Cancer Res, 1999 Dec 1, 59(23), 5908 - 11 Effects of p51/p63 missense mutations on transcriptional activities of p53 downstream gene promoters; Kato S et al.; The p51/p63 gene is a homologue of p53, the product of which acts as a transcriptional activator by binding to p53-responsive elements in the promoter regions of several p53 downstream genes . Recently, we identified four distinct mutations in the p51/p63 gene after screening >200 human tumors and cell lines . Because all of the detected p51/p63 mutations were missense mutations, the pathogenic effect of these mutations is difficult to determine without performing a functional analysis . In this study, we examined the transcriptional activity of tumor-derived p51/p63 missense mutations using a yeast-based assay and compared the data with that of artificial p51/p63 missense mutations at residues corresponding to the positions and substituted residues of p53 mutation "hotspots." Although most of the p51/p63 missense mutations at the p53 hotspot residues were unable to transactivate the promoters used in this study, the tumor-derived p51/p63 missense mutations retained their ability to transactivate the MDM2 and/or the BAX promoter but not the p21/WAF1 promoter . These results suggest that the p51/p63 mutation might be involved in an unknown tumor suppression pathway distinct from that of p53. Proc Nutr Soc, 1999 Aug, 58(3), 621 - 3 Regulation of gene expression by glucose; Ferre P; Fatty acid synthase (EC 2.3.1.85) is an enzyme involved in the lipogenic pathway allowing fatty acid synthesis from glucose . Glucose up-regulates the transcription of the fatty acid synthase gene in both adipocytes and hepatocytes, with insulin having only an indirect role . The signal metabolite could be glucose-6-phosphate rather than glucose itself . The glucose response element of the fatty acid synthase gene has not yet been precisely identified, although a -2 kb region of the fatty acid synthase promoter is sufficient to confer nutritional responsiveness to a reporter gene . ADD1/SREBP1, a b-HLH-LZ transcription factor belonging to the sterol regulatory element-binding protein family might be involved in the transduction of the glucose effect . Finally, the stimulatory effect of glucose on the expression of the fatty acid synthase gene is inhibited by the activation of AMP-activated protein kinase . Interestingly enough, AMP-activated protein kinase is structurally and functionally related to the yeast SNF1 protein kinase complex which is essential for the transcriptional activation of glucose-repressed genes in Saccharomyces cerevisiae. Trends Cell Biol, 2000 Jan, 10(1), 25 - 31 Protein translocation into mitochondria: the role of TIM complexes; Bauer MF et al.; Import of nuclear-encoded mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex . Both TIM complexes cooperate with the TOM complex but facilitate import of different classes of precursor proteins . Precursors with an N-terminal presequence are imported via the TIM23 complex, whereas mitochondrial carrier proteins require the TIM22 complex for insertion into the inner membrane . This review discusses recent advances in understanding the structure and function of the translocases of the inner membrane and the possible role of Tim proteins in the development of the Mohr-Tranebjaerg syndrome, a mitochondrial disorder leading to neurodegeneration. Oncogene, 1999 Dec 9, 18(52), 7566 - 75 Induction of apoptosis by SLK, a Ste20-related kinase; Sabourin LA et al.; We have cloned and characterized a novel murine Ste20-related kinase designated SLK . SLK displays high homology to the Ste20-related kinase LOK, and is more distantly related to MST1 and 2, both Ste20-like kinases . In addition, SLK displays high homology to microtubule and nuclear associated protein (M-NAP) and AT1-46, both of unknown function . SLK is ubiquitously expressed as multiple mRNAs in tissues and cell lines and is downregulated by mitogen depletion in differentiating myoblasts . Biochemical characterization showed that SLK overexpression activates c-Jun amino-terminal kinase 1 (JNK1) . However, in vitro kinase assays indicated that SLK was not activated in response to various growth factors or stress-inducing agents . Immunofluorescence studies revealed that SLK colocalized to distinct cytosolic domains, preferentially at the periphery of the cells . In addition, prolonged overexpression of SLK in cultured fibroblasts resulted in apoptosis as demonstrated by annexin-V and TUNEL staining . Our results suggest that SLK belongs to a new family of protein kinases, mediating activation of the stress response pathway through a novel signaling cascade. Oncogene, 1999 Dec 9, 18(52), 7495 - 505 Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal; Koike M et al.; Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation . Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations . Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70 . Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle . Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku . The nuclear translocation of Ku80 started during the late telophase/early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70 . Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70 . To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and defined to a region spanning nine amino acid residues (positions 561 - 569) . The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97 . All these findings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70. Chirality, 2000 Jan, 12(1), 26 - 9 Chemo-enzymatic synthesis of the antidepressant duloxetine and its enantiomer; Liu H et al.; Sodium borohydride reduction of 3-chloro-1-(2-thienyl)-1-propanone gave the corresponding racemic alcohol which was kinetically resolved with lipase B from Candida antarctica as catalyst to yield the chiral building blocks (S)-3-chloro-1-(2-thienyl)-1-propanol and the corresponding (R)-butanoate . The enantiopure chiral building blocks were converted into Duloxetine and its enantiomer . J Biol Chem, 1999 Dec 24, 274(52), 36987 - 94 The role of the Smad3 protein in phorbol ester-induced promoter expression; Biggs JR et al.; The Sp1 transcription factor plays an important role in mediating the p53-independent activation of the p21(WAF1) (WAF1) promoter by phorbol 12-myristate13-acetate (PMA) in hematopoietic cells . Using GAL4-Sp1 fusion proteins and a luciferase reporter, PMA is shown to activate the transcriptional activity of Sp1 independent of the WAF1 promoter . This activation does not require the Ser/Thr-rich region of Sp1 and can be mediated by 41 amino acids (152-193) of Sp1 that are important for the interaction with human TAF130 . Because transforming growth factor-beta enhances WAF1 promoter activity through both Sp1 and Smad proteins, the role of Smads in PMA transcriptional activation was examined . PMA addition to hematopoietic cells was found to activate a GAL4/Smad-dependent promoter and the transforming growth factor-beta-responsive promoter, p3TP-lux . Immunofluorescence data demonstrate that PMA addition to hematopoietic cells induces the translocation of Smad3 to the nucleus . However, Smad3 does not stimulate the WAF1 promoter, but rather slightly inhibits the PMA-mediated induction of transcription from this upstream region . Additionally, transfection of Smad3 did not enhance the activation of GAL4/Sp1 by PMA . These results demonstrate that, while PMA can activate Smad-mediated transcription, Smad proteins do not appear to play a major role in the PMA induction of the WAF1 promoter. J Biol Chem, 1999 Dec 24, 274(52), 36839 - 42 The xenopus Suc1/Cks protein promotes the phosphorylation of G(2)/M regulators; Patra D et al.; The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF) . In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1 . At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself . The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown . We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex . Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts . We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B . This observation could explain why overexpression of Pin1 inhibits mitotic initiation . These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2. Genes Dev, 1999 Dec 1, 13(23), 3115 - 24 Oppositely imprinted genes p57(Kip2) and igf2 interact in a mouse model for Beckwith-Wiedemann syndrome; Caspary T et al.; Beckwith-Wiedemann syndrome (BWS) is a clinically variable disorder characterized by somatic overgrowth, macroglossia, abdominal wall defects, visceromegaly, and an increased susceptibility to childhood tumors . The disease has been linked to a large cluster of imprinted genes at human chromosome 11p15.5 . A subset of BWS patients has been identified with loss-of-function mutations in p57(KIP2), a maternally expressed gene encoding a G(1) cyclin-dependent kinase inhibitor . Some patients display loss of imprinting of IGF2, a fetal-specific growth factor that is paternally expressed . To understand how the same disease can result from misregulation of two linked, but unrelated, genes, we generated a mouse model for BWS that both harbors a null mutation in p57(Kip2) and displays loss of Igf2 imprinting . These mice display many of the characteristics of BWS, including placentomegaly and dysplasia, kidney dysplasia, macroglossia, cleft palate, omphalocele, and polydactyly . Some, but not all, of the phenotypes are shown to be Igf2 dependent . In two affected tissues, the two imprinted genes appear to act in an antagonistic manner, a finding that may help explain how BWS can arise from mutations in either gene. EMBO J, 1999 Dec 15, 18(24), 6908 - 16 The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate; Christ F et al.; The monomeric homing endonuclease PI-SceI cleaves the two strands of its DNA substrate in a concerted manner, which raises the question of whether this enzyme harbours one or two catalytic centres . If PI-SceI has only one catalytic centre, one would expect that cross-linking enzyme and substrate should prevent reorientation of the enzyme required to perform the second cut after having made the first cut: PI-SceI, however, when cross-linked to its substrate, is able to cleave both DNA strands . If PI-SceI has two catalytic centres, one would expect that it should be possible to inactivate one catalytic centre by mutation and obtain a variant with preference for a substrate nicked in one strand; such variants have been found . The structural homology between the catalytic domain of PI-SceI having a pseudo 2-fold symmetry, and I-CreI, a homodimeric homing endonuclease, suggests that in PI-SceI active site I, which attacks the top strand, comprises Asp218, Asp229 and Lys403, while Asp326, Thr341 and Lys301 make up active site II, which cleaves the bottom strand . Cleavage experiments with modified oligodeoxynucleotides and metal ion mapping experiments demonstrate that PI-SceI interacts differently with the two strands at the cleavage position, supporting a model of two catalytic centres. Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 405 - 10 Sas3 is a histone acetyltransferase and requires a zinc finger motif; Takechi S et al.; SAS3 was originally isolated as a gene related to SAS2, which encodes a positive regulator of transcriptional silencing in yeast . The Sas3 protein possesses an evolutionally conserved domain that is shared by a group of SAS-like factors . This conserved domain contains an atypical zinc finger motif and a putative acetyl-CoA binding motif . We showed that recombinant Sas3 exhibits histone acetyltransferase (HAT) activity toward acetylate core histones H2A, H3, and H4 . This substrate specificity is similar to those of Tip60 and Esa1 . Analysis of a series of deletion mutants revealed that the minimum region required for HAT activity is located within amino acid residues 241-577, including the domain conserved in the MYST family proteins . Amino acid substitution mutant analysis showed that both the acetyl-CoA binding motif and the zinc finger motif are required for HAT activity . These results suggest that SAS3 and its family members require the zinc finger motif for their activity . Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 347 - 51 Identification and characterization of SorCS, a third member of a novel receptor family; Hermey G et al.; A novel receptor, SorCS, was isolated from murine brain . It shows homology to the mosaic receptor SorLA and the neurotensin receptor sortilin based on a common VPS10 domain which is the hallmark of this new receptor family . In the N-terminus of SorCS two putative cleavage sites for the convertase furin mark the beginning of the VPS10 domain, followed by a module of imperfect leucine-rich repeats and a transmembrane domain . The short intracellular C-terminus contains consensus signals for rapid internalization . The identified putative binding motifs for SH2 and SH3 domains are unique in the family of VPS10 domain receptors . SorCS is predominantly expressed in brain, but also in heart, liver, and kidney . SorCS transcripts detected by in situ hybridization in the murine central nervous system point to a neuronal expression . Protein Expr Purif, 1999 Dec, 17(3), 358 - 65 Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch; Shao F et al.; The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter . SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast . Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus . Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form . The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active . Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin . J Mol Biol, 1999 Dec 17, 294(5), 1311 - 25 Crystal structure of the histone acetyltransferase Hpa2: A tetrameric member of the Gcn5-related N-acetyltransferase superfamily; Angus-Hill ML et al.; We report the crystal structure of the yeast protein Hpa2 in complex with acetyl coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution . Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a family of enzymes with diverse substrates including histones, other proteins, arylalkylamines and aminoglycosides . In vitro, Hpa2 is able to acetylate specific lysine residues of histones H3 and H4 with a preference for Lys14 of histone H3 . Hpa2 forms a stable dimer in solution and forms a tetramer upon binding AcCoA . The crystal structure reveals that the Hpa2 tetramer is stabilized by base-pair interactions between the adenine moieties of the bound AcCoA molecules . These base-pairs represent a novel method of stabilizing an oligomeric protein structure . Comparison of the structure of Hpa2 with those of other GNAT superfamily members illustrates a remarkably conserved fold of the catalytic domain of the GNAT family even though members of this family share low levels of sequence homology . This comparison has allowed us to better define the borders of the four sequence motifs that characterize the GNAT family, including a motif that is not discernable in histone acetyltransferases by sequence comparison alone . We discuss implications of the Hpa2 structure for the catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone tail modification created by the tetrameric Hpa2 structure . Biochemistry, 1999 Dec 14, 38(50), 16613 - 9 Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1; Kvaratskhelia M et al.; Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae . Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange . In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques . Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer . Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium . The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold . This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments {Overmars and Altona (1997) J . Mol . Biol . 273, 519-524; Panyutin et al . (1995) EMBO J . 14, 1819-1826} . The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding. Biochemistry, 1999 Dec 14, 38(50), 16461 - 8 Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro; Ying J et al.; Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes . We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands . Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures . Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase . Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo. J Clin Endocrinol Metab, 1999 Dec, 84(12), 4362 - 70 Heritable disorders of pituitary development; Parks JS et al.; Basic and translational research achievements over the past 2 decades have disclosed the molecular mechanisms underlying several genetic forms of hypopituitarism . Disorders that are limited to the hypothalamic, pituitary, GH axis are caused by mutations in individual components of that axis . Disorders involving GH and one or more additional pituitary hormones are caused by mutations in the homeodomain transcription factors that direct embryological development of the anterior pituitary gland . Pit-1 has a POU-specific and a POU-homeo DNA-binding domain . The phenotype produced by mutations in the PIT1 gene involves deficiencies of GH, PRL, and TSH . Pituitary glands are either small or normally sized . The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain . Persons with inactivating mutations in PROP1 have deficiencies of LH and FSH, as well as GH, PRL, and TSH . Their pituitary glands may be small, normally sized, or extremely large and show suprasellar extension . Pituitary degeneration may produce acquired deficiency of ACTH . Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and it is much more widespread . The protein has a paired-like domain, and it competes with the product of PROP1 for DNA-binding . Homozygosity for inactivating mutations of HESX1 produces a complex phenotype that resembles septo-optic dysplasia . Much more needs to be learned about the role of HESX1 mutations in other forms of hypopituitarism. Hum Genet, 1999 Nov, 105(5), 489 - 95 Structure of the human Lanosterol synthase gene and its analysis as a candidate for holoprosencephaly (HPE1); Roessler E et al.; Holoprosencephaly (HPE) is the most common birth defect of the brain in humans . It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies . The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE . Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients . We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein . Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis. Mol Endocrinol, 1999 Dec, 13(12), 2151 - 62 Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2; Xie W et al.; In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TRE(pal)) but not the glucocorticoid response element (GRE) . This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor . In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner . Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors. Exp Lung Res, 1999 Oct-Nov, 25(7), 561 - 76 Developmental changes in phosphatidylinositol transfer protein concentration and phospholipid transfer activities in rat type II cells; Viscardi RM et al.; The phospholipid transfer proteins (PLTPs) are cytosolic proteins that have been characterized by their ability to facilitate the transfer of phospholipids between membranes in vitro . The goals of this study were to determine whether PITP alpha concentration and phospholipid transfer activities are enriched in type II cells compared with whole lung and to determine the developmental changes in PITP alpha concentration and phospholipid transfer activities during late gestation and newborn period . The concentration of PITP alpha in type II cell cytosol measured by enzyme-linked immunosorbent assay (ELISA) increased during late fetal gestation to 2.2-fold adult levels and declined 41% during the first postnatal day . However, compared to whole adult lung cytosol, type II cell cytosol was not significantly enriched with PITP alpha . Phospholipid transfer activities were determined by a vesicle-rat lung membrane transfer assay . In adult lung, transfer activities for all the phospholipids were enriched in adult type II cell cytosol compared to whole lung cytosol (phosphatidylglycerol {PG}, 12.5-fold; phosphatidylinositol {PI}, 9.2-fold; phosphatidylcholine {PC}, 6.5-fold; and phosphatidylethanolamine {PE}, 6.6-fold; P < .05 in each case) . The rate of phospholipid transfer in type II cell cytosol increased during late fetal gestation to levels 4.9 (PG), 3.7 (PI), and 2.8 (PC) times greater than adult levels . In cytosol from cells from different stages, the order of transfer rate was PG > PI > PC > PE . PITP alpha immunodepletion of adult type II cytosol did not significantly affect phospholipid transfer activities, suggesting that other PLTPs are responsible for the majority of the observed transfer activities in these cells . Developmental increases in PITP alpha concentration and other PLTPs parallel developmental changes in type II cell surfactant phospholipid metabolism, suggesting a possible role of these transfer proteins in the unique function of the type II cell. Plant Mol Biol, 1999 Oct, 41(3), 415 - 23 Selection of Arabidopsis genes encoding secreted and plasma membrane proteins; Goo JH et al.; Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms . Systematic cloning of the genes encoding these proteins is therefore of general interest . An effective method of trapping signal sequences was first described by Tashiro et al . (1993), and a similar yet more efficient method was reported by Klein et al . (1996) and Jacobs et al . (1997) . In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins . Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel . All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis . The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced . The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein . These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins. Semin Cell Dev Biol, 1999 Oct, 10(5), 465 - 72 Role and regulation of the ER chaperone BiP; Gething MJ; BiP, an HSP70 molecular chaperone located in the lumen of the endoplasmic reticulum (ER), binds newly-synthesized proteins as they are translocated into the ER and maintains them in a state competent for subsequent folding and oligomerization . BiP is also an essential component of the translocation machinery, as well as playing a role in retrograde transport across the ER membrane of aberrant proteins destined for degradation by the proteasome . BiP is an abundant protein under all growth conditions, but its synthesis is markedly induced under conditions that lead to the accumulation of unfolded polypeptides in the ER . This attribute provides a marker for disease states that result from misfolding of secretory and transmembrane proteins. Oncogene, 1999 Nov 18, 18(48), 6621 - 34 Mmip-2, a novel RING finger protein that interacts with mad members of the Myc oncoprotein network; Yin XY et al.; Mad proteins are basic-helix-loop-helix-leucine zipper (bHLH-ZIP)-containing members of the myc oncoprotein network . They interact with the bHLH-ZIP protein max, compete for the same DNA binding sites as myc-max heterodimers and down-regulate myc-responsive genes . Using the bHLH-ZIP domain of mad1 as a yeast two-hybrid 'bait', we identified Mmip-2, a novel RING finger protein that interacts with all mad members, but weakly or not at all with c-myc, max or unrelated bHLH or bZIP proteins . The mad1-Mmip-2 interaction is mediated by the ZIP domain in the former protein and by at least two regions in the latter which do not include the RING finger . Mmip-2 can disrupt max-mad DNA binding and can reverse the suppressive effects of mad proteins on c-myc-responsive target genes and on c-myc + ras-mediated focus formation in fibroblasts . Tagging with spectral variants of green fluorescent protein showed that Mmip-2 and mad proteins reside in separate cytoplasmic and nuclear compartments, respectively . When co-expressed, however, the proteins interact and translocate to the cellular compartment occupied by the more abundant protein . These observations suggest a novel way by which Mmip-2 can modulate the transcriptional activity of myc oncoproteins. Mol Cells, 1999 Oct 31, 9(5), 564 - 8 Selection of Drosophila genes encoding secreted and membrane proteins; Goo JH et al.; With the use of a yeast-based signal sequence trap (YSST) method, we screened a Drosophila cDNA library to isolate genes encoding secreted and membrane proteins . Of the 136 unique cDNA clones sequenced, 11 clones (8.1%) are identical to previously known Drosophila genes, 18 clones (13.2%) are homologous to other genes identified in various organisms, and 91 clones (66.9%) are novel . Most of these genes are secreted or membrane proteins, or appear to contain putative signal sequences at their amino termini . This indicates that YSST is an effective tool for the isolation and analysis of Drosophila genes that play roles in intercellular communication. J Agric Food Chem, 1999 Aug, 47(8), 3297 - 302 Response of the aroma fraction in sherry wines subjected to accelerated biological aging; Cortes MB et al.; The effect of an acceleration assay, carried out with a periodic aeration and an increased surface/volume ratio, on various aroma compounds of "fino" Sherry wines aging under a veil of a pure culture of Saccharomyces cerevisiae race capensis G1 flor film yeast was studied . The results were subjected to multifactor analysis of variance, and the compounds simultaneously depending on acceleration conditions and aging time at p < 0.01 were subjected to principal component analysis . The first component, accounting for 86.14% of the overall variance, was mainly defined by acetaldehyde and its derivatives 1,1-diethoxyethane and acetoin . These compounds reached higher concentrations in accelerated aging wines in a shorter time than they did in control wines, and no browning problems were detected . Taking into account that these compounds can be used as indicators for biological aging of "fino" Sherry wines, the acceleration condition assayed can be applied to shorten the time of this process. Mol Microbiol, 1999 Dec, 34(5), 1007 - 17 The MAP kinase kpp2 regulates mating and pathogenic development in Ustilago maydis; Muller P et al.; In the phytopathogenic fungus Ustilago maydis, fusion of compatible haploid cells is a prerequisite for infection . This process is genetically controlled by the biallelic a locus, encoding pheromone precursors and receptors . These are presumed to be coupled to a heterotrimeric G protein and a MAP kinase cascade, leading to activation of the HMG domain transcription factor Prf1 . Here, we have demonstrated that putative MAP kinase sites in Prf1 are required for its activity during mating . In addition, we have identified a gene, kpp2, which encodes a putative MAP kinase related to Pmk1 of Magnaporthe grisea and Fus3p of Saccharomyces cerevisiae . kpp2 deletion mutants are attenuated in several steps of development: cell fusion, induction of pheromone-responsive genes and pathogenicity . Epistasis analysis shows that kpp2 does not affect pheromone gene expression through the cAMP signalling cascade . Pathogenicity of kpp2 mutants can be partially restored by overexpressing the b genes, indicating a regulation of Prf1 by Kpp2 . These data support the hypothesis that the MAP kinase Kpp2 transmits the pheromone signal. Nat Struct Biol, 1999 Nov, 6(11), 1039 - 42 Solution structure of the hRPABC14.4 subunit of human RNA polymerases; del Rio-Portilla F et al.; The protein hRPABC14.4 is an essential subunit of human RNA polymerases I, II, and III and is required for the transcription of all human nuclear genes . The structure of hRPABC14.4 was determined by nuclear magnetic resonance spectroscopy . The protein fold comprises a highly conserved central domain forming two antiparallel alpha-helices flanked by the less conserved N- and C-terminal regions forming a five-stranded beta-sandwich . Amino acids from the two helices participate in the generation of a hydrophobic surface area which is conserved in all eukaryotic and archaeal homologous subunits, and likely constitutes a critical macromolecular interaction interface . The hRPABC14.4 structure accounts for mutagenesis results in Saccharomyces cerevisiae and provides a structural working model for elucidating the role of this subunit in the molecular architecture and function of the human nuclear RNA polymerases. Nat Struct Biol, 1999 Nov, 6(11), 1029 - 32 Crystal structure of the post-chaperonin beta-tubulin binding cofactor Rbl2p; Steinbacher S; The folding pathway of tubulins includes highly specific interactions with a series of cofactors (A, B, C, D and E) after they are released from the eukaryotic chaperonin CCT . The 2.2 A crystal structure of Rbl2p, the Saccharomyces cerevisiae homolog of beta-tubulin specific cofactor A, shows alpha-helical monomers forming a flat, slightly convex dimer . The surface of the molecule is dominated by polar and charged residues and lacks hydrophobic patches typically observed for chaperones that bind unfolded or partially folded proteins . This post-chaperonin cofactor is therefore clearly distinct from typical chaperones where hydrophobicity is a hallmark of substrate recognition. Nat Struct Biol, 1999 Nov, 6(11), 990 - 1 A chaperone with a hydrophilic surface; Cowan NJ et al.; The folding of native tubulin involves at least seven different chaperone proteins: prefoldin, the cytosolic chaperonin CCT and five tubulin-specific chaperone proteins named cofactors A-E . The structure of the yeast homolog of cofactor A, Rbl2p, shows it to be a dimer with largely hydrophilic surfaces, reflecting the fact that it interacts with quasi-native, not unfolded, beta-tubulin. J Microsc, 1999 Dec, 196 ( Pt 3), 279 - 87 Biological ultrastructure as revealed by high resolution cryo-SEM of block faces after cryo-sectioning; Walther P et al.; Ultrastructural information was obtained by imaging the block face of high-pressure-frozen cryo-sectioned biological samples in a high-resolution cryo-SEM . Cryo-sectioning leads to a well-defined flat artificial surface in contrast to cryo-fracturing . Typical artefacts of cryo-sections such as compression and crevasses were not visible on the block face . The ultrastructural features known from resin sections and from freeze-fractures could also be found on the block faces . The cytoplasms show particles of different size which most likely represent proteins . The effects of radiation damage could be reduced considerably by applying the double layer coating technique and backscattered electron imaging . High quality cryo-sections are only obtained from vitrified material . Reasonably flat block faces were, however, also obtained from adequately frozen microcrystalline samples, thereby facilitating ultrastructural studies in the frozen hydrated state. Plant Physiol, 1999 Dec, 121(4), 1127 - 42 Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system; Nebenfuhr A et al.; The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm . To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I . The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases . Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots . Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks . In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion . In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior . We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking. Mol Cell Biol, 2000 Jan, 20(1), 286 - 98 Essential function of the polo box of Cdc5 in subcellular localization and induction of cytokinetic structures; Song S et al.; Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation . In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic C-terminal domain, termed the polo box . Here we show that the budding-yeast polo kinase Cdc5, when fused to green fluorescent protein and expressed under its endogenous promoter, localizes at spindle poles and the mother bud neck . Overexpression of Cdc5 can induce a class of cells with abnormally elongated buds in a polo box- and kinase activity-dependent manner . In addition to localizing at the spindle poles and cytokinetic neck filaments, Cdc5 induces and localizes to additional septin ring structures within the elongated buds . Without impairing kinase activity, conservative mutations in the polo box abolish the ability of Cdc5 to functionally complement the defect associated with a cdc5-1 temperature-sensitive mutation, to localize to the spindle poles and cytokinetic neck filaments, and to induce elongated cells with ectopic septin ring structures . Consistent with the polo box-dependent subcellular localization, the C-terminal domain of Cdc5, but not its polo box mutant, is sufficient for subcellular localization, and its overexpression appears to inhibit cytokinesis . These data provide evidence that the polo box is required to direct Cdc5 to specific subcellular locations and induce or organize cytokinetic structures. Mol Cell Biol, 2000 Jan, 20(1), 242 - 8 Dbf4p, an essential S phase-promoting factor, is targeted for degradation by the anaphase-promoting complex; Ferreira MF et al.; The Dbf4p/Cdc7p protein kinase is essential for the activation of replication origins during S phase . The catalytic subunit, Cdc7p, is present at constant levels throughout the cell cycle . In contrast, we show here that the levels of the regulatory subunit, Dbf4p, oscillate during the cell cycle . Dbf4p is absent from cells during G(1) and accumulates during the S and G(2) phases . Dbf4p is rapidly degraded at the time of chromosome segregation and remains highly unstable during pre-Start G(1) phase . The rapid degradation of Dbf4p during G(1) requires a functional anaphase-promoting complex (APC) . Mutation of a sequence in the N terminus of Dbf4p which resembles the cyclin destruction box eliminates this APC-dependent degradation of Dbf4p . We suggest that the coupling of Dbf4p degradation to chromosome separation may play a redundant role in ensuring that prereplicative complexes, which assemble after chromosome segregation, do not immediately refire. Mol Cell Biol, 2000 Jan, 20(1), 104 - 12 Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II; Rodriguez CR et al.; The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II . Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important . We sought to identify the CTD kinase responsible for capping enzyme targeting . The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro . However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not . Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo . Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable . Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription. Mol Cell Biol, 2000 Jan, 20(1), 1 - 11 Scanning mutagenesis of Mcm1: residues required for DNA binding, DNA bending, and transcriptional activation by a MADS-box protein; Acton TB et al.; MCM1 is an essential gene in the yeast Saccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors . To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro . Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending . Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro . These positions have almost identical interactions with DNA in both the serum response factor-DNA and alpha2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA . Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding . These results suggest a general model of how MADS-box proteins recognize and bind DNA . We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites . Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype . This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell. J Biol Chem, 1999 Dec 17, 274(51), 36428 - 38 An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein; Golovkin M et al.; The U1 small nuclear ribonucleoprotein 70-kDa protein, a U1 small nuclear ribonucleoprotein-specific protein, has been shown to have multiple roles in nuclear precursor mRNA processing in animals . By using the C-terminal arginine-rich region of Arabidopsis U1-70K protein in the yeast two-hybrid system, we have identified an SC35-like (SR33) and a novel plant serine/arginine-rich (SR) protein (SR45) that interact with the plant U1-70K . The SR33 and SR45 proteins share several features with SR proteins including modular domains typical of splicing factors in the SR family of proteins . However, both plant SR proteins are rich in proline, and SR45, unlike most animal SR proteins, has two distinct arginine/serine-rich domains separated by an RNA recognition motif . By using coprecipitation assays we confirmed the interaction of plant U1-70K with SR33 and SR45 proteins . Furthermore, in vivo and in vitro protein-protein interaction experiments have shown that SR33 protein interacts with itself and with SR45 protein but not with two other members (SRZ21 and SRZ22) of the SR family that are known to interact with the Arabidopsis full-length U-70K only . A Clk/Sty protein kinase (AFC-2) from Arabidopsis phosphorylated four SR proteins (SR33, SR45, SRZ21, and SRZ22) . Coprecipitation studies have confirmed the interaction of SR proteins with AFC2 kinase, and the interaction between AFC2 and SR33 is modulated by the phosphorylation status of these proteins . These and our previous results suggest that the plant U1-70K interacts with at least four distinct members of the SR family including SR45 with its two arginine/serine-rich domains, and the interaction between the SR proteins and AFC2 is modulated by phosphorylation . The interaction of plant U1-70K with a novel set of proteins suggests the early stages of spliceosome assembly, and intron recognition in plants is likely to be different from animals. Curr Genet, 1999 Oct, 36(4), 222 - 31 Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes; Poggeler S; To gain a deeper insight into the evolution of mating-type genes from filamentous ascomycetes, a comprehensive sequence analysis of PCR-amplified sequences corresponding to A- and a-specific mating-type sequences was undertaken . The study included nine homothallic (compatible) and eight heterothallic (incompatible) members of the genera Neurospora and Sordaria . Distance and parsimony trees based on gene fragments from the mat a-1 and mat A-1 genes were compared with trees derived from partial DNA sequences of the gpd glyceraldehyde-3-phosphate dehydrogenase gene . In contrast to the sequences from the gpd gene, mating-type genes show striking sequence differences, suggesting that these genes evolve very rapidly . Strong inter-relationships were found among homothallic, as well as among heterothallic, members of both genera, indicating that in each genus a change from one reproductive strategy to another might result from one single event. Nucleic Acids Res, 2000 Jan 1, 28(1), 148 - 52 MitBASE : a comprehensive and integrated mitochondrial DNA database . The present status; Attimonelli M et al.; MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae . MitBASE reports all available information from different organisms and from intraspecies variants and mutants . Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc . The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE . Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities . The database is resident at the EBI and is available at the following site: e.pl . The impact of this project is intended for both basic and applied research . The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields . The database has been funded within the EU Biotechnology programme. Nucleic Acids Res, 2000 Jan 1, 28(1), 120 - 2 XREFdb: cross-referencing the genetics and genes of mammals and model organisms; Ploger R et al.; XREFdb supports the investigation of protein function in the context of information available through work in multiple organisms . In addition to facilitating the association of functional data among known genes from multiple organisms, XREFdb has developed strategies that provide access to information associated with as-yet unstudied genes . The database organizes protein similarity and genetic map positional information from diverse sources in the public domain to facilitate investigator evaluation of potential functional significance . XREFdb is found at URL www.ncbi.nlm.nih.gov/XREFdb Nucleic Acids Res, 2000 Jan 1, 28(1), 37 - 40 MIPS: a database for genomes and protein sequences; Mewes HW et al.; The Munich Information Center for Protein Sequences (MIPS-GSF), Martinsried, near Munich, Germany, continues its longstanding tradition to develop and maintain high quality curated genome databases . In addition, efforts have been intensified to cover the wealth of complete genome sequences in a systematic, comprehensive form . Bioinformatics, supporting national as well as European sequencing and functional analysis projects, has resulted in several up-to-date genome-oriented databases . This report describes growing databases reflecting the progress of sequencing the Arabidopsis thaliana (MATDB) and Neurospora crassa genomes (MNCDB), the yeast genome database (MYGD) extended by functional analysis data, the database of annotated human EST-clusters (HIB) and the database of the complete cDNA sequences from the DHGP (German Human Genome Project) . It also contains information on the up-to-date database of complete genomes (PEDANT), the classification of protein sequences (ProtFam) and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database . These databases can be accessed through the MIPS WWW server . Nature, 1999 Dec 2, 402(6761), 555 - 60 The RCAF complex mediates chromatin assembly during DNA replication and repair; Tyler JK et al.; Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome . In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin . Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro . RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4 . The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones . Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo. Yakugaku Zasshi, 1999 Nov, 119(11), 822 - 34 {Analysis of xenobiotic detoxification system mediated by efflux transporters}; Suzuki H; The excretion of drugs mediated by transporters plays an important role in the detoxification of xenobiotics . In this article, I will summarize recent progress we have made in this field, particularly focusing on the roles of transporters responsible for exporting drugs . As far as the biliary excretion of xenobiotics is concerned, it has been suggested that canalicular multispecific organic anion transporter/multidrug resistance associated protein 2 (cMOAT/MRP2) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane . By comparing the transport across this membrane between normal rats and Eisai hyperbilirubinemic rats whose cMOAT/MRP2 function is hereditarily defective, we were able to demonstrate the substrate specificity of cMOAT/MRP2 . This includes non-conjugated anionic drugs, and glutathione- and glucuronide-conjugates of xenobiotics . The role of cMOAT/MRP2 in drug disposition has also been clarified . Moreover, the cDNA of cMOAT/MRP2 has been cloned and its functional analysis has been completed . Thus, it may be possible to predict in vivo transport across the bile canalicular membrane from in vitro data using the recombinant transporter . We also cloned MRP3 as an inducible transporter in the liver under the cholestatic conditions . Although MRP3 mediates the cellular export of non-conjugated organic anions and glucuronide-conjugates, the substrate specificity of MRP3 is different from that of cMOAT/MRP2 in that glutathione-conjugates are poor substrates for MRP3 . It is possible that MRP3 plays an important role under certain pathological conditions in the liver . Since it has been shown that cMOAT/MRP2 and MRP 3 are expressed in the small intestine under physiological conditions, it seems reasonable that these transporters are responsible for the previously reported cellular extrusion of organic anions . We also found that there was MRP activity in the blood-brain and blood-cerebrospinal fluid barriers . RT-PCR resulted in the amplification of MRP1, 5 and 6 from freshly isolated rat cerebral endothelial cells . It has been suggested that there is basolateral localization of MRP1 in the choroid plexus . In conjunction with the P-glycoprotein located on the luminal membrane of cerebral endothelial cells, these transporters play significant roles in restricting the entry of xenobiotics from the circulating blood into the central nervous system . Regulation of the activity of these efflux transporters allows the disposition of drugs to be altered. Adv Biochem Eng Biotechnol, 2000, 66, 209 - 31 Metabolic network analysis . A powerful tool in metabolic engineering; Christensen B et al.; Metabolic network analysis is a tool for investigating the features that identify the topology of a metabolic network and the relative activities of its individual branches . The pillars of metabolic network analysis are mathematical modeling, allowing for a quantitative analysis, biochemical knowledge of, for example, reaction stoichiometry, and the experimental techniques, providing input for the modeling part . The modeling part includes metabolite balancing, usually the basis for metabolic flux analysis, and isotope balancing . Isotope balancing can be used for both identification of active pathways and for estimation of the relative fluxes through two pathways leading to the same metabolite, aspects that are difficult to investigate using metabolite balancing . The combination of metabolite and isotope balancing is very powerful and constitutes the basis of metabolic network analysis . With the main focus being on investigating the metabolic network structure, this review describes how central metabolic features, for example, pathway identification, flux distribution, and compartmentation, can be addressed using a combination of metabolite balancing and labeling experiments. Mutat Res, 1999 Nov 29, 430(1), 131 - 44 Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background; Sengstag C et al.; Mutations in the TP53 tumor suppressor gene are the most common alteration in cancer, and human primary liver cancers related to previous dietary exposure to the mycotoxin aflatoxin B1 (AFB1) exhibit a specific hot spot mutation at TP53 codon 249 . We have asked whether the 249 hot spot is related to a particular susceptibility to AFB1 of this TP53 region or whether it is related to a phenotype of the 249S p53 mutant protein . This was addressed by constructing a metabolically competent variant of Saccharomyces cerevisiae strain yIG397 expressing human cytochrome P450 1A2 and P450-reductase and isolating AFB1-induced mutants that failed to express the genomic ADE2 reporter gene . Molecular analysis revealed that only 8/40 mutants had a mutation in the TP53 target gene, whereas 32/40 mutants were due to a recombination event eliminating the ADE2 reporter gene . None of 19 mutations identified in the eight mutant TP53 plasmids altered codon 249, thus this codon was no hot spot if the TP53 gene was in the heterologous background yeast . The genotoxic action of AFB1 was completely different from that of the alkylating agent ethyl-methane-sulfonate, where 28/30 induced mutations were linked to the TP53 target gene. Yeast, 1999 Dec, 15(16), 1761 - 8 New tools for protein linkage mapping and general two-hybrid screening; Durfee T et al.; The two-hybrid system has proved to be a facile method for detecting and analyzing protein-protein interactions . An expanded application of this system, protein linkage mapping, provides a means of identifying interactions on a global scale and should prove a powerful tool in analyzing whole genomes as their sequences become available . To overcome some of the inherent difficulties in such a large-scale approach, we have constructed a set of new strains and vectors that will allow for more efficient screening . The strains contain a GAL1-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ reporter . The strains are of opposite mating types, permitting libraries present in one strain to be easily screened against a second library in the companion strain . We also constructed a family of CEN-based vectors for expression of both Gal4 DNA-binding and activation domain fusions . These plasmids include a hemagglutinin epitope tag and different polylinkers to increase the ease of subcloning . CEN-based vectors are maintained at 1-2 copies per cell, limiting the number of individual cells containing multiple plasmids that can confuse further analyses, and ensuring that fusions are not expressed at toxic levels . Using these vectors, both homo- and heterodimeric interactions resulted in up to 10-fold higher reporter gene transcription than obtained with 2micro;-based plasmids, despite significantly lower protein levels . In addition to protein linkage mapping, these reagents should be generally useful in standard two-hybrid applications . Mol Biol Cell, 1999 Dec, 10(12), 4149 - 61 Promiscuity in Rab-SNARE interactions; Grote E et al.; Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins . We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles . Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain . However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells . In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles . In an alternate approach to look for selective Rab-SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop . However, Rab-SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp . We conclude that Rabs and SNAREs do not cooperate to specify the target membrane. Mol Biol Cell, 1999 Dec, 10(12), 4121 - 33 The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity; Adamo JE et al.; Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane . This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus . We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes . Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane . We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70 . These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface. Nat Cell Biol, 1999 Dec, 1(8), 514 - 21 Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans; Kitagawa R et al.; The spindle-assembly checkpoint ensures that, during mitosis and meiosis, chromosomes do not segregate until they are properly attached to the microtubules of the spindle . Here we show that mdf-1 and mdf-2 are components of the spindle-assembly checkpoint in Caenorhabditis elegans, and are essential for the long-term survival and fertility of this organism . Loss of function of either of these genes leads to the accumulation of a variety of defects, including chromosome abnormalities, X-chromosome non-disjunction or loss, problems in gonad development, and embryonic lethality . Antibodies that recognize the MDF-2 protein localize to nuclei of the cleaving embryo in a cell-cycle-dependent manner . mdf-1, a gene encoding a product that interacts with MDF-2, is required for cell-cycle arrest and proper chromosome segregation in premeiotic germ cells treated with nocodazole, a microtubule-depolymerizing agent . In the absence of mdf gene products, errors in chromosome segregation arise and accumulate, ultimately leading to genetic lethality. Biophys J, 1999 Dec, 77(6), 3293 - 304 Sampling field heterogeneity at the heme of c-type cytochromes by spectral hole burning spectroscopy and electrostatic calculations; Laberge M et al.; We report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes-namely yeast and horse heart cytochromes c-using a combination of hole burning Stark spectroscopy and electrostatic calculations . The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes . In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed . The yeast species, on the other hand, displays a single electronic origin . Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space . The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of "electrostatic substates" that they can sample, thus accounting for the different spectral populations observed in the two cytochromes. Biochem J, 1999 Dec 15, 344 Pt 3, 903 - 14 Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Delta3-Delta2-enoyl-CoA isomerase in beta-oxidation of unsaturated fatty acids; Gurvitz A et al.; Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3, 2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the beta-oxidation of unsaturated fatty acids . However, their function during this process has not been demonstrated . Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment . The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since beta-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3, 2-isomerase (Eci1p) . A yeast sps19Delta mutant expressing human 2, 4-reductase ending with the native C-terminus could not grow on petroselinic acid {cis-C(18:1(6))} medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu) . We therefore reason that the human protein is a physiological 2, 4-reductase but that it is probably not peroxisomal . Rat 3, 2-isomerase expressed in a yeast eci1Delta strain was able to re-establish growth on oleic acid {cis-C(18:1(9))} medium irrespective of an SKL extension . Since we had shown that Delta(2,4) double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3) unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes . Immunoblotting of fractionated yeast cells expressing rat 3, 2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes . The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase. Anal Biochem, 1999 Dec 1, 276(1), 18 - 26 Usefulness of statistic experimental designs in enzymology: example with recombinant hCYP3A4 and 1A2; Bournique B et al.; First, the effects of 10 incubation factors were screened altogether on nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD) activities catalyzed by recombinant human CYP3A4 and 1A2, respectively . Using a statistic experimental design, only 36 assays were needed to be exhaustive . Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH, temperature, Mg/EDTA, cytochrome b5, NADPH-P450 reductase, NADH, and solvent . Two factors had no significant effect: total ionic concentration and catalase/reduced glutathione . Six factors influenced CYP1A2-mediated MROD rates: buffer type, pH, temperature, Mg/EDTA, NADH, and glycerol . Four factors had no significant effect: total ionic concentration, cytochrome b5, reductase, and NAD+ . Secondly, the combined effects of ionic strength and Mg concentration on NIF/CYP3A4 were studied and easily modeled using another statistic experimental design . The effect of Mg was strong at a constant ionic strength of 100 mM and negligible at a constant ionic strength of 500 mM . Thirdly, the effects of influencing factors and their interactions on MROD/CYP1A2 were modeled after 40 assays using a third statistic experimental design . Later experiments confirmed the predictivity of the models and the efficiency of optimized conditions . This approach can be applied to other biochemistry areas . Mol Gen Genet, 1999 Oct, 262(3), 426 - 36 A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA; Senbongi H et al.; We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient rho- or rho0 mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methane-sulfonate or ultraviolet light . The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter . When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia . Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron . We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells . Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair . These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. J Lipid Res, 1999 Dec, 40(12), 2244 - 54 Phytanoyl-CoA hydroxylase from rat liver . Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation; Jansen GA et al.; Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway . Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes . In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial . In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein . We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE . Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38 . 6 kDa protein . The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2) . Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa . This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2 . This type of processing has been reported in several other peroxisomal proteins that contain a PTS2 . Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat . The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human. J Lipid Res, 1999 Dec, 40(12), 2204 - 11 Family of human oxysterol binding protein (OSBP) homologues . A novel member implicated in brain sterol metabolism; Laitinen S et al.; Oxysterol binding protein (OSBP) is a cytosolic protein that undergoes ligand-induced binding to the Golgi apparatus and has been implicated in the regulation of cellular cholesterol metabolism . In the yeast Saccharomyces cerevisiae an OSBP homologue is involved in membrane trafficking through the Golgi complex . Prompted by the multitude of OSBP-related genes in the yeast genome, we carried out a search for human expressed sequence tags (ESTs) displaying homology to the sterol-binding domain of OSBP . This revealed a minimum of six novel OSBP-related proteins, designated ORP-1 to ORP-6 . ORP cDNA probes were generated by reverse transcription-PCR from human liver mRNA, and used for Northern blot analysis of human tissue transcript panels . This verified that each of them represents a different gene product and showed that they display distinct tissue-specific expression patterns . The ORP-1 and -2 mRNA expression levels were similar to or higher than that of OSBP while the ORP-3 to -6 mRNAs were detected at lower levels in specific tissues . The most abundantly expressed new gene, ORP-1, was transcribed at strikingly high levels in the cortical areas of human brain and displayed sterol-regulated expression in a cultured human neuroblastoma cell line . This indicates that ORP-1 may play an important role in maintaining the sterol balance in cells of the central nervous system . Together with OSBP, the identified gene products constitute a novel human protein family that may provide a link between organellar sterol status and membrane dynamics. J Mol Biol, 1999 Dec 10, 294(4), 1041 - 9 Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53; Liao H et al.; The forkhead-associated (FHA) domain is a 55-75 amino acid residue module found in >20 proteins from yeast to human . It has been suggested to participate in signal transduction pathways, perhaps via protein-protein interactions involving recognition of phosphopeptides . Neither the structure nor the ligand of FHA is known . Yeast Rad53, a checkpoint protein involved in DNA damage response, contains two FHA domains, FHA1 (residues 66-116) and FHA2 (residues 601-664), the second of which recognizes phosphorylated Rad9 . We herein report the solution structure of an "FHA2-containing domain" of Rad53 (residues 573-730) . The structure consists of a beta-sandwich containing two antiparallel beta-sheets and a short, C-terminal alpha-helix . Binding experiments suggested that the FHA2-containing domain specifically recognizes pTyr and a pTyr-containing peptide from Rad9, and that the binding site involves residues highly conserved across FHA domains . The results, along with other recent reports, suggest that FHA domains could have pTyr and pSer/Thr dual specificity . Dev Biol, 1999 Dec 1, 216(1), 210 - 29 Wingless modulates the effects of dominant negative notch molecules in the developing wing of Drosophila; Brennan K et al.; The development and patterning of the wing in Drosophila relies on a sequence of cell interactions molecularly driven by a number of ligands and receptors . Genetic analysis indicates that a receptor encoded by the Notch gene and a signal encoded by the wingless gene play a number of interdependent roles in this process and display very strong functional interactions . At certain times and places, during wing development, the expression of wingless requires Notch activity and that of its ligands Delta and Serrate . This has led to the proposal that all the interactions between Notch and wingless can be understood in terms of this regulatory relationship . Here we have tested this proposal by analysing interactions between Delta- and Serrate-activated Notch signalling and Wingless signalling during wing development and patterning . We find that the cell death caused by expressing dominant negative Notch molecules during wing development cannot be rescued by coexpressing Nintra . This suggests that the dominant negative Notch molecules cannot only disrupt Delta and Serrate signalling but can also disrupt signalling through another pathway . One possibility is the Wingless signalling pathway as the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signalling . Furthermore, we observe that the outcome of the interactions between Notch and Wingless signalling differs when we activate Wingless signalling by expressing either Wingless itself or an activated form of the Armadillo . For example, the effect of expressing the activated form of Armadillo with a dominant negative Notch on the patterning of sense organ precursors in the wing resembles the effects of expressing Wingless alone . This result suggests that signalling activated by Wingless leads to two effects, a reduction of Notch signalling and an activation of Armadillo . Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14647 - 51 Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain; Nikolskaya T et al.; A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3'-tail, was used to complement a yeast ACC1 mutation . These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins . Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5'-end, complement the yeast mutation . Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 microM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases . This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo . The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase . Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors . The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors. FEMS Microbiol Lett, 1999 Dec 15, 181(2), 225 - 8 Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus; Kim BG et al.; Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA) . Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei . Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts . Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA . Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy . These results indicate the integrated DNA was stably inherited. J Biol Chem, 1999 Dec 10, 274(50), 36009 - 14 The assembly of the CAAT-box binding complex at a photosynthesis gene promoter is regulated by light, cytokinin, and the stage of the plastids; Kusnetsov V et al.; A functionally important region in the promoter of the spinach photosynthesis gene AtpC, which encodes the subunit gamma of the chloroplast ATP synthase, is located immediately upstream of the CAAT-box . A single nucleotide exchange in this region (AAAATTCAAT --> AAGATCAAT) uncouples the expression of an AtpC promoter::uidA gene fusion from the regulation by light, cytokinin, and functional plastids and results in a high constitutive expression of the reporter gene . By screening an Arabidopsis thaliana expression library with a double-stranded wild-type oligonucleotide from this promoter region, we have isolated cDNAs from Arabidopsis libraries that code for plant homologs of the CAAT-box binding factor (CBF)-C . Binding occurs only in the presence of nuclear extracts, consistent with reports from metazoa CBFs that the subunits A and B in addition to C are required for the formation of the CBF-DNA complex . At least eight genes with homologies to CBF-C are present in the Arabidopsis genome; one of them exhibits striking similarities to the gene for the human global transcriptional repressor Drap1 . In gel mobility shift assays, low binding activity of CBF to the wild-type AtpC promoter sequence was observed with nuclear extracts from tissue with low AtpC expression levels, i.e . extracts from etiolated and photobleached seedlings, whereas high binding activity was detectable with extracts from tissues with high AtpC expression levels, i.e . extracts from light-grown seedlings and etiolated seedlings treated with cytokinin . Binding to the mutant sequence, which directs constitutive high level uidA expression in vivo, is significantly stronger than to the wild-type sequence . The data are consistent with the idea that the assembly of CBF at the AtpC promoter is regulated in response to light and cytokinin and that the low level of expression in etiolated and photobleached material is caused by an inhibitory effect . The structure/function relationships of the Arabidopsis CBFs are discussed in relation to their regulatory function in AtpC gene expression. J Biol Chem, 1999 Dec 10, 274(50), 35999 - 6008 Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription; Ogino T et al.; In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors) . Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin . In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain . This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46) . From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK) . Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46 . Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation . On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated . The enzymatic activity per se of PGK did not seem to be required for its activity . West-Western blot analysis showed that PGK could directly interact with tubulin . These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex. J Biol Chem, 1999 Dec 10, 274(50), 35991 - 8 Translational regulation of ribonucleotide reductase by eukaryotic initiation factor 4E links protein synthesis to the control of DNA replication; Abid MR et al.; Ribonucleotide reductase synthesizes dNDPs, a specific and limiting step in DNA synthesis, and can participate in neoplastic transformation when overexpressed . The small subunit (ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a subtraction library from eukaryotic initiation factor 4E (eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)) . CHO-4E cells have 20-40-fold elevated RNR2 protein, reflecting an increased distribution of RNR2 mRNA to the heavy polysomes . CHO-4E cells display an altered cell cycle with shortened S phase, similar to cells selected for RNR2 overexpression with hydroxyurea . The function of ribonucleotide reductase as a checkpoint component of S progression was studied in yeast in which elevated eIF4E rescued S-arrested rnr2-68(ts) cells, by increasing recruitment of its mRNA to polysomes . Crosses between rnr2-68(ts) and mutant eIF4E (cdc33-1(ts)) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl . The double mutant (cdc33-1 rnr2-68) also identified a unique terminal phenotype, arrested with small bud and a randomly distributed single nucleus, which is distinct from those of both parental single mutants . This phenotype defines eIF4E and RNR2 as determinants in an important cell cycle checkpoint, in early/mid-S phase . These results also provide a link between protein and DNA synthesis and provide an explanation for cell cycle alterations induced by elevated eIF4E. J Biol Chem, 1999 Dec 10, 274(50), 35734 - 40 How an inhibitor of the HIV-I protease modulates proteasome activity; Schmidtke G et al.; The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years . Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease . In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM) . In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced . In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome . Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir . At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase . Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded . Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response. J Biol Chem, 1999 Dec 10, 274(50), 35583 - 90 Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening; Ayers NA et al.; The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions . By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region . Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237 . The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059 . Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971 . Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate . This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase . Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed. J Biol Chem, 1999 Dec 10, 274(50), 35434 - 40 Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates; Yang X et al.; Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)) . Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP . Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site . Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding . GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP . Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95% . More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84) . This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases. J Biol Chem, 1999 Dec 10, 274(50), 35393 - 9 Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A; Snoek GT et al.; In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein alpha (PI-TPalpha), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPalpha . Two stable cell lines, i.e . SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPalpha . Overexpression of PI-TPalpha resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13-14 h for SPI6 and SPI8 cells . Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G(1) phase is mainly responsible for this decrease . The saturation density of the cells increased from 0.20 x 10(5) cells/cm(2) for wtNIH3T3 cells to 0.53 x 10(5) cells/cm(2) for SPI6 and SPI8 cells . However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar . Upon equilibrium labeling of the cells with myo-{(3)H} inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells . A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4, 5-bisphosphate were the same as those in control cells . The addition of PI-TPalpha to a total lysate of myo-{(3)H}inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns . The addition of Ca(2+) further increased this formation . Based on these observations, we propose that PI-TPalpha is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns . The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells . The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate. J Biol Chem, 1999 Dec 10, 274(50), 35337 - 42 Binding of nucleotides to guanylate kinase, p21(ras), and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry; Prinz H et al.; The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions . The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution . For guanylate kinase (EC 2.7.4 . 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed . Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP . Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP . Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism . All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate . The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-). Exp Cell Res, 1999 Dec 15, 253(2), 637 - 48 Transient overexpression of murine dishevelled genes results in apoptotic cell death; Strovel ET et al.; The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are implicated in Wnt signal transduction . In mammals, the manner in which Wnt signals are transduced remains unclear . The biochemical and molecular mechanisms defining the Wnt-1 pathway are of great interest because of its important role in development and its activation in murine breast tumors . In order to elucidate Dvl's role in Wnt signaling, we attempted to overexpress Dvl in cells, but were unable to obtain stable cell lines . We show here that the overexpression of Dvl genes alters nuclear and cellular morphology of COS-1 and C57MG cells and causes cell death due to the induction of apoptosis . Deletion studies demonstrate that all three conserved domains of Dvl (DIX, PDZ, and DEP) are required for Dvl-mediated cell death . Coexpression of protein phosphatase 2Calpha, a Dvl-interacting protein identified in yeast two-hybrid studies, protects cells from the cell death observed in cells overexpressing Dvl alone . Furthermore, the adenomatous polyposis coli (APC) gene product appears to be required for Dvl-mediated cell death . The relevance of these findings to Wnt signal transduction, as well as to developmental processes and disease, are discussed . Philos Trans R Soc Lond B Biol Sci, 1999 Sep 29, 354(1389), 1533 - 50 SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis; Willems AR et al.; Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes . SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome . SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53 . Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30 . The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2 . Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes . As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis. Nat Struct Biol, 1999 Dec, 6(12), 1081 - 3 If the loop fits..; Frankel AD; The structure of the yeast L30 ribosomal protein bound to its autoregulatory RNA site has been determined by NMR spectroscopy . The intricate architecture of the RNA internal loop and the structure of the binding region of the protein both are stabilized in the complex, highlighting the importance of mutually-induced fit in RNA-protein interactions. Appl Environ Microbiol, 1999 Dec, 65(12), 5546 - 53 The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases; Iwashita K et al.; We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1 . The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein . Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1 . The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B . We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast . A . kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase . A . kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis . Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A . kawachii. Appl Environ Microbiol, 1999 Dec, 65(12), 5252 - 6 Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides; McCormick SP et al.; We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene . Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F . sporotrichioides homolog of the Fusarium graminearum TRI101 gene . Mutants of F . sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3, 15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain . Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin. J Neurochem, 1999 Dec, 73(6), 2240 - 9 Identification of a mammalian homologue of the fungal Tom70 mitochondrial precursor protein import receptor as a thyroid hormone-regulated gene in specific brain regions; Alvarez-Dolado M et al.; Thyroid hormone is an important regulator of mammalian brain maturation . By differential display PCR, we isolated a cDNA clone (S2) that is specifically up-regulated in the striatum of neonatal hypothyroid rats . S2 was identified as KIAA0719, the first human gene distantly homologous to the fungal Tom70, which encodes a member of the translocase mitochondrial outer membrane complex involved in the import of preproteins into the mitochondria . By northern and in situ hybridization studies, KIAA0719 was found to be up-regulated in the striatum, nucleus accumbens, and discrete cortical layers of 15-day-old hypothyroid rats . In contrast, lower expression was found in the olfactory tubercle, whereas no differences were detected in other brain regions . Significantly, treatment of hypothyroid animals with single injections of thyroxine restored the normal levels of KIAA0719 expression . Moreover, treatment of control animals with thyroxine led to a reduced expression, demonstrating a negative hormonal regulation in vivo . Thus, KIAA0719 gene expression is regulated by thyroid hormone in the neonatal rat brain in a region-specific fashion . Given the role of the homologous Tom70 gene, the alteration of KIAA0719 expression may contribute to the changes in mitochondrial morphology and physiology caused by hypothyroidism in the developing rat brain. EMBO J, 1999 Dec 1, 18(23), 6832 - 44 The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain; Xie Y et al.; We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae . The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p . However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates . In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain . Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p . These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system. EMBO J, 1999 Dec 1, 18(23), 6816 - 22 Carbohydrate deficient glycoprotein syndrome type IV: deficiency of dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase; Korner C et al.; Type IV of the carbohydrate deficient glycoprotein syndromes (CDGS) is characterized by microcephaly, severe epilepsy, minimal psychomotor development and partial deficiency of sialic acids in serum glycoproteins . Here we show that the molecular defect in the index patient is a missense mutation in the gene encoding the mannosyltransferase that transfers mannose from dolichyl-phosphate mannose on to the lipid-linked oligosaccharide (LLO) intermediate Man(5)GlcNAc(2)-PP-dolichol . The defect results in the accumulation of the LLO intermediate and, due to its leaky nature, a residual formation of full-length LLOs . N-glycosylation is abnormal because of the transfer of truncated oligosaccharides in addition to that of full-length oligosaccharides and because of the incomplete utilization of N-glycosylation sites . The mannosyltransferase is the structural and functional orthologue of the Saccharomyces cerevisiae ALG3 gene. EMBO J, 1999 Dec 1, 18(23), 6744 - 51 Hsp26: a temperature-regulated chaperone; Haslbeck M et al.; Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far . Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro . Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone . Like other sHsps, Hsp26 forms large oligomeric complexes . At heat shock temperatures, however, the 24mer chaperone complex dissociates . Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity . Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers . In this complex one monomer of substrate is bound per Hsp26 dimer . The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone. EMBO J, 1999 Dec 1, 18(23), 6672 - 81 Gal83 mediates the interaction of the Snf1 kinase complex with the transcription activator Sip4; Vincent O et al.; The Snf1/AMPK protein kinase family is widely conserved in eukaryotes . In Saccharomyces cerevisiae, the Snf1 kinase is an essential element of the glucose response pathway and has diverse regulatory roles . The Snf1 complex contains one of the related proteins Sip1, Sip2 and Gal83, which are also conserved in higher eukaryotes . Previous studies showed that the Sip1/Sip2/Gal83 component plays a structural role in the complex . We present evidence that this component also mediates the interaction of the Snf1 kinase complex with specific targets . We show that Gal83 mediates the association of the kinase with Sip4, a Snf1-regulated transcription activator of gluconeogenic genes . Gal83 interacts with Sip4 in two-hybrid assays in vivo, and bacterially expressed proteins bind in vitro . Moreover, Gal83 is required for the two-hybrid interaction of Sip4 with the Snf1 kinase . Gal83 also facilitates the rapid Snf1-dependent phosphorylation and activation of Sip4 in response to glucose limitation, indicating that Gal83 mediates the functional interaction of Snf1 with Sip4 . Evidence indicates that Sip1 and Sip2 do not interact with Sip4 . We propose that members of the Sip1/Sip2/Gal83 family confer specificity to the kinase complex in its interactions with target proteins. EMBO J, 1999 Dec 1, 18(23), 6662 - 71 A new regulatory domain on the TATA-binding protein; Cang Y et al.; Recognition of the TATA box by the TATA-binding protein (TBP) is a highly regulated step in RNA polymerase II-dependent transcription . Several proteins have been proposed to regulate TBP activity, yet the TBP domains responsive to all these regulators have not been defined . Here we describe a new class of TBP mutants that increase transcription from core promoters in vivo . The majority of these mutations alter amino acids that cluster tightly on the TBP surface, defining a new TBP regulatory domain . The mutant TBP proteins are defective for binding the transcriptional regulator yNC2, are resistant to inhibition by yNC2 in vitro and exhibit allele-specific genetic interactions with yNC2 . These results provide strong biochemical and genetic evidence that TBP is directly repressed in vivo, and define a new TBP domain important for transcriptional regulation. EMBO J, 1999 Dec 1, 18(23), 6619 - 29 Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells; Yamaguchi-Iwai Y et al.; Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand break (DSB) repair pathways . Vertebrate Mre11 is essential . Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification . Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies . Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair . Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11-independent component of metazoan NHEJ. Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 147 - 51 MIDA1, an Id-associating protein, has two distinct DNA binding activities that are converted by the association with Id1: a novel function of Id protein; Inoue T et al.; Id proteins not only regulate cell differentiation negatively, but they also promote growth, immortalization, and apoptosis . To know the mechanism of how Id regulates cell fate, we previously isolated an Id-associating protein, MIDA1, which positively regulates cell growth (1) . Its predicted amino acid sequence consists of a Zuotin (a Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats (Tryp-med repeats) . MIDA1 exhibits a sequence-specific DNA binding activity through the Tryp-med repeats (manuscript in preparation) . In this study, we revealed that, like Zuotin, MIDA1 can specifically bind to Z-DNA . This suggested that MIDA is a novel DNA binding protein that has two different DNA binding activities . Furthermore, association of Id1 with MIDA1 stimulated the sequence-specific DNA binding activity, while it inhibited the Z-DNA binding activity . Therefore, we concluded that MIDA1 may act as a mediator of the growth-promoting function of Id, by switching the two DNA binding activities of MIDA1 . Nature, 1999 Nov 18, 402(6759), 320 - 2 Sustained oscillations in living cells; Dano S et al.; Glycolytic oscillations in yeast have been studied for many years simply by adding a glucose pulse to a suspension of cells and measuring the resulting transient oscillations of NADH . Here we show, using a suspension of yeast cells, that living cells can be kept in a well defined oscillating state indefinitely when starved cells, glucose and cyanide are pumped into a cuvette with outflow of surplus liquid . Our results show that the transitions between stationary and oscillatory behaviour are uniquely described mathematically by the Hopf bifurcation . This result characterizes the dynamical properties close to the transition point . Our perturbation experiments show that the cells remain strongly coupled very close to the transition . Therefore, the transition takes place in each of the cells and is not a desynchronization phenomenon . With these two observations, a study of the kinetic details of glycolysis, as it actually takes place in a living cell, is possible using experiments designed in the framework of nonlinear dynamics . Acetaldehyde is known to synchronize the oscillations . Our results show that glucose is another messenger substance, as long as the glucose transporter is not saturated. J Hepatol, 1999 Nov, 31(5), 783 - 90 Involvement of phosphatidylserine and non-phospholipid components of the hepatitis B virus envelope in human Annexin V binding and in HBV infection in vitro; De Meyer S et al.; BACKGROUND/AIMS: We have previously demonstrated that human liver Annexin V (hAV), a Ca2+-dependent phospholipid binding protein, binds specifically to small HBsAg (SHBsAg) . Because of the propensity of AV to bind phospholipids, we here examine the role of phospholipids, as component of the HBV envelope, in binding to hAV and in HBV infection . METHODS: The influence of phospholipids (phosphatidylserine and phosphatidylcholine) on the binding of hAV to SHBsAg or to anti-hAV monoclonals was determined by ELISA . Their influence on HBV infection was investigated using an in vitro HBV infection assay . RESULTS: Two monoclonals, specific against hAV, were able to block the binding of hAV to SHBsAg and recognized different epitopes of hAV . The binding of one of these monoclonals to hAV could be inhibited by phosphatidylserine, but not by phosphatidylcholine . Further experiments revealed that phosphatidylserine could also inhibit the binding of hAV to SHBsAg and could even prevent HBV infection in vitro . Phosphatidylcholine had no effect on the binding of hAV to SHBsAg and could not prevent HBV infection in vitro . However, since phosphatidylserine was not able to abolish the binding of the other blocking monoclonal to hAV, a non-phospholipid component of the HBV envelope must also be involved in hAV binding . CONCLUSIONS: These results indicate that phosphatidylserine and a non-phospholipid component of the HBV envelope are involved in hAV binding and in HBV infection. Genes Dev, 1999 Nov 15, 13(22), 3027 - 33 Regulation of CDK4 activity by a novel CDK4-binding protein, p34(SEI-1); Sugimoto M et al.; The p16(INK4a) tumor suppressor inhibits cyclin-dependent kinases (CDK4 and CDK6) . Here we report the isolation of a novel gene, SEI-1, whose product (p34(SEI-1)) appears to antagonize the function of p16(INK4a) . Addition of p34(SEI-1) to cyclin D1-CDK4 renders the complex resistant to inhibition by p16(INK4a) . Expression of SEI-1 is rapidly induced on addition of serum to quiescent fibroblasts, and ectopic expression of p34(SEI-1) enables fibroblasts to proliferate even in low serum concentrations . p34(SEI-1) seems to act as a growth factor sensor and may facilitate the formation and activation of cyclin D-CDK complexes in the face of inhibitory levels of INK4 proteins. Genes Dev, 1999 Nov 15, 13(22), 2946 - 57 Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases; Cheng A et al.; Activating phosphorylation of cyclin-dependent protein kinases (CDKs) is necessary for their kinase activity and cell cycle progression . This phosphorylation is carried out by the Cdk-activating kinase (CAK); in contrast, little is known about the corresponding protein phosphatase . We show that type 2C protein phosphatases (PP2Cs) are responsible for this dephosphorylation of Cdc28p, the major budding yeast CDK . Two yeast PP2Cs, Ptc2p and Ptc3p, display Cdc28p phosphatase activity in vitro and in vivo, and account for approximately 90% of Cdc28p phosphatase activity in yeast extracts . Overexpression of PTC2 or PTC3 results in synthetic lethality in strains temperature-sensitive for yeast CAK1, and disrupti