J Lipid Res, 1999 Nov, 40(11), 2099 - 110 Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation; Raspe E et al.; 3-Thia fatty acids are modified fatty acids that promote hepatic peroxisome proliferation and decrease serum triacylglycerol, cholesterol and free fatty acid levels in rats . In vivo administration of tetradecylthioacetic acid (TTA) to rats led to a significant decrease in liver apolipoproteins apoA-I, A-II, A-IV, and C-III mRNA levels, and to an increase of liver acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-II, and 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA synthase) mRNA levels and activities . By contrast, no significant changes of lipoprotein lipase (LPL) mRNA levels were detected in rat epididymal adipose tissue . Liver carnitine palmitoyltransferase-I, apoB, apoE, and LDL receptor mRNA levels were not significantly affected . When tested in vitro, TTA increased rat ACO and carnitine palmitoyltransferase-I mRNA levels in primary rat hepatocytes and also LPL mRNA levels in 3T3-L1 preadipocytes . TTA also enhanced the transcriptional activity of chimeras containing the DNA binding domain of the yeast transcription factor Gal4 fused to the ligand binding domain of either human PPARalpha or human PPARgamma . The effect depended on the concentration tested and the cell type.In conclusion, our data suggest that in vitro, TTA activates both PPARalpha and PPARgamma, but the latter with much lower affinity . TTA affects serum lipid levels in vivo in rats by acting mainly on the liver via PPARalpha where it decreases the liver expression of genes involved in vascular lipid transport and increases the expression of genes involved in intracellular fatty acid metabolism . -Raspe, E., L . Madsen, A-M . Lefebvre, I . Leitersdorf, L . Gelman, J . Peinado-Onsurbe, J . Dallongeville, J-C . Fruchart, R . Berge, and B . Staels . Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation. Am J Pathol, 1999 Dec, 155(6), 1817 - 21 Exclusive detection of the t(11;18)(q21;q21) in extranodal marginal zone B cell lymphomas (MZBL) of MALT type in contrast to other MZBL and extranodal large B cell lymphomas; Rosenwald A et al.; Extranodal mucosa-associated lymphoid tissue (MALT)-type lymphomas and nodal and splenic marginal zone B cell lymphomas (MZBL) share morphological and immunophenotypic features with marginal zone B cells of reactive lymphoid tissues . Although displaying a similar immunophenotype, recent investigations suggest fundamental genetic differences among these subgroups . To determine the prevalence of the t(11;18) in a larger series of MALT-type lymphomas and to investigate a possible occurrence in other lymphomas, we screened 106 non-Hodgkin's lymphomas (NHL) by interphase cytogenetics using yeast artificial chromosome (YAC) probes flanking the breakpoint at 11q21 . A signal constellation indicating a disruption in 11q21 and thus pointing to the presence of the t(11;18) was observed in 9 of 33 (27%) low-grade lymphomas of MALT type . The complete absence of t(11;18)-positive cells in 32 primary and secondary extranodal high-grade lymphomas suggests that low-grade lymphomas of MALT type characterized by the t(11;18) are unlikely to transform into high-grade tumors . The absence of tumor cells carrying the t(11;18) in nodal MZBL challenges the assumption that most, if not all, of these tumors represent the nodal manifestation of a so far undetected low-grade lymphoma of MALT type . The t(11;18) was not detected in a single case of 29 splenic MZBL investigated . This observation strengthens the view that splenic MZBL are biologically different from extranodal MZBL of MALT type. Protein Sci, 1999 Nov, 8(11), 2487 - 93 Locating and identifying posttranslational modifications by in-source decay during MALDI-TOF mass spectrometry; Lennon JJ et al.; A technique is described for identifying and locating posttranslational modifications (PTMs) in peptides and proteins of known sequence by interpretation of c(n) ion signals generated by in-source decay during delayed ion extraction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . Sites of phosphorylation in seven synthetic peptides were determined, as was the location of both the heme group and N,N,N-trimethyllysine in yeast cytochrome c . A semi-automated data analysis process facilitates the identification of segments of the sequence on each side of the PTM, permitting its placement at the junction of the segments and definition of the added mass . A graphical display facilitates illustration of both the location and mass of the PTM. Mol Cell Biol, 2000 Jan, 20(1), 329 - 39 Characterization of a Ustilago maydis gene specifically induced during the biotrophic phase: evidence for negative as well as positive regulation; Basse CW et al.; The phytopathogenic basidiomycete Ustilago maydis requires its host plant, maize, for completion of its sexual cycle . To investigate the molecular events during infection, we used differential display to identify plant-induced U . maydis genes . We describe the U . maydis gene mig1 (for "maize-induced gene"), which is not expressed during yeast-like growth of the fungus, is weakly expressed during filamentous growth in axenic culture, but is extensively upregulated during plant infection . mig1 encodes a small, highly charged protein of unknown function which contains a functional N-terminal secretion sequence and is not essential for pathogenic development . Adjacent to mig1 is a second gene (mdu1) related to mig1, which appears to result from a gene duplication . mig1 gene expression during the infection cycle was assessed by fusing the promoter to eGFP . Expression of mig1 was absent in hyphae growing on the leaf surface but was detected after penetration and remained high during subsequent proliferation of the fungus until teliospore formation . Successive deletions as well as certain internal deletions in the mig1 promoter conferred elevated levels of reporter gene expression during growth in axenic culture, indicative of negative regulation . During fungal growth in planta, sequence elements between positions -148 and -519 in the mig1 promoter were specifically required for high levels of induction, illustrating additional positive control . We discuss the potential applications of mig1 for the identification of inducing compounds and the respective regulatory genes. J Biol Chem, 1999 Dec 17, 274(51), 36796 - 800 Identification of COUP-TF as a transcriptional repressor of the c-mos proto-oncogene; Lin HB et al.; The c-mos proto-oncogene is specifically expressed in the male and female germ cells of the mouse and other vertebrates . We previously identified a 15-base pair sequence element (B2) as the binding site of a candidate repressor of c-mos transcription in somatic cells . In the present study, we used the yeast one-hybrid system to isolate HeLa cell cDNAs encoding proteins that specifically bound to the c-mos B2 element . Nucleotide sequencing identified several of the clones isolated in this screen as the orphan nuclear receptors COUP-TFI and COUP-TFII . A COUP-TF-binding site was then identified within the B2 sequence . Complexes formed between purified COUP-TFs and the c-mos B2 probe comigrated in electrophoretic mobility shift assays with those formed using whole nuclear extracts of NIH 3T3 or HeLa cells . Moreover, the complexes formed with NIH 3T3 nuclear extracts and B2 probe were supershifted with antibody against COUP-TF, identifying COUP-TF as the candidate repressor previously detected in these somatic cell extracts . Substitution of a consensus COUP-TF-binding site for the c-mos negative regulatory element suppressed expression from the c-mos promoter in transfected somatic cells, demonstrating the functional activity of COUP-TF as a repressor of c-mos transcription. J Biol Chem, 1999 Dec 17, 274(51), 36774 - 80 Binding of 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947); Fuglsang AT et al.; 14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription . The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H(+)-ATPase . The 14-3-3 binding site in the Arabidopsis plasma membrane H(+)-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr(946)-Thr-Val) . Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM) . Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947) . We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein. J Biol Chem, 1999 Dec 17, 274(51), 36663 - 9 Functional interaction between Galpha(z) and Rap1GAP suggests a novel form of cellular cross-talk; Meng J et al.; G(z) is a member of the G(i) family of trimeric G proteins whose primary role in cell physiology is still unknown . In an ongoing effort to elucidate the cellular functions of G(z), the yeast two-hybrid system was employed to identify proteins that specifically interact with a mutationally activated form of Galpha(z) . One of the molecules uncovered in this screen was Rap1GAP, a previously identified protein that specifically stimulates GTP hydrolytic activity of the monomeric G protein Rap1 and thus is believed to function as a down-regulator of Rap1 signaling . Like G(z), the precise role of Rap1 in cell physiology is poorly understood . Biochemical analysis using purified recombinant proteins revealed that the physical interaction between Galpha(z) and Rap1GAP blocks the ability of RGSs (regulators of G protein signaling) to stimulate GTP hydrolysis of the alpha subunit, and also attenuates the ability of activated Galpha(z) to inhibit adenylyl cyclase . Structure-function analyses indicate that the first 74 amino-terminal residues of Rap1GAP, a region distinct from the catalytic core domain responsible for the GAP activity toward Rap1, is required for this interaction . Co-precipitation assays revealed that Galpha(z), Rap1GAP, and Rap1 can form a stable complex . These data suggest that Rap1GAP acts as a signal integrator to somehow coordinate and/or integrate G(z) signaling and Rap1 signaling in cells. J Biol Chem, 1999 Dec 17, 274(51), 36609 - 15 Identification of arfophilin, a target protein for GTP-bound class II ADP-ribosylation factors; Shin OH et al.; Yeast two-hybrid screening of a human kidney cDNA library using the GTP-bound form of a class II ADP-ribosylation factor (ARF5) identified a novel ARF5-binding protein with a calculated molecular mass of 82.4 kDa, which was named arfophilin . Northern hybridization analysis showed high level arfophilin mRNA expression in human heart and skeletal muscle . Arfophilin bound only to the active, GTP-bound form of ARF5 and did not bind to GTP-ARF3, which is a class I ARF . The N terminus of ARF5 (1-17 amino acids) was essential for binding to arfophilin . The GTP-bound form of ARF5 with amino acid residues in the N terminus mutated to those in ARF4 (another class II ARF) also bound to arfophilin, suggesting it is a target protein for GTP-bound forms of class II ARFs . The binding site for ARF on arfophilin was localized to the C terminus (residues 612-756), which contains putative coiled-coil structures . Recombinant arfophilin overexpressed in CHO-K1 cells was localized in the cytosol and translocated to a membrane fraction in association with GTP-bound ARF5 . ARF5 containing the N terminus of ARF3 did not promote translocation indicating that class II ARFs are specific carriers for arfophilin. J Biol Chem, 1999 Dec 17, 274(51), 36592 - 600 Identification and characterization of cvHsp . A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues; Krief S et al.; Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp . cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart . The cvHsp gene mapped to 1p36.23-p34.3 between markers D1S434 and D1S507 . The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues . In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding) . In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart . Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart . cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with alpha-filamin or actin-binding protein 280 . Within cvHsp, amino acid residues 56-119 were shown to be important for its specific interaction with the C-terminal tail of alpha-filamin. J Biol Chem, 1999 Dec 17, 274(51), 36288 - 92 MIR is a novel ERM-like protein that interacts with myosin regulatory light chain and inhibits neurite outgrowth; Olsson PA et al.; The ERM protein family members ezrin, radixin, and moesin are cytoskeletal effector proteins linking actin to membrane-bound proteins at the cell surface . Here we report on the cloning of myosin regulatory light chain interacting protein (MIR), a protein with an ERM-homology domain and a carboxyl-terminal RING finger, that is expressed, among other tissues, in brain . MIR is distributed in cultured COS cells, in a punctuated manner as shown using enhanced green fluorescent protein (EGFP)-tagged MIR and by staining with a specific antibody for MIR . In the yeast two-hybrid system and in transfected COS cells, MIR interacts with myosin regulatory light chain B, which in turn regulates the activity of the actomyosin complex . Overexpression of MIR cDNA in PC12 cells abrogated neurite outgrowth induced by nerve growth factor (NGF) without affecting TrkA signaling . The results show that MIR, a novel ERM-like protein, affects cytoskeleton interactions regulating cell motility, such as neurite outgrowth. J Biol Chem, 1999 Dec 17, 274(51), 36159 - 67 MRG1 binds to the LIM domain of Lhx2 and may function as a coactivator to stimulate glycoprotein hormone alpha-subunit gene expression; Glenn DJ et al.; Tissue-specific expression of the alpha-subunit gene of glycoprotein hormones involves an enhancer element designated the pituitary glycoprotein basal element, which interacts with the LIM homeodomain transcription factor, Lhx2 . In the present studies we have explored the function of the LIM domain of Lhx2 in stimulating alpha-subunit transcription . When fused to the GAL4 DNA-binding domain, the LIM domain of Lhx2 was shown to contain a transcriptional activation domain . Furthermore, in the context of an alpha-subunit reporter gene in which a GAL4-binding site replaced the pituitary glycoprotein basal element, the LIM domain enhanced both basal and Ras-mediated transcription . In addition, a synergistic response to Ras activation was observed when the Lhx2 LIM domain and the transactivation domain of Elk1 are directed to a minimal reporter gene . A yeast two-hybrid screen identified the recently described melanocyte-specific gene-related gene 1 (MRG1) as an Lhx2 LIM-interacting protein . MRG1 was shown to bind Lhx2 in vitro, and a co-immunoprecipitation assay provided evidence that endogenous MRG1 forms a complex with Lhx2 in alphaT3-1 cells . Expression of MRG1 in alphaT3-1 cells enhanced alpha-subunit reporter gene activity . MRG1 was also shown to bind in vitro to the TATA-binding protein and the transcriptional coactivator, p300 . These data suggest a model in which the Lhx2 LIM domain activates transcription through interaction with MRG1 leading to recruitment of p300/CBP and the TATA-binding protein. J Biol Chem, 1999 Dec 17, 274(51), 36089 - 96 Isolation, characterization, and functional expression of cDNAs encoding NADH-dependent methylenetetrahydrofolate reductase from higher plants; Roje S et al.; Methylenetetrahydrofolate reductase (MTHFR) is the least understood enzyme of folate-mediated one-carbon metabolism in plants . Genomics-based approaches were used to identify one maize and two Arabidopsis cDNAs specifying proteins homologous to MTHFRs from other organisms . These cDNAs encode functional MTHFRs, as evidenced by their ability to complement a yeast met12 met13 mutant, and by the presence of MTHFR activity in extracts of complemented yeast cells . Deduced sequence analysis shows that the plant MTHFR polypeptides are of similar size (66 kDa) and domain structure to other eukaryotic MTHFRs, and lack obvious targeting sequences . Southern analyses and genomic evidence indicate that Arabidopsis has two MTHFR genes and that maize has at least two . A carboxyl-terminal polyhistidine tag was added to one Arabidopsis MTHFR, and used to purify the enzyme 640-fold to apparent homogeneity . Size exclusion chromatography and denaturing gel electrophoresis of the recombinant enzyme indicate that it exists as a dimer of approximately 66-kDa subunits . Unlike mammalian MTHFR, the plant enzymes strongly prefer NADH to NADPH, and are not inhibited by S-adenosylmethionine . An NADH-dependent MTHFR reaction could be reversible in plant cytosol, where the NADH/NAD ratio is 10(-3) . Consistent with this, leaf tissues metabolized {methyl-(14)C}methyltetrahydrofolate to serine, sugars, and starch . A reversible MTHFR reaction would obviate the need for inhibition by S-adenosylmethionine to prevent excessive conversion of methylene- to methyltetrahydrofolate. J Biol Chem, 1999 Dec 17, 274(51), 36035 - 8 MEKK3 directly regulates MEK5 activity as part of the big mitogen-activated protein kinase 1 (BMK1) signaling pathway; Chao TH et al.; Big mitogen-activated protein (MAP) kinase (BMK1), also known as ERK5, is a member of the MAP kinase family whose cellular activity is elevated in response to growth factors, oxidative stress, and hyperosmolar conditions . Previous studies have identified MEK5 as a cellular kinase directly regulating BMK1 activity; however, signaling molecules that directly regulate MEK5 activity have not yet been defined . Through utilization of a yeast two-hybrid screen, we have identified MEKK3 as a molecule that physically interacts with MEK5 . This interaction appears to take place in mammalian cells as evidenced by the fact that cellular MEK5 and MEKK3 co-immunoprecipitate . In addition, we show that a dominant active form of MEKK3 stimulates BMK1 activity through MEK5 . Moreover, we demonstrate that MEKK3 activity is required for growth factor mediated cellular activation of endogenous BMK1 . Taken together, these results identify MEKK3 as a kinase that regulates the activity of MEK5 and BMK1 during growth factor-induced cellular stimulation. Nippon Rinsho, 1999 Oct, 57(10), 2211 - 7 {Nicotinic acid and nicotinamide}; Kobayashi M et al.; Nicotinic acid and nicotinamide are called niacin . They are the antipellagra vitamin essential to many animals for growth and health . In human being, niacin is believed necessary together with other vitamins for the prevention and cure of pellagra . Niacin is widely distributed in nature; appreciable amounts are found in liver, fish, yeast and cereal grains . Nicotinamide is a precursor of the coenzyme NAD and NADP . Some of the most understood metabolic processes that involve niacin are glycolysis, fatty acid synthesis and respiration . Niacin is also related to the following diseases: Hartnup disease; blue diaper syndrome; tryptophanuria; hydroxykynureninuria; xanthurenic aciduria; Huntington's disease. Nucleic Acids Res, 2000 Jan 1, 28(1), 163 - 5 MitoNuc and MitoAln: two related databases of nuclear genes coding for mitochondrial proteins; Pesole G et al.; Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases . All functions of mitochondria depend on the interaction of nuclear and organellar genomes . Mitochondrial genomes have been extensively sequenced and analysed and the data collected in several specialised databases . In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc and MitoAln, two related databases containing, respectively, detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa and yeast, and the multiple alignments of the relevant homologous protein coding regions . MitoNuc and MitoAln retrieval through SRS at can easily allow the extraction of sequence data, subsequences defined by specific features and nucleotide or amino acid multiple alignments. Nucleic Acids Res, 2000 Jan 1, 28(1), 97 - 101 INE: a rice genome database with an integrated map view; Sakata K et al.; The Rice Genome Research Program (RGP) launched a large-scale rice genome sequencing in 1998 aimed at decoding all genetic information in rice . A new genome database called INE (INtegrated rice genome Explorer) has been developed in order to integrate all the genomic information that has been accumulated so far and to correlate these data with the genome sequence . A web interface based on Java applet provides a rapid viewing capability in the database . The first operational version of the database has been completed which includes a genetic map, a physical map using YAC (Yeast Artificial Chromosome) clones and PAC (P1-derived Artificial Chromosome) contigs . These maps are displayed graphically so that the positional relationships among the mapped markers on each chromosome can be easily resolved . INE incorporates the sequences and annotations of the PAC contig . A site on low quality information ensures that all submitted sequence data comply with the standard for accuracy . As a repository of rice genome sequence, INE will also serve as a common database of all sequence data obtained by collaborating members of the International Rice Genome Sequencing Project (IRGSP) . The database can be accessed at dna.affrc.go.jp:82/giot/INE . html or its mirror site at http://www.staff.or.jp/giot/INE.html J Cell Sci, 2000 Jan, 113 ( Pt 1), 103 - 12 Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella; Fujita A et al.; The small GTPase Rho; functions as a molecular switch that regulates various cellular processes such as cell adhesion, motility, gene expression and cytokinesis . We previously isolated several putative Rho; targets including rhophilin which bound selectively to the GTP-bound form of Rho; . Rhophilin is expressed highly in testis and is localized specifically in sperm flagella . The presence of a PDZ domain at the carboxy terminus of rhophilin suggested that rhophilin works as an adaptor molecule . To test this hypothesis, we employed a yeast two hybrid system using the rhophilin PDZ domain as a bait, and screened a mouse testis cDNA library . We isolated several positive clones containing the same insert . The open reading frame of the cDNA encoded a novel protein of 212 amino acids designated as ropporin from a Japanese word 'oppo' (the tail) . The amino-terminal 40 amino acid sequence of ropporin showed high homology to that of the regulatory subunit of type II cAMP-dependent protein kinase, which is involved in dimerization and binding to A-kinase anchoring proteins . Consistently, a yeast two hybrid assay and gel filtration of recombinant ropporin indicated that ropporin dimerizes through this domain . Deletion analysis indicated that the carboxy-terminal four amino acids are essential for binding of ropporin to rhophilin, and ropporin and RhoV14 coprecipitated in the presence of rhophilin in vitro . Northern blot analysis showed that ropporin is exclusively expressed in testis, and induced at the late stage of spermatogenesis . This induction paralleled that of rhophilin . Immunocytochemistry using anti-ropporin antibody showed that ropporin is localized in the principal piece and the end piece of sperm flagella . Electronmicroscopy revealed that ropporin is mostly localized in the inner surface of the fibrous sheath while rhophilin is present in the outer surface of the outer dense fiber . These results suggest that rhophilin and ropporin may form a complex in sperm flagella. Rinsho Byori, 1999 Oct, 47(10), 976 - 9 {Subcutaneous phaeohyphomycosis of the right thumb}; Maekura S et al.; Black fungi are a group of fungi that are characterized by the development of a pale brown to black color in the cell walls of their vegetative cells, conidia, or both . A mycotic infection caused by a member of black fungi can be subdivided into three clinical entities: phaeohyphomycosis, chromoblastomycosis, and mycetoma . Phaeohyphomycosis is distinguished from mycetoma by the absence of grain (organized, interwoven mycelial aggregates) formation, and from chromoblastomycosis by the absence of sclerotic bodies (thick-walled muriform cells) . Phaeohyphomycosis is a rare disease and has been sporadically reported . In the present report, phaeohyphomycosis of the right thumb of a 72-year-old man was presented . A precipitating trauma of two months earlier at the site was recalled . A solitary mass, 10 mm in diameter, was gradually formed in the palm side of the distal right thumb and finally resected . Histological examination disclosed a solitary granulomatous lesion surrounded by an incomplete fibrous capsule . The lesion mainly involved subcutaneous tissue and was composed of multiple pyogranulomas . Pigmented branched septate hyphae and yeast-like cells were sparsely found in the periphery of the abscess and within histiocytic cells of the granulomas . No sclerotic cells were detected . When pigmentation of black fungi in tissue is as faint as in the present case, Fontana-Masson staining is useful to accentuate the presence of melanin-like pigment of fungal cell walls. Mycoses, 1999, 42 Suppl 1, 12 - 21 {Taxonomy and ecology of the genus Candida}; Schauer F et al.; Candida is a heterogeneous genus which contains about a quarter of all yeast species . It includes not only species of uncertain affiliation but also unrelated strains whose phylogenetic relationships have not been resolved . A great variety of CoQ types are present in the genus, the mol % G + C ranges from 30-63%, and species that were found to sporulate have teleomorphic counterparts in 11 different genera . Candida species are mainly associated with plants, rotting vegetation, with insects which feed on plants or with food . In line with this, 71% of Candida species utilize xylose (wood degradation), 57% of species use cellobiose (cellulose degradation), 29% oxidize aliphatic hydrocarbons (components of plant cuticula), 27% of species degrade starch as a plant storage material, and 7% utilize methanol as a possible metabolite from pectin catabolism . 85% of species require individual vitamins produced mainly in plant materials . 65% of Candida species are not able to grow at temperatures of 37 degrees C . In comparison only relatively few species occur normally in humans and other warm blooded animals . About 16% of type strains and selected strains for comparative purposes (CBS) were isolated from human specimens . Perhaps up to 10% of Candida species may be of medical importance, though this has so far only been clearly demonstrated for less than 5% of currently known species. Pharmacogenetics, 1999 Oct, 9(5), 641 - 50 Enhanced proteasomal degradation of mutant human thiopurine S-methyltransferase (TPMT) in mammalian cells: mechanism for TPMT protein deficiency inherited by TPMT*2, TPMT*3A, TPMT*3B or TPMT*3C; Tai HL et al.; Inheritance of the TPMT*2, TPMT*3A and TPMT*3C mutant alleles is associated with deficiency of thiopurine S-methyltransferase (TPMT) activity in humans . However, unlike TPMT*2 and TPMT*3A, the catalytically active protein coded by TPMT*3C does not undergo enhanced proteolysis when heterologously expressed in yeast, making it unclear why this common mutant allele should be associated with inheritance of TPMT-deficiency . To further elucidate the mechanism for TPMT deficiency associated with these alleles, we characterized TPMT proteolysis following heterologous expression of wild-type and mutant proteins in mammalian cells . When expressed in COS-1 cells, proteins encoded by TPMT*2, TPMT*3A, and TPMT*3C cDNAs had significantly reduced steady-state levels and shorter degradation half-lives compared with the wild-type protein . Similarly, in rabbit reticulocyte lysate (RRL), these mutant TPMT proteins were degraded significantly faster than the wild-type protein . Thus, enhanced proteolysis of TPMT*3C protein in mammalian cells is in contrast to its stability in yeast, but consistent with TPMT-deficiency in humans . Proteolysis was ATP-dependent and sensitive to proteasomal inhibitors MG115, MG132 and lactacystin, but not to calpain inhibitor II . We conclude that all of these mutant TPMT proteins undergo enhanced proteolysis in mammalian cells, through an ATP-dependent proteasomal pathway, leading to low or undetectable levels of TPMT protein in humans who inherit these mutant alleles. J Bioenerg Biomembr, 1999 Jun, 31(3), 177 - 90 Structure of the avian mitochondrial cytochrome bc1 complex; Berry EA et al.; There are now four structures of vertebrate mitochondrial bc1 complexes available in the protein databases and structures from yeast and bacterial sources are expected soon . This review summarizes the new information with emphasis on the avian cytochrome bc1 complex (PDB entries 1BCC and 3BCC) . The Rieske iron-sulfur protein is mobile and this has been proposed to be important for catalysis . The binding sites for quinone have been located based on structures containing inhibitors and, in the case of the quinone reduction site Qi, the quinone itself. Plant Cell, 1999 Dec, 11(12), 2379 - 91 Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein; Svennelid F et al.; The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin . A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified . A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin . Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV . Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo . Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase . Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth. Mol Biol Cell, 1999 Dec, 10(12), 4403 - 17 The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells; Pitts KR et al.; The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments . The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%) . Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells . To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells . Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern . Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy . Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER . These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells . Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER . How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed. J Agric Food Chem, 1999 Oct, 47(10), 4323 - 6 Release of deuterated nonenal during beer aging from labeled precursors synthesized in the boiling kettle; Noel S et al.; The use of labeled nonenal enabled the demonstration that the appearance of the cardboard flavor in finished beer comes from lipid auto-oxidation during wort boiling and not from lipoxygenasic activity during mashing . Free trans-2-nonenal produced by linoleic acid auto-oxidation in the kettle disappears, owing to retention by wort amino acids and proteins . This binding linkage protects trans-2-nonenal from yeast reduction but is reversible, allowing release of the compound at lower pH during aging . Labeled trans-2-nonenal is detected after aging when deuterated precursors form in the boiling kettle . The amount of alkenal released correlates with the concentration of reversible associations in the pitching wort . This work brings new illumination to the formation of trans-2-nonenal and overturns many previous hypotheses . It also explains why a reduction in the beer pH intensifies the cardboard flavor. J Virol, 2000 Jan, 74(1), 245 - 53 Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein; Gonzalez A et al.; The E1 protein of bovine papillomavirus (BPV) is a site-specific DNA binding protein that recognizes an 18-bp inverted repeat element in the viral origin of replication . Sequence-specific DNA binding function maps to the region from approximately amino acids 140 to 300, and isolated polypeptides containing this region have been shown to retain origin binding in vitro . To investigate the sequence and structural characteristics which contribute to sequence-specific binding, the primary sequence of this region was examined for conserved features . The BPV E1 DNA binding domain (E1DBD) contains three major hydrophilic domains (HR1, amino acids 179-191; HR2, amino acids 218 to 230; and HR3, amino acids 241 to 252), of which only HR1 and HR3 are conserved among papillomavirus E1 proteins . E1DBD proteins with lysine-to-alanine mutations in HR1 and HR3 were severely impaired for DNA binding function in vitro, while a lysine-to-alanine mutation in HR2 had a minimal effect on DNA binding . Mutation of adjacent threonine residues in HR1 (T187 and T188) revealed that these two amino acids made drastically different contributions to DNA binding, with the T187 mutant being severely defective for origin binding whereas the T188 mutant was only mildly affected . Helical wheel projections of HR1 predict that T187 is on the same helical face as the critical lysine residues whereas T188 is on the opposing face, which is consistent with their respective contributions to DNA binding activity . To examine E1 binding in vivo, a yeast one-hybrid system was developed . Both full-length E1 and the E1DBD polypeptide were capable of specifically interacting with the E1 binding site in the context of the yeast genome, and HR1 was also critical for this in vivo interaction . Overall, our results indicate that HR1 is essential for origin binding by E1, and the features and properties of HR1 suggest that it may be part of a recognition sequence that mediates specific E1-nucleotide contacts. J Exp Med, 1999 Dec 6, 190(11), 1657 - 68 RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation; Rajagopal K et al.; A novel T cell-specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library . RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk . Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation . RIBP-disrupted knockout mice displayed apparently normal T cell development . However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired . Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon gamma, but not IL-4 . These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation. Ann N Y Acad Sci, 1999 Sep 14, 883, 60 - 4 Distal hereditary motor neuropathy type II (distal HMN type II): phenotype and molecular genetics; Timmerman V et al.; The distal hereditary motor neuropathies (distal HMN) are clinically and genetically heterogeneous and are subdivided in seven subtypes according to the mode of inheritance, age at onset and clinical evolution . We studied a multigenerational Belgian pedigree with autosomal dominant distal HMN type II . The clinical phenotype closely resembles classical Charcot-Marie-Tooth (CMT) disease with an age at onset between 15 and 25 years . Linkage studies have shown that distal HMN II is not linked to the known CMT1 and CMT2 loci . A genome-wide search was performed and significant linkage was obtained between markers D12S86 and D12S340, suggesting that a gene causing distal HMN II is located on chromosome 12q24.3 . The gene encoding the human pancreatic phospholipase A2 (PLA2A), which is expressed in peripheral nerves during degeneration, is a positional candidate gene . Because no disease-specific mutations were detected in the coding region, however, PLA2A is most likely not the disease causing gene . A yeast artificial chromosome (YAC) contig map spanning the candidate region has been constructed to isolate the gene responsible for distal HMN II . Positional and functional candidate genes are currently being screened for the presence of mutations in distal HMN II patients. Genomics, 1999 Nov 15, 62(1), 108 - 12 The copper chaperone Atox1 in canine copper toxicosis in Bedlington terriers; Nanji MS et al.; Copper toxicosis, resulting in liver disease, commonly occurs in Bedlington terriers . This recessively inherited disorder, similar in many respects to Wilson disease, is of particular interest because the canine Atp7b gene, homologous to ATP7B defective in Wilson disease, is not responsible for canine copper toxicosis as has been expected . Atox1, a copper chaperone delivering copper to Atp7b, therefore became a potential candidate . We cloned canine Atox1, which shows conserved motifs of the copper-binding domain (MTCXXC) and of the lysine-rich region (KTGK), and showed 88, 80, and 41% amino acid sequence identity with the orthologous mouse, human, and yeast proteins . No gross deletions of Atox1 could be identified in the affected Bedlington terriers by Southern blot analysis of genomic DNA . The canine Atox1 gene spans about 4 kb, with a 204-bp open reading frame cDNA contained within two exons . Sequence analysis of the coding regions, including intron/exon boundaries, showed no mutations in Atox1 from genomic DNA of an affected dog . We have also identified an apparently nontranscribed canine Atox1 pseudogene, with 12 sequence changes and no intron . Mapping of Atox1 and a marker closely linked to the canine copper toxicosis locus indicated lack of synteny . Atox1 is therefore excluded as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene must be responsible for this copper storage disease in dogs and also suggesting the possibility of a similar gene responsible for a copper storage disease in humans . Genomics, 1999 Nov 15, 62(1), 74 - 81 Multiple MSP pseudogenes in a local repeat cluster on 1p36.2: An expanding genomic graveyard? van der Drift P, Chan A, Zehetner G, Westerveld A, Versteeg R. Chromosomal region 1p36.2 harbors an intriguing gene cluster of about 1 Mb . In addition to normal high-copy-number repeats, this cluster consists entirely of locally repeated sequences among which there are tRNA and small nuclear RNA (snRNA) genes . In 23 PACs and YACs from the 1p36.2 cluster, we identified eight different copies of a sequence with about 97% homology to the macrophage stimulating protein (MSP) gene located on chromosomal band 3p21 . These MSP-like (MSPL) sequences on 1p36.2 are scattered over the repeat region . Nucleotide substitutions and single nucleotide deletions in exons of all identified MSPL genes on 1p36.2 mark them as pseudogenes . We constructed a phylogenetic tree of these sequences with their most likely order of origin in evolution . MSP from 3p21 could be identified as the ancestral sequence, a copy of which was captured into the cluster of tRNA and snRNA genes on 1p36.2 about 6 million years (MY) ago . MSP subsequently coamplified with the other sequences in the cluster . Analysis of the DNA of 18 individuals shows that the MSPL copy number is polymorphic, with a range of four to seven or more copies per haploid genome . Analysis of corresponding clusters in macaque chromosomes indicated an age for the tRNA/snRNA cluster of at least 30 MY . The MSPL sequence thus functions as a probe for the more recent primate evolution of this cluster and suggests a continuation of its unusual activity over the last 6 MY . Genomics, 1999 Nov 15, 62(1), 1 - 10 Pseudoxanthoma elasticum maps to an 820-kb region of the p13.1 region of chromosome 16; Le Saux O et al.; We have performed linkage analysis on 21 families with pseudoxanthoma elasticum (PXE) using 10 polymorphic markers located on chromosome 16p13.1 . The gene responsible for the PXE phenotype was localized to an 8-cM region of 16p13.1 between markers D16S500 and D16S3041 with a maximum lod score of 8.1 at a recombination fraction of 0.04 for marker D16S3017 . The lack of any locus heterogeneity suggests that the major predisposing allele for the PXE phenotype is located in this region . Haplotype studies of a total of 36 PXE families identified several recombinations that further confined the PXE gene to a region (< 1 cM) between markers D16S3060 and D16S79 . This PXE locus was identified within a single YAC clone and several overlapping BAC recombinants . From sequence analysis of these BAC recombinants, it is clear that the distance between markers D16S3060 and D16S79 is about 820 kb and contains a total of nine genes including three pseudogenes . We predict that mutations in one of the expressed genes in the locus will be responsible for the PXE phenotype in these families . J Biol Chem, 1999 Dec 10, 274(50), 35741 - 8 Molecular cloning of six novel Krüppel-like zinc finger genes from hematopoietic cells and identification of a novel transregulatory domain KRNB; Han ZG et al.; To clone zinc finger genes expressed in hematopoietic system, we designed primers based on conserved Cys(2)/His(2) zinc finger sequences to amplify corresponding domains from mRNA of normal bone marrow and leukemia cell line NB4 . DNA fragments of novel zinc finger genes were chosen and used as probe pool to screen cDNA libraries or subject to rapid amplification of cDNA ends in order to obtain full-length cDNA . Six cDNAs including whole open reading frame of zinc finger proteins, named as ZNF191, ZNF253 (BMZF-1), ZNF255 (BMZF-2), ZNF256 (BMZF-3), ZNF257 (BMZF-4), and ZNF254 (BMZF-5) were obtained . All six belong to the Kruppel-like zinc finger gene family, and typical transcriptional regulatory motifs exist in the N-terminal moiety, such as the SCAN box in ZNF191, and the KRAB domains in ZNF253, ZNF254, ZNF256, and ZNF257 . A previously undefined sequence nominated as Kruppel-related novel box, which may represent a new transregulatory motif, was revealed at the N terminus of ZNF255 . The transregulatory function of non-zinc finger regions of ZNF191, ZNF253, and ZNF255 were addressed in yeast and mammalian cells . The results indicated that ZNF255 might be a conditional transactivator, whereas ZNF253 and ZNF191 displayed a suppressive effect on the transcription in yeast and/or mammalian systems. Genetics, 1999 Dec, 153(4), 1929 - 48 The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley; Wei F et al.; Powdery mildew of barley, caused by Erysiphe graminis f . sp . hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens . A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H) . The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm . AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities . These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster . Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones . Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval . Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster. Lett Appl Microbiol, 1999 Oct, 29(4), 238 - 41 Inhibitory effects of some spice essential oils on Aspergillus ochraceus NRRL 3174 growth and ochratoxin A production; Basilico MZ et al.; Inhibitory effects of essential oils of oregano (Origanum vulgare), mint (Menta arvensis), basil (Ocimum basilicum), sage (Salvia officinalis) and coriander (Coriandrum sativum), on the mycelial growth and ochratoxin A production by Aspergillus ochraceus NRRL 3174 were studied . Cultures were incubated on yeast extract-sucrose (YES) broth, at concentrations of 0, 500, 750 and 1000 p.p.m . of essential oils during 7, 14 and 21 d at 25 degrees C . At 1000 p.p.m., oregano and mint completely inhibited the fungal growth and ochratoxin A production up to 21 d, while basil was only effective up to 7 d . At 750 p.p.m., oregano was completely effective up to 14 d, whereas mint allowed fungal growth but no ocratoxin A production up to 14 d . At 500 p.p.m., no evident inhibition could be in observed with any of the essential oils under analysis . Sage and coriander showed no important effect at any of the concentrations studied . These inhibitory effects are interesting in connection with the prevention of mycotoxin contamination in many foods and they could be used instead of synthetic antifungal products. Genes Cells, 1999 Oct, 4(10), 563 - 72 Presence of telomeric G-strand tails in the telomerase catalytic subunit TERT knockout mice; Yuan X et al.; BACKGROUND: Telomerase consists of two essential subunits, the template RNA (TR; telomerase RNA) and the catalytic subunit TERT (telomerase reverse transcriptase) . Knockout mice with a mTR (mouse TR) deletion have been described and well characterized . However, mice with a mTERT (mouse TERT) deletion have not been reported . RESULTS: mTERT-knockout mice have been constructed . The first generation mTERT -/- mice were fertile, and did not show any noticeable macroscopic or microscopic phenotypic change . All tissue cells derived from mTERT -/- mice that were examined lacked telomerase activity, indicating that mTERT is the only gene encoding the telomerase catalytic subunit . Pulse field gel electrophoresis (PFGE) and nondenaturing in-gel hybridization analyses showed that mouse telomeric DNA has G-strand 5'-overhangs, as demonstrated for human and yeast cells . This telomeric single-stranded G-tail was also observed in MEF (mouse embryonic fibroblast) and liver cells derived from mTERT -/- mice . CONCLUSIONS: mTERT-knockout mice show phenotypes that are apparently normal at least during the early generations . This observation is similar to that obtained with the mTR-knockout mice . The presence of the telomeric G-strand tails in mTERT -/- mice suggests that these telomeric 5'-overhangs are produced by telomerase-independent mechanisms, as has been proposed for yeast and human. Cancer Res, 1999 Nov 15, 59(22), 5785 - 92 Two antigens recognized by autologous cytolytic T lymphocytes on a melanoma result from a single point mutation in an essential housekeeping gene; Chiari R et al.; We have pursued our analysis of antigens recognized by autologous cytolytic T lymphocytes (CTLs) on the melanoma cells of patient LB33 . This patient enjoys an unusually favorable evolution, which is associated with a strong and sustained antitumor CTL response . We reported previously the analysis of two melanoma cell lines, MEL.A and MEL.B, which were derived from metastases removed from the patient at 5 years' distance . Autologous CTL clones derived from blood lymphocytes recognized several antigens presented by different HLA class I molecules on MEL.A . The MEL.B cells resisted lysis by these CTLs because they have lost expression of most HLA molecules, suggesting that they were selected in vivo by the anti-MEL.A CTL response . One of the MEL.A antigens was shown to result from a point mutation in the tumor . Here we report the cloning of a gene that encodes two other MEL.A antigens . This new gene, MUM-2, is expressed ubiquitously . In the melanoma cells of patient LB33, it contains a point mutation that changes one amino acid in the translated protein . Two different antigenic peptides, one presented to CTL by HLA-B44 molecules and another by HLA-C6 molecules, overlap and contain the mutated residue . Gene MUM-2 is homologous to an essential yeast gene, bet5, that was recently shown to be implicated in the vesicular transport of proteins from the endoplasmic reticulum to the Golgi . In a mutant yeast with a disrupted bet5 gene, both the wild-type and the mutated MUM-2 genes could complement for bet5 function . These results indicate that the antigenic mutation does not destroy the function of the protein, a function that is conserved in eukaryotic cells . The identification of these antigens suggests that point mutations could be the major cause of the strong immunogenicity of MEL.A cells. Prog Biophys Mol Biol, 1999, 72(3), 299 - 328 The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction; Craig KL et al.; The ubiquitin system of intracellular protein degradation controls the abundance of many critical regulatory proteins . Specificity in the ubiquitin system is determined largely at the level of substrate recognition, a step that is mediated by E3 ubiquitin ligases . Analysis of the mechanisms of phosphorylation directed proteolysis in cell cycle regulation has uncovered a new class of E3 ubiquitin ligases called SCF complexes, which are composed of the subunits Skp1, Rbx1, Cdc53 and any one of a large number of different F-box proteins . The substrate specificity of SCF complexes is determined by the interchangeable F-box protein subunit, which recruits a specific set of substrates for ubiquitination to the core complex composed of Skp1, Rbx1, Cdc53 and the E2 enzyme Cdc34 . F-box proteins have a bipartite structure--the shared F-box motif links F-box proteins to Skp1 and the core complex, whereas divergent protein-protein interaction motifs selectively bind their cognate substrates . To date all known SCF substrates are recognised in a strictly phosphorylation dependent manner, thus linking intracellular signalling networks to the ubiquitin system . The plethora of different F-box proteins in databases suggests that many pathways will be governed by SCF-dependent proteolysis . Indeed, genetic analysis has uncovered roles for F-box proteins in a variety of signalling pathways, ranging from nutrient sensing in yeast to conserved developmental pathways in plants and animals . Moreover, structural analysis has revealed ancestral relationships between SCF complexes and two other E3 ubiquitin ligases, suggesting that the combinatorial use of substrate specific adaptor proteins has evolved to allow the regulation of many cellular processes . Here, we review the known signalling pathways that are regulated by SCF complexes and highlight current issues in phosphorylation dependent protein degradation. Virus Res, 1999 Dec 15, 65(2), 141 - 54 Two distinct regions of the BPV1 E1 replication protein interact with the activation domain of E2; Moscufo N et al.; Papillomavirus E1 and E2 proteins co-operation in viral DNA replication is mediated by protein-protein interactions that lead to formation of an E1-E2 complex . To identify the domains involved, portions of the two proteins were expressed as fusions to the DNA-binding protein LexA or the transactivation domain of VP16 and analyzed by the yeast two-hybrid system . The C-terminal 266 amino acids of BPV1 E1 (E1C266) interacted strongly with E2 in the yeast system and in a mammalian two-hybrid assay . VP16-E1C266 interacted with a region encompassing amino acids 1-200 of the transactivation domain of E2 that was fused to LexA . The interaction between E1 full length and E2 was clearly observed only when E1 was expressed as LexA-E1 chimera . In addition, we found that in the LexA context also the N-terminal region encompassing the first 340 amino acids of E1 (E1N340) interacted with E2 full length . The interactions of E1N340 and E1C266 with E2 were confirmed also by in vitro binding studies . These observations demonstrate that two distinct regions of E1 mediate the interaction with E2 in vivo. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 395 - 404 The ABCA subclass of mammalian transporters; Broccardo C et al.; We describe here a subclass of mammalian ABC transporters, the ABCA subfamily . This is a unique group that, in contrast to any other human ABC transporters, lacks a structural counterpart in yeast . The structural hallmark of the ABCA subfamily is the presence of a stretch of hydrophobic amino acids thought to span the membrane within the putative regulatory (R) domain . As for today, four ABCA transporters have been fully characterised but 11 ABCA-encoding genes have been identified . ABCA-specific motifs in the nucleotide binding folds can be detected when analysing the conserved sequences among the different members . These motifs may reveal functional constraints exclusive to this group of ABC transporters. Genetics, 1999 Dec, 153(4), 1909 - 18 Induction and characterization of Ph1 wheat mutants; Roberts MA et al.; The cloning of genes for complex traits in polyploid plants that possess large genomes, such as hexaploid wheat, requires an efficient strategy . We present here one such strategy focusing on the homologous pairing suppressor (Ph1) locus of wheat . This locus has been shown to affect both premeiotic and meiotic processes, possibly suggesting a complex control . The strategy combined the identification of lines carrying specific deletions using multiplex PCR screening of fast-neutron irradiated wheat populations with the approach of physically mapping the region in the rice genome equivalent to the deletion to reveal its gene content . As a result, we have located the Ph1 factor controlling the euploid-like level of homologous chromosome pairing to the region between two loci (Xrgc846 and Xpsr150A) . These loci are located within 400 kb of each other in the rice genome . By sequencing this region of the rice genome, it should now be possible to define the nature of this factor. Genetics, 1999 Dec, 153(4), 1873 - 83 Long inverted repeats are an at-risk motif for recombination in mammalian cells; Waldman AS et al.; Certain DNA sequence motifs and structures can promote genomic instability . We have explored instability induced in mouse cells by long inverted repeats (LIRs) . A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence . The tk gene was introduced into the genome of mouse Ltk(-) fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined . Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10(-5) events per cell per generation . Of the tk(+) segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk(+) segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot . Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements. Genetics, 1999 Dec, 153(4), 1789 - 97 Genetic characterization of cytological region 77A-D harboring the presenilin gene of Drosophila melanogaster; Lukinova NI et al.; We performed a systematic lethal mutagenesis of the genomic region uncovered by Df(3L)rdgC-co2 (cytological interval 77A-D) to isolate mutations in the single known Presenilin (Psn) gene of Drosophila melanogaster . Because this segment of chromosome III has not been systematically characterized before, inter se complementation testing of newly recovered mutants was carried out . A total of 79 lethal mutations were isolated, representing at least 17 lethal complementation groups, including one corresponding to the Psn gene . Fine structure mapping of the genomic region surrounding the Psn transcription unit by transgenic rescue experiments allowed us to localize two of the essential loci together with Psn within an approximately 12-kb genomic DNA region . One of these loci, located 3' to Psn, encodes a Drosophila protein related to the yeast 60S ribosomal protein L10 precursor . We also determined which of the newly recovered lethal mutant groups correspond to previously isolated lethal P-element insertions, lethal inversion breakpoints, and lethal polo gene mutants . Point mutations were identified in all five recovered Psn alleles, one of which results in a single amino acid substitution G-E at a conserved residue in the C-terminal cytoplasmic tail of the protein, suggesting an important functional role for this C-terminal domain of Presenilin . In addition, some viable mutations were recovered in the screen, including new alleles of the clipped and inturned loci. Genetics, 1999 Dec, 153(4), 1641 - 54 SEL-5, a serine/threonine kinase that facilitates lin-12 activity in Caenorhabditis elegans; Fares H et al.; Ligands present on neighboring cells activate receptors of the LIN-12/Notch family by inducing a proteolytic cleavage event that releases the intracellular domain . Mutations that appear to eliminate sel-5 activity are able to suppress constitutive activity of lin-12(d) mutations that are point mutations in the extracellular domain of LIN-12, but cannot suppress lin-12(intra), the untethered intracellular domain . These results suggest that sel-5 acts prior to or during ligand-dependent release of the intracellular domain . In addition, sel-5 suppression of lin-12(d) mutations is tissue specific: loss of sel-5 activity can suppress defects in the anchor cell/ventral uterine precursor cell fate decision and a sex myoblast/coelomocyte decision, but cannot suppress defects in two different ventral hypodermal cell fate decisions in hermaphrodites and males . sel-5 encodes at least two proteins, from alternatively spliced mRNAs, that share an amino-terminal region and differ in the carboxy-terminal region . The amino-terminal region contains the hallmarks of a serine/threonine kinase domain, which is most similar to mammalian GAK1 and yeast Pak1p. EMBO J, 1999 Dec 1, 18(23), 6752 - 61 Two activities of cofilin, severing and accelerating directional depolymerization of actin filaments, are affected differentially by mutations around the actin-binding helix; Moriyama K et al.; The biochemical activities of cofilin are controversial . We demonstrated that porcine cofilin severs actin filaments and accelerates monomer release at the pointed ends . At pH 7.1, 0.8 microM cofilin cut filaments (2.2 microM actin) about every 290 subunits and increased the depolymerization rate 6.4-fold . A kink in the major alpha-helix of cofilin is thought to constitute a contact site for actin . Side chain hydroxyl groups of Ser119, Ser120 and Tyr82 in cofilin form hydrogen bonds with main chain carbonyl moieties from the helix, causing the kink . We eliminated side chain hydroxyls by Ser-->Ala and/or Tyr-->Phe mutagenesis . Severing and depolymerization-enhancing activities were reduced dramatically in an Ala120 mutant, whereas the latter was decreased in a Phe82 mutant with a relatively small effect on severing, suggesting different structural bases for the two activities of cofilin . The Ala120-equivalent mutation in yeast cofilin affected cell growth, whereas that of the Phe82-equivalent had no effect in yeast . These results indicate the physiological significance of the severing activity of cofilin that is brought about by the kink in the helix. Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 216 - 21 Identification of EPS8 as a Dvl1-associated molecule; Inobe M et al.; Dishevelled (Dsh) is involved in both Wingless (Wg) and Frizzled (Fz) signaling pathways . To further determine the function of Dsh, we have performed yeast two-hybrid screening and isolated several genes encoding the molecules associated with the PDZ domain of Dvl1, one of the murine Dsh homologs . During the screening, we found that EPS8, which is a substrate for activated EGF receptor (EGFR), specifically interacted with Dvl1 . This interaction was also confirmed in vitro . Through transfection studies, we observed the mutual action between Dvl1 and EPS8 . Dvl1 was hyperphosphorylated in the presence of EPS8, whereas the tyrosine phosphorylation of EPS8 by activated EGFR was inhibited in the presence of Dvl1 . Immunohistochemistry showed that Dvl1 and EPS8 expression overlap in particular tissues during organogenesis . These results indicate that interaction between Dvl1 and receptor tyrosine kinase signal plays certain roles in developmental events . Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 115 - 22 A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins; Mitsui K et al.; Cyclin E-Cdk2 is an evolutionary conserved cyclin-dependent kinase (CDK) complex that drives the G1 to S phase transition of the cell cycle . A novel cDNA encoding a HECT family protein also containing RCC1-like repeats was isolated by a yeast two-hybrid screening using both cyclin E and its inhibitor p21 . The protein product of this cDNA, Ceb1, interacts with various cyclin subunits of CDKs in mammalian cells . Expression of Ceb1 is specifically detected in testis and ovary and is highly elevated when the functions of the tumor suppressor proteins, p53 and RB, are compromised by mutations or viral oncoproteins . The present results suggest that Ceb1 may play a critical role when its expression and the CDK activity are upregulated by inactivation of p53 and RB . Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 24 - 7 Interaction between GPIbalpha and FcgammaIIA receptor in human platelets; Sun B et al.; Glycoprotein (GP) Ib (alpha and beta) in platelets forms a noncovalent hetero-oligomeric complex with GPIX and GPV and serves as a receptor for von Willebrand factor (vWF), which mediates the initial adhesion of platelets to the subendothelium after vascular damage and also plays a role in thrombin-induced platelet activation . We investigated the interaction between GPIbalpha and FcgammaIIA receptor using a yeast two-hybrid system and mutagenesis, and we identified residues R542G543R544 in GPIbalpha and D298D299D300 in FcgammaIIA receptor as the primary interaction sites . These results further confirmed the interaction between GPIbalpha and FcgammaIIA receptor and support the hypothesis that the signal transduction of GPIb-IX-V that leads to platelet activation may be partially mediated through FcgammaIIA receptor . Gene, 1999 Nov 29, 240(2), 297 - 305 Fine physical and transcript mapping of a 1.8 Mb region spanning the locus for childhood acute lymphoblastic leukemia on chromosome 12p12 . 3; Aissani B et al.; Rearrangements of the short arm of chromosome 12 are frequently observed in hematological disorders . Previous studies of loss of heterozygosity identified a small genetic interval on chromosome 12p12.3 that is frequently deleted in childhood acute lymphoblastic leukemia (ALL) . Two genes, ETV6/TEL and p27/KIP1, are located in this interval . Evidence has accumulated that an as-yet unidentified tumor suppressor gene is closely linked to these . To facilitate the identification of candidate genes, a long-range high-resolution restriction map of the ALL locus was constructed using a contig of YAC clones . Several marker loci, including 11 STS, three newly developed YAC end-based STS, six EST, and seven genes were unambiguously positioned in the new map . The map covers 1.8Mb and extends from the distal salivary proline-rich protein gene cluster to the proximal p27/KIP1 gene . The data confirmed the order tel-D12S358-p27/KIP1-cen and excluded p27/KIP1 as a candidate tumor suppressor gene . The critical region delimited by D12S89 and D12S358 is a 750kb CpG-island rich region that includes the 240kb TEL/ETV6 gene as well as CLAPS3 (clathrin-adaptor small chain 3) . The new map provides a molecular framework for the identification of novel genes and transcriptional units in the ALL interval. Biochim Biophys Acta, 1999 Dec 6, 1473(1), 237 - 46 O-mannosyl glycans in mammals; Endo T; Most proteins within living organisms contain glycans . Glycan structures can modulate the biological properties and functions of glycoproteins . The major glycans of glycoproteins can be classified into two groups, N-glycans and O-glycans, according to their glycan-peptide linkage regions . Developments in glycobiology have revealed a new type of glycosidic linkage to the peptide portion, the O-mannosyl linkage, in mammals, while so far it had been thought to be specific to yeast . This review will give an outline of the O-mannosyl glycans of mammalian glycoproteins . Since one of the most well known O-mannosyl-modified mammalian glycoproteins is dystroglycan, the functional aspects of the O-mannosyl glycan of dystroglycan will be described to help understand this new glycobiological field. FEBS Lett, 1999 Nov 26, 462(1-2), 192 - 8 The antioxidant functions of cytochrome c; Korshunov SS et al.; Low (C(1/2) = 1.5 x 10(-7) M) concentrations of horse cytochrome c strongly inhibit H(2)O(2) production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD(+) . The effect is abolished by binding of cytochrome c with liposomes and is not prevented by SOD . Yeast cytochrome c is much less effective than the horse protein whereas acetylated horse cytochrome c is without effect . H(2)O(2) formation stimulated by antimycin A is resistant to added cytochrome c . In inside-out submitochondrial vesicles, H(2)O(2) production is suppressed by all three cytochrome c samples tested, but at higher concentrations (C(1/2) is about 5 x 10(-7) M) . In vesicles, SOD abolishes the cytochrome c inhibition . We conclude that extramitochondrial cytochrome c is competent in down-regulation of the Complex I H(2)O(2) production linked to the reverse electron transfer . Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome c function can be observed, i.e . the oxidation of O(2-*) to O(2) . A possible role of cytochrome c in the antioxidant defence is discussed. FEBS Lett, 1999 Nov 26, 462(1-2), 79 - 84 Secondary structure in the 5'-leader or 3'-untranslated region reduces protein yield but does not affect the functional interaction between the 5'-cap and the poly(A) tail; Niepel M et al.; The 5'-cap structure and poly(A) tail of eukaryotic mRNAs cooperate to promote translation initiation but whether this functional interaction benefits certain classes of mRNAs has not been investigated . In this study, we investigate whether a structured 5'-leader or 3'-untranslated region (UTR) affects the cap/poly(A) tail interaction . A structured leader reduced the degree to which the 5'-cap promoted translation in plant cells and inhibited translation from capped and uncapped mRNAs equally in yeast . Secondary structure within the 3'-UTR reduced translational efficiency when adjacent to the stop codon but had little effect on the cap/poly(A) tail synergy . The functional interaction between the cap and poly(A) tail was as important for an mRNA with a structured leader or 3'-UTR as it was for an unstructured mRNA in either species, suggesting that these structures can reduce translation without affecting the functional interaction between the cap and poly(A) tail . However, the loss of Xrn1p, the major 5'-->3' exoribonuclease in yeast, abolished cap-dependent translation and the functional interaction between the cap and poly(A) tail, suggesting that the cap/poly(A) tail synergy is of particular importance under conditions of active RNA turnover. J Gen Virol, 1999 Nov, 80 ( Pt 11), 2809 - 12 Effect of mutations within the cys-rich region of potyvirus helper component-proteinase on self-interaction; Urcuqui-Inchima S et al.; The first approximately 60 amino acids of the N-terminal part of the potyvirus helper component-proteinase (HC-Pro) include highly conserved residues comprising a Cys-rich region . In the present study, the domain in Potato virus Y sufficient for self-interaction was mapped using the yeast two-hybrid system to the 83 N-terminal amino acids of HC-Pro . Mutations in the conserved His and two Cys residues within the Cys-rich region have a strong debilitating effect on self-interaction when introduced in the full-length HC-Pro, but not when introduced in the N-terminal fragment. Methods, 1999 Nov, 19(3), 425 - 33 In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment; Kuo MH et al.; Chromatin structure plays important roles in regulating many DNA-templated processes, such as transcription, replication, and recombination . Considerable progress has recently been made in the identification of large, multisubunit complexes dedicated to these nuclear processes, all of which occur on nucleosomal templates . Mapping specific genomic loci relative to the position of selectively modified or unique histone variants or nonhistone components provides valuable insights into how these proteins (and their modifications) function in their normal chromatin context . Here we describe a versatile and high-resolution method which involves two basic steps: (1) in vivo formaldehyde cross-linking of intact cells followed by (2) selective immunoprecipitation of protein-DNA complexes with specific antibodies . This method allows for detailed analyses of protein-DNA interactions in a native chromatin environment . Recently, this technique has been successfully employed to map the boundaries of specifically modified (e.g., acetylated) histones along target genes, to define the cell cycle-regulated assembly of origin-dependent replication and centromere-specific complexes with remarkable precision, and to map the in vivo position of reasonably rare transcription factors on cognate DNA sites . Thus, the basic chromatin immunoprecipitation technique is remarkably versatile and has now been used in a wide range of cell types, including budding yeast, fly, and human cells . As such, it seems likely that many more studies, centered around chromatin structure and protein-DNA interactions in its native setting, will benefit from this technique . In this article, a brief review of the history of this powerful approach and a discussion of the basic method are provided . Procedures for protein recovery as well as limitations and extensions of the method are also presented . Exp Cell Res, 1999 Nov 25, 253(1), 186 - 209 The EH network; Santolini E et al.; The EH domain is an evolutionary conserved protein-protein interaction domain present in a growing number of proteins from yeast to mammals . Even though the domain was discovered just 5 years ago, a great deal has been learned regarding its three-dimensional structure and binding specificities . Moreover, a number of cellular ligands of the domain have been identified and demonstrated to define a complex network of protein-protein interactions in the eukaryotic cell . Interestingly, many of the EH-containing and EH-binding proteins display characteristics of endocytic "accessory" proteins, suggesting that the principal function of the EH network is to regulate various steps in endocytosis . In addition, recent evidence suggests that the EH network might work as an "integrator" of signals controlling cellular pathways as diverse as endocytosis, nucleocytosolic export, and ultimately cell proliferation . Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1999 Sep, 124(1), 1 - 5 A simple procedure for isolation of Bufo arenarum C3 complement fraction and preparation of antiserum; Llanos RJ et al.; Because of the need for antibodies in our studies involving the third component of complement in Bufo arenarum, we performed a simple procedure to purify C3 from B . arenarum serum to use as antigen in the preparation of the antiserum . The strategy was based on the well-known ability of C3 to bind to zymosan (Zy), a yeast cell wall extract comprised of polysaccharides . The Zy-bound fraction showed cross reactivity with a commercial antibody to human C3 as well as a similar electrophoretic profile (SDS-PAGE) to C3 from other species . The Zy-C3 complex resulting from binding Zy to B . arenarum serum was injected into rabbits and the antiserum against this C3-like fraction was purified by protein A-Sepharose chromatography . The purified C3 antibody was found to be suitable for immunochemical studies. Plant Mol Biol, 1999 Sep, 41(2), 259 - 68 Cloning and expression of amino acid transporters from broad bean; Montamat F et al.; This work describes the isolation of a full-length (VfAAP2) and three partial amino acid transporter genes (VfAAPa, VfAAPb, VfAAPc) from broad bean (Vicia faba L.) . The function of VfAAP2 was tested by heterologous expression in a yeast mutant deficient in proline uptake . VfAAP2 mediates proton-dependent proline uptake with an apparent Km of about 1 mM . Analysis of substrate specificity by competition experiments showed that aromatic amino acids, neutral aliphatic acids and L-citrulline are the best competitors, whereas basic amino acids do not compete with proline . Northern analysis indicates that all VfAAPs exhibit different patterns of expression . VfAAP2 is most strongly expressed in the stem and at a lower level in sink leaves and pods . VfAAPa, VfAAPb and VfAAPc are most strongly expressed in the flowers, but their expression in the other organs varies. Endocrinology, 1999 Dec, 140(12), 5469 - 77 Characterization of the antiapoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) and the stimulation of its message by gonadotropins in the rat ovary; Leo CP et al.; The majority of ovarian follicles undergo atresia mediated by apoptosis . Bcl-2-related proteins act as regulators of apoptosis via the formation of dimers with proteins inside and outside the Bcl-2 family . Previous studies have identified BAD as a proapoptotic Bcl-2 family member expressed in the ovary . It is known that BAD phosphorylation induced by survival factors leads to its preferential binding to 14-3-3 and suppression of the death-inducing function of BAD . To identify ovarian binding partners for hypophosphorylated BAD, we performed a yeast two-hybrid screening of a rat ovary complementary DNA library using as bait a mutant BAD incapable of binding to 14-3-3 . Screening of yeast transformants yielded positive clones encoding the rat ortholog of Mcl-1 (myeloid cell leukemia-1), an antiapoptotic Bcl-2 protein . Amino acid sequence analysis revealed that rat and human Mcl-1 showed a complete conservation of the Bcl-2 homology domains BH1, BH2, and BH3 . In the yeast two-hybrid system, Mcl-1 binds to the hypophosphorylated mutant of BAD and interacts preferentially with different proapoptotic (Bax, Bak, Bok, Bik, and BOD) compared with antiapoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, CED-9, and BHRF-1) . Northern blot hybridization demonstrated expression of Mcl-1 transcripts of 2.3 and 3.7 kb in the ovary and diverse other rat tissues . In immature rats, PMSG treatment led to a transient increase in the 2.3-kb Mcl-1 transcript, peaking at 6 h after injection and returning to baseline levels after 24 h . Moreover, the same transcript was induced in the PMSG-primed preovulatory rat ovary 6 h after the administration of ovulatory doses of either hCG or FSH . In situ hybridization studies revealed that the gonadotropin stimulation of ovarian Mcl-1 message occurs in both granulosa and thecal cells . In conclusion, rat Mcl-1 was identified as an ovarian BAD-interacting protein and the message for the antiapoptotic Mcl-1 protein was induced after treatment with gonadotropins in granulosa and thecal cells of growing follicles. Biol Cell, 1999 May-Jun, 91(4-5), 313 - 20 Dictyostelium discoideum: a promising centrosome model system; Daunderer C et al.; The centrosome of the slime mould Dictyostelium discoideum displays a morphology markedly different from centriolar centrosomes or yeast spindle pole bodies, while fulfilling the same conserved functions in the organization of the microtubule cytoskeleton . Recent advances suggest that the Dictyostelium centrosome may offer an interesting model system, usefully complementing other well-studied centrosome models . The establishment of an isolation procedure and the generation of a range of monoclonal antibodies have been achieved, which are important pre-requisites for biochemical investigation . Furthermore, the role of the centrosome in cell motility and centrosome duplication process have been investigated using cells with GFP-labelled centrosomes. Microsc Microanal, 1999 May, 5(3), 197 - 207 Topographic Imaging of Chromium-Coated Frozen-Hydrated Cell and Macromolecular Complexes by In-Lens Field Emission Scanning Electron Microscopy; Apkarian RP et al.; : An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information . High-resolution topographic images at high primary magnification (>/=200,000x) were digitally recorded with very short dwell times and without beam damage . Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues . The "vitreous" nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be </=2-3 nm (the approximate instrument resolution on cryo-specimens) . Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected specimens immersed in featureless ice . The time necessary to cryo-immobilize the specimen and record the image is <3 hr . The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed . A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed . These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems. Cytogenet Cell Genet, 1999, 86(3-4), 263 - 6 Transcriptional map of chromosome region 6q16-->q21; Karayianni E et al.; We present the transcription map of chromosome region 6q16-->q21 by mapping fifteen known genes within this region . Five genes lay in the subregion containing a tumor suppressor gene, eight genes are located in the subregion harboring a senescence gene, and two genes are distal to the latter region . The precise location of the genes was obtained using a previously described translocation and deletion mouse/human hybrid panel . An even more accurate definition was possible for the genes spanning the senescence gene region, since a previously described YAC contig with its restriction map was available . From this transcription map it is possible to derive a large region of synteny with mouse chromosome 10. Cytogenet Cell Genet, 1999, 86(3-4), 214 - 8 Exon structure and promoter identification of STIM1 (alias GOK), a human gene causing growth arrest of the human tumor cell lines G401 and RD; Sabbioni S et al.; The stromal interaction molecular 1 gene (STIM1) encodes a type I trans-membrane protein of unknown function, which induces growth arrest and degeneration of the human tumor cell lines G401 and RD but not HBL100 and CaLu-6, suggesting a role in the pathogenesis of rhabdomyosarcomas and rhabdoid tumors . Here, we describe the STIM1 genomic organization including the identification of the promoter region . The gene consists of 12 exons that span a region larger than 250 kb between the genes RRM1 and NUP98 . Nucleotide sequences of all exon-intron boundaries were determined and oligonucleotide primers for the amplification of individual exons were designed . The promoter region was identified within a 1.8-kb SacI fragment at the 5' end of the gene . In vitro CpG methylation of the promoter region indicated that transcription can be downregulated by this mechanism . The genetic tools developed in the present work will help to determine whether pathogenetic mechanisms that associate STIM1 with tumorigenesis involve mutations in coding sequences and/or promoter, and whether methylation could determine STIM1 transcriptional down-regulation in tumor samples. J Biol Chem, 1999 Dec 3, 274(49), 35152 - 8 Characterization of the amino-terminal activation domain of peroxisome proliferator-activated receptor alpha . Importance of alpha-helical structure in the transactivating function; Hi R et al.; The transactivating function of the A/B region of mouse peroxisome proliferator-activated receptor alpha (PPARalpha; NR1C1) was characterized . The truncated version of PPARalpha lacking the A/B region had 60-70% lower transactivating function than full-length PPARalpha in both the presence and absence of the peroxisome proliferator ciprofibrate . When tethered to the yeast Gal4 DNA-binding domain, the A/B region exhibited the significant ligand-independent transactivating function, AF-1 activity . The first 44 amino acid residues were necessary for maximal transactivation, and the minimally essential region was further delimited to amino acids 15-44 . This region is highly enriched with acidic residues, but mutational analyses showed that the protein structure, rather than the negative charge itself, was important for the AF-1 activity . An alpha-helical configuration was predicted for this region, and a CD spectrum analysis of the synthetic peptides showed that mutant sequences with higher AF-1 activity have higher helical contents and vice versa . The most active mutant, in which Met(31) was replaced with Leu, was approximately 5-fold more potent than the wild-type A/B region . These findings indicate that the AF-1 region of PPARalpha is an acidic activation domain and that the helix-forming property is implicated in the transactivating function. J Biol Chem, 1999 Dec 3, 274(49), 35113 - 8 Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons; Meyer D et al.; c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK . More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3 . We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain . To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system . One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA . Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells . The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells . Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF . This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine . Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines . Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells . The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells. J Biol Chem, 1999 Dec 3, 274(49), 35080 - 8 A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter; Hayashi Y et al.; The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites . In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE . Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies . A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1) . The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts . Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts . From these results we conclude that GRH is identical to UREF . Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene. J Biol Chem, 1999 Dec 3, 274(49), 34646 - 56 The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase; Caldwell BD et al.; The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14 . 17.3) contains multiple signals determining its subcellular localization . Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M . R., Caldwel, B . D., Johnson, R . C., Darlington, D . N., Mains, R . E., and Eipper, B . A . (1996) J . Biol . Chem . 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase . In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons . Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic . P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D . Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin . Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2 . Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted . P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein. J Biol Chem, 1999 Dec 3, 274(49), 34527 - 30 Cyclin K functions as a CDK9 regulatory subunit and participates in RNA polymerase II transcription; Fu TJ et al.; Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit . The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function . To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo . Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9 . The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions. J Biol Chem, 1999 Dec 3, 274(49), 34519 - 22 Novel human N-acetyltransferase 2 alleles that differ in mechanism for slow acetylator phenotype; Leff MA et al.; Three novel human NAT2 alleles (NAT2*5D, NAT2*6D, and NAT2*14G) were identified and characterized in a yeast expression system . The common rapid (NAT2*4) and slow (NAT2*5B) acetylator human NAT2 alleles were also characterized for comparison . The novel recombinant NAT2 allozymes catalyzed both N- and O-acetyltransferase activities at levels comparable with NAT2 5B and significantly below NAT2 4, suggesting that they confer slow acetylation phenotype . In order to investigate the molecular mechanism of slow acetylation in the novel NAT2 alleles, we assessed mRNA and protein expression levels and protein stability . No differences were observed in NAT2 mRNA expression among the novel alleles, NAT2*4 and NAT2*5B . However NAT2 5B and NAT2 5D, but not NAT2 6D and NAT2 14G protein expression were significantly lower than NAT2 4 . In contrast, NAT2 6D was slightly (3.4-fold) and NAT2 14G was substantially (29-fold) less stable than NAT2 4 . These results suggest that the 341T --> C (Ile(114) --> Thr) common to the NAT2*5 cluster is sufficient for reduction in NAT2 protein expression, but that mechanisms for slow acetylator phenotype differ for NAT2 alleles that do not contain 341T --> C, such as the NAT2*6 and NAT2*14 clusters . Different mechanisms for slow acetylator phenotype in humans are consistent with multiple slow acetylator phenotypes. Structure Fold Des, 1999 Nov 15, 7(11), 1427 - 37 Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme-membrane association control; Rosano C et al.; BACKGROUND: Hexokinase I sets the pace of glycolysis in the brain, catalyzing the ATP-dependent phosphorylation of glucose . The catalytic properties of hexokinase I are dependent on product inhibition as well as on the action of phosphate . In vivo, a large fraction of hexokinase I is bound to the mitochondrial outer membrane, where the enzyme adopts a tetrameric assembly . The mitochondrion-bound hexokinase I is believed to optimize the ATP/ADP exchange between glucose phosphorylation and the mitochondrial oxidative phosphorylation reactions . RESULTS: The crystal structure of human hexokinase I has been determined at 2.25 A resolution . The overall structure of the enzyme is in keeping with the closed conformation previously observed in yeast hexokinase . One molecule of the ATP analogue AMP-PNP is bound to each N-terminal domain of the dimeric enzyme in a surface cleft, showing specific interactions with the nucleotide, and localized positive electrostatic potential . The molecular symmetry brings the two bound AMP-PNP molecules, at the centre of two extended surface regions, to a common side of the dimeric hexokinase I molecule . CONCLUSIONS: The binding of AMP-PNP to a protein site separated from the catalytic centre of human hexokinase I can be related to the role played by some nucleotides in dissociating the enzyme from the mitochondrial membrane, and helps in defining the molecular regions of hexokinase I that are expected to be in contact with the mitochondrion . The structural information presented here is in keeping with monoclonal antibody mapping of the free and mitochondrion-bound forms of the enzyme, and with sequence analysis of hexokinases that differ in their mitochondria binding properties. Plant J, 1999 Sep, 19(6), 645 - 53 The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene; Zou J et al.; In Arabidopsis thaliana (ecotype Columbia) mutant line AS11, an EMS-induced mutation at a locus on chromosome II results in a reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol, an altered seed fatty acid composition, and delayed seed development . A mutation has been identified in AS11 in a gene, which we designated as TAG1, that encodes a protein with an amino acid sequence which is similar to a recently reported mammalian DGAT, and, to a lesser extent, to acyl CoA:cholesterol acyltransferases . Molecular analysis revealed that the mutant allele in AS11 has a 147 bp insertion located at the central region of intron 2 . At the RNA level, an 81 bp insertion composed entirely of an exon 2 repeat was found in the transcript . While the seed triacylglycerol content is reduced by the lesion in AS11, there is no apparent effect on sterol ester content in the mutant seed . The TAG1 cDNA was over-expressed in yeast, and its activity as a microsomal DGAT confirmed . Therefore, the TAG1 locus encodes a diacylglycerol acyltransferase, and the insertion mutation in the TAG1 gene in mutant AS11 results in its altered lipid phenotype. J Interferon Cytokine Res, 1999 Nov, 19(11), 1245 - 52 Subcellular localization of interferon-inducible Myc/stat-interacting protein Nmi is regulated by a novel IFP 35 homologous domain; Lee ND et al.; Nmi was initially identified through a yeast two-hybrid interaction with N-Myc but it also interacts with c-Myc, Max, Fos, and several other transcription factors, including signal transducer and activator of transcription (Stat) proteins . Nmi is an interferon (IFN)-inducible protein with 25% amino acid identity to the IFN-inducible protein IFP 35 . We have found that this homology consists of a novel domain of approximately 90-92 amino acids (aa) that is repeated in tandem in each protein . This region, termed Nmi/IFP 35 domain (NID), is important for subcellular localization of Nmi . Full-length Nmi protein or deletion constructs containing a single NID are localized to the cytoplasm, but amino-terminal Nmi fragments of up to 92 aa containing neither NID are nuclear . Fusion of the amino-terminal end of Nmi to pyruvate kinase, an exclusively cytoplasmic protein, results in a cytoplasmic fusion protein, suggesting that the amino-terminal end of Nmi does not contain a classic nuclear localization signal (NLS) . Fusion of the amino-terminal end of Nmi to green fluorescent protein (GFP), which is normally found in both nuclear and cytoplasmic compartments, does not alter GFP distribution, whereas fusion of a single NID to GFP targets the fusion to the cytoplasm . Fusion of a nuclear localization signal (NLS) to full-length Nmi or NID repeats targets the hybrid to the nucleus, suggesting that a strong NLS is dominant to the cytoplasmic localization function of NID . NID may mediate cytoplasmic localization of the full-length Nmi protein through NID-NID protein interactions as demonstrated by yeast two-hybrid assay, immunoprecipitation, and the presence of Nmi in a high molecular weight protein complex . These results suggest that Nmi is composed of a modular structure with an amino-terminal domain that when separated from the rest of the protein is nuclear . The carboxy-terminal two thirds of the protein is composed of two NID that mediate cytoplasmic localization of the full-length protein. RNA, 1999 Oct, 5(10), 1357 - 63 Identity elements in bovine tRNA(Trp) required for the specific stimulation of gelonin, a plant ribosome-inactivating protein; Brigotti M et al.; Ribosome-inactivating proteins (RIPs) are RNA-N-glycosidases widely present in plants that depurinate RNA in ribosomes at a specific universally conserved position, A4324, in the rat 28S rRNA . A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes . Among cofactor-dependent RIPs, gelonin is specifically and uniquely stimulated by tRNA(Trp) . The active species are avian (chicken) and mammalian (beef, rat, and rabbit) tRNA(Trp), whereas yeast tRNA(Trp) is completely devoid of stimulating activity . In the present article, bovine and yeast tRNA(Trp) with unmodified bases were prepared by assembly of the corresponding genes from synthetic oligonucleotides followed by PCR and T7 RNA polymerase transcription of the amplified products . The two synthetic tRNAs were fully active (bovine) or inactive (yeast) as the wild-type tRNAs . Construction of chimeric tRNA(Trp) transcripts identified the following bovine nucleotides as recognition elements for gelonin-stimulating activity: G26 and bp G12-C23 in the D arm and G57, A59, and bp G51-C63 and U52-A62 in the T arm . Among single-stranded nucleotides, A59 has a prominent role, but full expression of the gelonin-stimulating activity requires an extensive cooperation between nucleotides in both arms. J Cell Biochem, 1999 Dec 15, 75(4), 698 - 709 Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3; Bonetto F et al.; An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system . Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region . In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1 . In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb . This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages. Blood, 1999 Dec 1, 94(11), 3737 - 47 A novel BTB/POZ transcriptional repressor protein interacts with the Fanconi anemia group C protein and PLZF; Hoatlin ME et al.; Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome . The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities . At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack recognizable motifs, and their functions are unknown . We have isolated a binding partner for the Fanconi anemia group C protein (FANCC) by yeast two-hybrid screening . We show that the novel gene, FAZF, encodes a 486 amino acid protein containing a conserved amino terminal BTB/POZ protein interaction domain and three C-terminal Kruppel-like zinc fingers . FAZF is homologous to the promyelocytic leukemia zinc finger (PLZF) protein, which has been shown to act as a transcriptional repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and HDAC1 complex) . Consistent with a role in FA, BTB/POZ-containing proteins have been implicated in oncogenesis, limb morphogenesis, hematopoiesis, and proliferation . We show that FAZF is a transcriptional repressor that is able to bind to the same DNA target sequences as PLZF . Our data suggest that the FAZF/FANCC interaction maps to a region of FANCC deleted in FA patients with a severe disease phenotype . We also show that FAZF and wild-type FANCC can colocalize in nuclear foci, whereas a patient-derived mutant FANCC that is compromised for nuclear localization cannot . These results suggest that the function of FANCC may be linked to a transcriptional repression pathway involved in chromatin remodeling. Biochemistry, 1999 Oct 26, 38(43), 14146 - 56 Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation; Yudt MR et al.; The human estrogen receptor (hER) is a ligand-activated transcription factor which, like many other members of the nuclear receptor protein family, exhibits a dimerization-dependent transcriptional activation . Several previous reports have provided evidence of the phosphorylation of the hER at tyrosine 537 (Y537) . However, the exact function of a putative phosphorylation at this site remains controversial . Using a yeast transactivation assay, and in vitro biochemical approaches, we show that phosphorylation of tyrosine 537 is not required for the hER to bind hormone, or to activate transcription . An hER tyrosine 537 to phenylalanine (Y537F) mutant retains 70-75% of the transactivation potential of wild type hER in a yeast reporter system . Furthermore, the mutated receptor exhibits wild type hormone and DNA binding affinities . However, this mutation results in a decrease in receptor stability as measured by a decrease in the extent of hormone binding over time . The most striking difference between the wild type and Y537F hER is in the estradiol binding kinetics . Whereas the off-rate for estradiol exhibits a two-state binding mechanism, the Y537F mutant hER exhibits a monophasic estradiol off-rate . On the basis of these data and other reports describing the structure and activity of Y537 mutations, as well as knowledge of the three-dimensional structure of the hER ligand binding domain, we propose an alternate model wherein Y537F mutation favors an "open" pocket conformation, affecting the estrogen binding kinetics and stability of the hormone-bound, transcriptionally active "closed" pocket conformation . Although its phosphorylation is not essential for function of the hER, Y537 is nevertheless a critical residue intricately involved with the conformational changes of the hER and its ability to activate transcription. Plant J, 1999 Oct, 20(2), 183 - 95 UPL1 and 2, two 405 kDa ubiquitin-protein ligases from Arabidopsis thaliana related to the HECT-domain protein family; Bates PW et al.; The ubiquitin/26S proteasome pathway is a major route for degrading abnormal and important short-lived regulatory proteins in eukaryotes . Covalent attachment of ubiquitin, which triggers entry of target proteins into the pathway, is accomplished by an ATP-dependent reaction cascade involving the sequential action of three enzymes, E1s, E2s and E3s . Although much of the substrate specificity of the pathway is determined by E3s (or ubiquitin-protein ligases, UPLs), little is known about these enzymes in plants and how they choose appropriate targets for ubiquitination . Here, we describe two 405 kDa E3s (UPL1 and 2) from Arabidopsis thaliana related to the HECT-E3 family that is essential in yeast and animals . UPL1 and 2 are encoded by 13 kbp genes 26 cM apart on chromosome I, that are over 95% identical within both the introns and exons, suggesting that the two loci arose from a recent gene duplication . The C-terminal HECT domain of UPL1 is necessary and sufficient to conjugate ubiquitin in vitro in a reaction that requires the positionally conserved cysteine within the HECT domain, E1, and an E2 of the UBC8 family . Given that HECT E3s help define target specificity of the ubiquitin conjugation, a continued characterization of UPL1 and 2 should be instrumental in understanding the functions of ubiquitin-dependent protein turnover in plants and for identifying pathway substrates. FEBS Lett, 1999 Oct 22, 460(1), 117 - 22 Isolation and characterization of the E2F-like gene in plants; Sekine M et al.; The transcription factor E2F regulates the expression of genes involved in the progression of G1/S transition and DNA replication in mammalian cells . We cloned and characterized a cDNA (NtE2F) corresponding to a E2F homolog of tobacco (Nicotiana tabacum) . The transcription of NtE2F was induced as cells progressed from G1 to the S phase and expressed much earlier than that of the proliferating cell nuclear antigen (PCNA) gene . We demonstrated that NtE2F can interact with the tobacco retinoblastoma (Rb)-related protein in a yeast two-hybrid assay . To further characterize NtE2F, the trans-activation activity of NtE2F was examined by using a transient assay in the tobacco Bright Yellow-2 (BY-2) cells with NtE2F fused to the DNA-binding domain of the veast transcriptional activator GAL4 . NtE2F activated the transcription of the beta-glucuronidase (GUS) reporter gene driven by a cauliflower mosaic virus (CaMV) 35S core promoter containing the GAL4-binding sequence . This is the first report of the identification of a functionally equivalent E2F-like gene in plants. FEBS Lett, 1999 Oct 22, 460(1), 93 - 8 BH-protocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain; Yoshida K et al.; Using a yeast two-hybrid system, we isolated eight cDNA clones which interacted with BH-protocadherin-c (BH-Pcdh-c) from the human brain cDNA library . One clone encoded protein phosphatase type I isoform alpha (PP1alpha) and another two PP1alpha2 . PP1alpha was co-immunoprecipitated from the extract of a gastric adenocarcinoma cell line MKN-28 with anti-BH-Pcdh-c antibody . PP1alpha activity towards glycogen phosphorylase was inhibited by the intracellular domain of BH-Pcdh-c . Inhibition of the phosphatase required more than the minimal domain of BH-Pcdh-c which could associate with PP1alpha . In situ hybridization revealed that BH-Pcdh-c mRNA was predominantly expressed in cerebral cortex neurons in the adult mouse brain. FEBS Lett, 1999 Oct 22, 460(1), 41 - 5 Identification of proteins that interact with NF-YA; Yamada K et al.; We used the yeast two-hybrid system to show that the serum response factor (SRF) and zinc-fingers and homeobox 1 (ZHXI) proteins interact with the A subunit of nuclear factor-Y (NF-YA) . GST pulldown assays revealed that both proteins interact specifically with NF-YA in vitro . Amino acids located between 272 and 564, a region that contains two homeodomains, are required for the interaction of ZHX1 with NF-YA . Two different domains of NF-YA, a glutamine-rich region and a serine/threonine-rich region, are necessary for the interactions with ZHX1 and SRF, respectively. Gene, 1999 Oct 18, 239(1), 39 - 48 A PSTTLRE-form of cdc2-like gene in the marine microalga Dunaliella tertiolecta; Lin S et al.; To understand the genetic control of algal cell division cycle that pertains to phytoplankton bloom dynamics in the sea, we cloned and analyzed a gene coding for a cyclin-dependent kinase (CDK) for the chlorophyte Dunaliella tertiolecta . The cDNA cloned, 1061 bp long, contained an open reading frame of 314 amino acids . FASTA and GAP analyses showed that this sequence was most homologous to cdc2 out of all known cdks, with an identity of 54-68% and a similarity of 65-76% to cdc2 in higher plants, animals, and yeast . Several signature domains of cdc2 were identified from this sequence, although the PSTAIRE and GDSEID motifs were replaced with PSTTLRE and GDCELQ, respectively . Southern blot hybridization demonstrated that this gene occurred as a single copy in this species, and quantitative RT-PCR showed that the transcription of this gene was constitutive . The present results suggest that the universal cdc2 is conserved in the lower eukaryote with unique structural characteristics. Biochim Biophys Acta, 1999 Oct 8, 1455(2-3), 167 - 78 Molecular basis of carbohydrate-deficient glycoprotein syndromes type I with normal phosphomannomutase activity; Freeze HH et al.; Carbohydrate deficient glycoprotein syndromes (CDGS) are inherited disorders in glycosylation . Isoelectric focusing of serum transferrin is used as a biochemical indicator of CDGS; however, this technique cannot diagnose the molecular defect . Even though phosphomannomutase (PMM) deficiency accounts for the great majority of known CDGS cases (CDGS type Ia), newly discovered cases have significantly different clinical presentations than the PMM-deficient patients . These differences arise from other defects affecting the biosynthesis of N-linked oligosaccharides in the endoplasmic reticulum and in the Golgi compartment . The most notable is the loss of phosphomannose isomerase (PMI) (CDGS type Ib) . It causes severe hypoglycemia, protein-losing enteropathy, vomiting, diarrhea, and congenital hepatic fibrosis . In contrast to PMM-deficiency, there is no developmental delay nor neuropathy . Most symptoms