair of BADE-I and BADE-II adducts were significantly slower in human cell extracts . Damage by DBPDE at 3 microM in vitro yielded approximately 5-fold higher DNA adducts than BPDE as determined by quantitative PCR . This potent DNA reactivity may account in part for the potent carcinogenicity of dibenzo{a,l}pyrene . The correlation of these results to the carcinogenic properties of the PAH compounds is discussed . Furthermore, we show that NER plays a role in AP site repair in vivo in the eukaryotic model organism yeast .

J Mol Biol, 1999 Mar 12, 286(5), 1311 - 23
MADS-box transcription factors adopt alternative mechanisms for bending DNA; West AG et al.; Transcription factor-induced DNA bending is important in determining local promoter architecture and it is thought to be a key determinant of their function . The human MADS-box transcription factors serum response factor and MEF2A exhibit different propensities to bend their binding sites . Here, we have investigated the ability of several family members from different species to bend DNA and the molecular mechanisms underlying this process . Differential DNA bending is observed in yeast and plant MADS-box proteins . Like MEF2A, the yeast proteins Rlm1 and Smp1 exhibit low DNA bending propensities . A comparison of serum response factor and SQUA reveals that the basic mechanisms of DNA bending appear to be conserved between these proteins, although several key differences do exist . In contrast to serum response factor, SQUA bends DNA in a DNA sequence-dependent manner . In both proteins, protein-DNA contacts made between residues in the beta-loop and the N-terminal end of the recognition helices in the MADS-box are the major determinants of DNA bending . However, although residues which are involved in DNA bending are predicted to be located in similar positions in their tertiary structures, different residues dictate bending by each protein . Further complexities are uncovered in the links between the DNA bending propensity and the binding specificity . In combination with structural studies, our results provide a model to explain how differential bending by MADS-box proteins is achieved at the molecular level and provide insights into how this might affect their biological function .

EMBO J, 1999 Mar 1, 18(5), 1159 - 71
The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15; Sengar AS et al.; Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins . We have cloned two mammalian genes which code for a number of endocytic adaptor proteins . Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains . Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces . Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively . Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold . The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.

Diabetologia, 1999 Feb, 42(2), 195 - 203
Glucose induces early growth response gene (Egr-1) expression in pancreatic beta cells; Josefsen K et al.; A copy deoxyribonucleic acid (cDNA) clone of the immediate early growth response gene, egr-1 (Krox-24, Zif268, NGFI-1), was isolated through subtractive hybridization screening to identify glucose-induced genes in pancreatic beta cells . Glucose rapidly and transiently induced egr-1 mRNA in the SV40-transformed murine beta-cell line, MIN6 . Glucose also increased egr-1 mRNA expression in INS-1, betaTC3 and RINm5F beta-cell lines, although with different kinetics . Expression of the 82 kDa Egr-1 protein was induced both in MIN6 cells stimulated with glucose in vitro and in primary rat islet cells stimulated in vivo or in vitro . This response is unique to beta cells since glucose did not affect egr-1 expression in NIH-3T3 fibroblasts or glucose-sensitive hepatocytes . In beta cells egr-1 induction is specifically associated with insulin secretion, as it was not observed after stimulation with serum or insulin but was elicited by insulin secretagogues, including membrane depolarizing agents and cAMP agonists . Moreover, induction of egr-1 by glucose was inhibited by EDTA, indicating dependence on influx of extracellular Ca2+ . Other immediate early response genes, c-fos and junB, were also induced following glucose stimulation with kinetics similar to egr-1, whereas c-jun and junD expression were not affected . Since the zinc-finger protein encoded by egr-1 is highly homologous to transcription factors that control expression of glucose-regulated genes in yeast, Egr-1 could mediate delayed adaptive responses of beta cells to sustained glucose stimulation through transcriptional regulation.

Int J Pharm, 1999 Mar 15, 179(2), 267 - 71
Flourescence-assay on traces of protein on re-usable medical devices: cleaning efficiency; Verjat D et al.; The cleaning of re-usable medical devices before disinfection or sterilization is recognized as being an essential phase . Detection of residual proteins can be used to validate the process, provided a sufficiently sensitive method is employed . A fluorescent method is presented, using orthophtalaldehyde (OPA) bound to N,N dimethyl-2-mercaptoethylammonium, to demonstrate the presence of amino acids on a medical device following cleaning . The sensitivity of this method (10-5 g/l) was assessed and the applicability of this detection technique is verified, using three types of carriers (steel blades, glass tubes or ceramic penicylinders), three types of contaminants (yeast extract, bovine albumin with native sheep's blood and formaldehyde fixed fibrin) . In this context, studies involving formaldehyde-fixed fibrin are more sensitive and are to be recommended.Copyright

Genomics, 1999 Mar 1, 56(2), 221 - 3
Elongated telomeres in scid mice; Hande P et al.; Severe combined immunodeficiency (scid) mice are deficient in the enzyme DNA-PK (DNA-dependent protein kinase) as a result of the mutation in the gene encoding the catalytic subunit (DNA-PKcs) of this enzyme . DNA-PKcs is a member of the phosphatidylinositol 3-kinase superfamily, which includes the human protein ATM (ataxia telangiectasia mutated) and the yeast protein Tel1 . Using Q-FISH (quantitative fluorescence in situ hybridization), we show here that scid mice from four different genetic backgrounds have, on average, 1.5-2 times longer telomeres than those of corresponding wild-type mice . Our results point to the possibility that DNA-PKcs may, directly or indirectly, be involved in telomere length regulation in mammalian cells .

Genomics, 1999 Mar 1, 56(2), 197 - 202
Genomic organization and chromosomal localization of the human histone deacetylase 3 gene; Mahlknecht U et al.; Reversible acetylation of histone proteins plays a critical role in transcriptional regulation, cell cycle progression, and developmental events . The steady state of histone acetylation is controlled by the enzymatic activities of multiple histone acetyltransferases and histone deacetylases (HDACs) . Three distinct human HDACs are homologous to RPD3, a yeast transcriptional regulator . We have isolated and sequenced a genomic clone for the human HDAC3 gene . This is a single-copy gene spanning a region of at least 13 kb . Determination of the intron-exon splice junctions established that the gene is encoded by 15 exons ranging in size from 56 to 657 bp . Fluorescence in situ hybridization studies localized this gene to 5q31 . Double-target experiments in which both HDAC3 and the early-growth response 1 gene (EGR1), which is localized in the 5q31.2 region, were used as probes showed that the HDAC3 gene lies in region 5q31.3, immediately distal to EGR1 with respect to the centromere .

Genomics, 1999 Mar 1, 56(2), 149 - 59
Physical and transcriptional map of a 3-Mb region of mouse chromosome 1 containing the gene for the neural tube defect mutant loop-tail (Lp); Eddleston J et al.; The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis . To identify the Lp gene, a positional cloning approach has been adopted . Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1 . Here we report a detailed physical map of this region . The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb . Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval . Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags . Added to other known genes in the region, a total of 29 genes were located and ordered within the contig . Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene . The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb . Within this region, 10 potential candidate genes have been mapped . The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q .

Mamm Genome, 1999 Mar, 10(3), 289 - 93
High-resolution, human-bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus; Pirottin D et al.; A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends . The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb . A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region . Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human . Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments.

Mamm Genome, 1999 Mar, 10(3), 229 - 34
High-resolution comparative physical mapping of mouse chromosome 10 in the region of homology with human chromosome 21; Cole SE et al.; Comparative mapping of human and mouse chromosomes can be used to predict locations of homologous loci between the species, provides the substrate to examine the process of chromosomal evolution, and facilitates the continuing development of mouse genetic models for human disorders . A YAC contig of the region of mouse Chromosome (Chr) 10 (MMU10) that demonstrates conserved linkage with the distal portion of human Chr 21 (HSA21) has been constructed . The contig contains all known genes mapped in both species, defines the proximal region of homology between MMU10 and HSA22, and contains the evolutionary junction between HSA21 and HSA22 on MMU10 . It consists of 23 YACs and 2 PACs, and covers 3.2 Mb of MMU10 . The average marker density for this region is 1 marker/69 kb . Nine of 22 expressed sequences are mapped here for the first time in mouse, and two are newly characterized expressed sequences . The contig also contains 12 simple sequence repeats (SSRs) and 16 YAC and PAC endclone markers . YAC fragmentation analysis was used to create a physical map for the proximal 2.2 Mb of the contig . Cloning of the corresponding region of HSA21 has proven difficult, and the mouse contig includes segments absent from previously described sequence ready maps of HSA21.

Oncogene, 1999 Feb 18, 18(7), 1495 - 501
Physical and functional interactions between the transcription factor PU.1 and the coactivator CBP; Yamamoto H et al.; Yeast two-hybrid system was employed to isolate novel proteins that physically interact with PU.1, a member of Ets family transcription factors . Sequence analyses of several isolated clones positive for beta-galactosidase activity revealed that one of these clones was confirmed to encode a transcriptional coactivator, CREB binding protein (CBP) . GST binding assay showed that the interacting sites were located at the transcriptional activation domain of PU.1 through 74-122 and the region spanning residues 1283-1915 of CBP . CBP potentiated PU.1-mediated transcription of the reporter gene driven by the multimerized PU.1-binding sites, suggesting that CBP functions as a coactivator for PU.1 . Considering that CBP is a limited cellular component to function as a coactivator for several transcription factors, CBP may mediate synergistic and antagonistic interactions between PU.1 and other transcription factors during the process of hematopoietic cell differentiation.

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