S. cerevisiae

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7773 - 8 Epub 2003 Jun 13.
Inducible nitric oxide synthase expression inhibition by adenovirus E1A; Cao W et al.; Nitric oxide (NO) is an antiviral effector of the innate immune system . Viruses that can interfere with NO synthesis may be able to replicate more rapidly than viruses that cannot limit NO synthesis . We show that the adenovirus E1A protein inhibits NO production by decreasing expression of the inducible NO synthase (NOS2) . The amino-terminal portion of E1A decreases transactivation of the NOS2 5'-flanking region, limiting the DNA binding activity of NF-kappaB and inhibiting NOS2 expression . E1A is thus able to deactivate a critical component of the host defense against viral infection . Viral inhibition of NO production is a mechanism that may enable certain viruses to evade the host innate immune system.

Mol Cell Biol, 2003 Jul, 23(13), 4449 - 60
Sequential protein association with nascent 60S ribosomal particles; Saveanu C et al.; Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles . These intermediate particles follow a maturation pathway in which important changes in their protein composition occur . The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood . We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis . The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2 . The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins . Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

Mol Biol Cell, 2003 Jun, 14(6), 2559 - 69 Epub 2003 Feb 21.
Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins; Kimata Y et al.; In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2 . It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism . In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation . To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene . When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress . Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway . Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate . We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2.

Mol Biol Cell, 2003 Jun, 14(6), 2543 - 58 Epub 2003 Feb 21.
Nuclear export and plasma membrane recruitment of the Ste5 scaffold are coordinated with oligomerization and association with signal transduction components; Wang Y et al.; The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gbetagamma dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5 . Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown . Herein, we show that oligomerization affects Ste5 activity and localization . The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated . Increasing the pool of Ste5 oligomers increases association with Ste11 . Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade . Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization . Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation . Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site.

Mol Biol Cell, 2003 Jun, 14(6), 2357 - 71 Epub 2003 Mar 07.
A novel Golgi membrane protein is a partner of the ARF exchange factors Gea1p and Gea2p; Chantalat S et al.; The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking . The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors . However, these interactions remain largely unknown . Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs . Gmh1p is an evolutionarily conserved protein . We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p . This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins . A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain . We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER . Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function . These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.

Mol Biol Cell, 2003 Jun, 14(6), 2342 - 56 Epub 2003 Feb 06.
Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane; Sesaki H et al.; In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p . We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion . Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells . Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division . In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents . Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion . Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes . Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures . However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria . Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane . Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.

Mol Biol Cell, 2003 Jun, 14(6), 2292 - 302 Epub 2003 Apr 04.
MTG1 codes for a conserved protein required for mitochondrial translation; Barrientos A et al.; The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence . A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria . Mtg1p is a member of a family of GTPases with largely unknown functions . The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis . Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants . This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action . The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs . More likely, Mtg1p functions in assembly of the large ribosomal subunit . This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.

Mol Biol Cell, 2003 Jun, 14(6), 2206 - 15 Epub 2003 Mar 07.
Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation; Christensen TW et al.; Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae . We have cloned MCM10 from Drosophila melanogaster and show that it complements a ScMCM10 null mutant . Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2 . Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1 . RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content . Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation . The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants . Our results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked.

J Biol Chem, 2003 Sep 5, 278(36), 34373 - 9 Epub 2003 Jun 14.
The structure of Aip1p, a WD repeat protein that regulates Cofilin-mediated actin depolymerization; Voegtli WC et al.; Actin-interacting protein 1 (Aip1p) is a 67-kDa WD repeat protein known to regulate the depolymerization of actin filaments by cofilin and is conserved in organisms ranging from yeast to mammals . The crystal structure of Aip1p from Saccharomyces cerevisiae was determined to a 2.3-A resolution and a final crystallographic R-factor of 0.204 . The structure reveals that the overall fold is formed by two connected seven-bladed beta-propellers and has important implications for the structure of Aip1 from other organisms and WD repeat-containing proteins in general . These results were unexpected because a maximum of 10 WD repeats had been reported in the literature for this protein using sequence data . The surfaces of the beta-propellers formed by the D-A and B-C loops are positioned adjacent to one another, giving Aip1p a shape that resembles an open "clamshell." The mapping of conserved residues to the structure of Aip1p reveals dense patches of conserved residues on the surface of one beta-propeller and at the interface of the two beta-propellers . These two patches of conserved residues suggest a potential binding site for F-actin on Aip1p and that the orientation of the beta-propellers with respect to one another plays a role in binding an actin-cofilin complex . In addition, the conserved interface between the domains is mediated by a number of interactions that appear to impart rigidity between the two domains of Aip1p and may make a large substrate-induced conformational change difficult.

J Biol Chem, 2003 Sep 5, 278(36), 34347 - 55 Epub 2003 Jun 14.
Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated; Vartiainen MK et al.; Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals . In addition to the previously characterized mammalian twinfilin-1, a second protein with approximately 65% sequence identity to twinfilin-1 exists in mouse and humans . However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M . (1999) Eur . J . Biochem . 263, 518-525) . Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein . Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 microM) than ATP-G-actin (KD = 1.96 microM) and efficiently inhibits actin filament assembly in vitro . Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein . Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate . Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns . Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles . Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms . The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2 . Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways.

J Biol Chem, 2003 Aug 29, 278(35), 33436 - 44 Epub 2003 Jun 13.
ATP uptake in the Golgi and extracellular release require Mcd4 protein and the vacuolar H+-ATPase; Zhong X et al.; Extracellular nucleotides signal via a large group of purinergic receptors . Although much is known about these receptors, the mechanism of nucleotide transport out of the cytoplasm is unknown . We developed a functional screen for ATP release to the extracellular space and identified Mcd4p, a 919-amino acid membrane protein with 14 putative transmembrane domains, as a participant in glucose-dependent ATP release from Saccharomyces cerevisiae . This release occurred through the vesicular trafficking pathway initiated by ATP uptake into the Golgi compartment . Both the compartmental uptake and the extracellular release of ATP were regulated by the activity of the vacuolar H+-ATPase . It is likely that the Mcd4p pathway is generally involved in non-mitochondrial ATP movement across membranes, it is essential for Golgi and endoplasmic reticulum function, and its occurrence led to the appearance of P2 purinergic receptors.

Carcinogenesis, 2003 Jul, 24(7), 1247 - 55 Epub 2003 Jun 05.
Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo{a}pyrene-7R-trans-7,8-dihydrodiol; Mammen JS et al.; Genetic differences that underlie inter-individual variation in the metabolism of common carcinogens are important potential sources of cancer susceptibility . Cytochrome P450 1B1 (CYP1B1), a central enzyme in the activation of the ubiquitous environmental carcinogen benzo{a}pyrene (B{a}P), has several genetic variants . This study investigated six rare mutations and four common polymorphisms for their effects on B{a}P metabolism . Five missense mutations associated with congenital glaucoma (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu and Arg469Tryp) dramatically decreased the capacity of CYP1B1 to convert (-)benzo{a}pyrene-7R-trans-7,8-dihyrodiol (B{a}P-7,8-diol) to (+/-)benzo{a}pyrene-r-7,t-8-dihydrodiol-9,10-epoxides . These five mutations resulted in enzymes with 3-12% of normal activity when assayed in vitro using an Saccharomyces cerevisiae microsomal expression system . A 10 bp deletion mutation produced no detectable protein or activity . In contrast, proteins containing all possible combinations of four common single nucleotide polymorphisms (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on B{a}P-7,8-diol metabolism . Michaelis-Menten analysis suggested that two alleles, Arg48, Ala119, Val432, Ser453 (RAVS) and Arg48, Ala119, Leu432, Ser453 (RALS), have KM values 2-fold lower than Arg48, Ala119, Val432, Ser453 (RAVN): 1.4+/-0.3 and 1.3+/-0.4 microM, respectively, compared with 2.8+/-0.8 microM (P<0.05) . However, these differences could not be confirmed with direct measurements of rate at low substrate concentration . There were no significant differences for either of two other kinetic parameters, kcat or kcat/KM . Allele frequency analysis in three populations reveals the Ser453 variant is rare among those of Asian (<1%) and African ancestry (<4%), and more common in individuals of European ancestry (16%) . Haplotypes containing the Ser453 variant were uncommon; only RALS was detectable in our small populations . The RALS allele occurred between 0.5% in Asians and 15% in Europeans . Our study demonstrates that rare, disease-associated mutations in CYP1B1 significantly decrease the enzyme's metabolism of B{a}P-7,8-diol; however, our results do not identify any major differences in this metabolism due to four common single amino acid polymorphisms.

Toxicol Sci, 2003 Sep, 75(1), 40 - 6 Epub 2003 Jun 12.
Antiandrogenic effects of bisphenol A and nonylphenol on the function of androgen receptor; Lee HJ et al.; By using a yeast detection system for androgenic and antiandrogenic effects of chemicals, we identified bisphenol A (BPA) and nonylphenol (NP) as antiandrogens . In this study, we report molecular mechanisms for the antiandrogenic action of BPA and NP . In the ARhLBD-activating signal cointegrator 1 (ASC1) yeast two-hybrid system, which reflects the androgen-dependent interaction between androgen receptor (AR) and its coactivator, ASC1, BPA and NP acted as potent AR antagonists comparable to a known strong antagonist, cyproterone acetate . Ligand competition assays revealed that {3H}5alpha-dihydroxytestosterone (DHT) binding to AR is inhibited a maximum of 30 and 40% at approximately 5 nM of NP and 50 nM of BPA, respectively . In addition, the nuclear translocation of green fluorescent protein (GFP)-AR fusion protein in the presence of testosterone was affected by the addition of BPA and NP, which cause rather dispersed distribution of GFP-AR between the nuclear and the cytoplasmic compartments . Furthermore, in transient transfection assays, BPA and NP inhibited androgen-induced AR transcriptional activity . Taken together, the results suggest that BPA and NP affect multiple steps of the activation and function of AR, thereby inhibiting the binding of native androgens to AR, AR nuclear localization, AR interaction with its coregulator, and its subsequent transactivation . These data may help us better understand the biological alterations induced by these environmental compounds.

Plant Physiol, 2003 Jun, 132(2), 757 - 67 Epub 2003 Apr 17.
Arabidopsis hot mutants define multiple functions required for acclimation to high temperatures; Hong SW et al.; Plants acquire thermotolerance to lethal high temperatures if first exposed to moderately high temperature or if temperature is increased gradually to an otherwise lethal temperature . We have taken a genetic approach to dissecting acquired thermotolerance by characterizing loss-of-function thermotolerance mutants in Arabidopsis . In previous work, we identified single recessive alleles of four loci required for thermotolerance of hypocotyl elongation, hot1-1, hot2-1, hot3-1, and hot4-1 . Completed screening of M2 progeny from approximately 2500 M1 plants has now identified new alleles of three of these original loci, along with three new loci . The low mutant frequency suggests that a relatively small number of genes make a major contribution to this phenotype or that other thermotolerance genes encode essential or redundant functions . Further analysis of the original four loci was performed to define the nature of their thermotolerance defects . Although the HOT1 locus was shown previously to encode a major heat shock protein (Hsp), Hsp101, chromosomal map positions indicate that HOT2, 3, and 4 do not correspond to major Hsp or heat shock transcription factor genes . Measurement of thermotolerance at different growth stages reveals that the mutants have growth stage-specific heat sensitivity . Analysis of Hsp accumulation shows that hot2 and hot4 produce normal levels of Hsps, whereas hot3 shows reduced accumulation . Thermotolerance of luciferase activity and of ion leakage also varies in the mutants . These data provide the first direct genetic evidence, to our knowledge, that distinct functions, independent of Hsp synthesis, are required for thermotolerance, including protection of membrane integrity and recovery of protein activity/synthesis.

Plant Physiol, 2003 Jun, 132(2), 544 - 55
The SAC domain-containing protein gene family in Arabidopsis; Zhong R et al.; The SAC domain was first identified in the yeast (Saccharomyces cerevisiae) Sac1p phosphoinositide phosphatase protein and subsequently found in a number of proteins from yeast and animals . The SAC domain is approximately 400 amino acids in length and is characterized by seven conserved motifs . The SAC domains of several proteins have been recently demonstrated to possess phosphoinositide phosphatase activities . Sac1p has been shown to regulate the levels of various phosphoinositides in the phosphoinositide pool and affect diverse cellular functions such as actin cytoskeleton organization, Golgi function, and maintenance of vacuole morphology . The Arabidopsis genome contains a total of nine genes encoding SAC domain-containing proteins (AtSACs) . The SAC domains of the AtSACs possess the conserved amino acid motifs that are believed to be important for the phosphoinositide phosphatase activities of yeast and animal SAC domain proteins . AtSACs can be divided into three subgroups based on their sequence similarities, hydropathy profiles, and phylogenetic relationship . Gene expression analysis demonstrated that the AtSAC genes exhibited differential expression patterns in different organs and, in particular, the AtSAC6 gene was predominantly expressed in flowers . Moreover, the expression of the AtSAC6 gene was highly induced by salinity . These results provide a foundation for future studies on the elucidation of the cellular functions of SAC domain-containing proteins in Arabidopsis.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7581 - 6 Epub 2003 Jun 12.
The coiled-coil of the human Rad50 DNA repair protein contains specific segments of increased flexibility; van Noort J et al.; Protein structural features are usually determined by defining regularities in a large population of homogeneous molecules . However, irregular features such as structural variation and flexibility are likely to be missed, despite their vital role for their biological function . In this paper, we report the observation of striking irregularities in the flexibility of the coiled-coil region of the human Rad50 DNA repair protein . Existing methods to quantitatively analyze flexibility are applicable to homogeneous polymers only . Because protein coiled-coils cannot be assumed to be homogeneous, we develop a method to quantify the local flexibility from high-resolution atomic force microscopy images . Indeed, in Rad50 coiled-coils, two positions of increased flexibility are observed . We discuss how this dynamic structural feature is integral to Rad50 function.

J Biol Chem, 2003 Aug 15, 278(33), 30421 - 4 Epub 2003 Jun 12.
Diverse but overlapping functions of the two forkhead-associated (FHA) domains in Rad53 checkpoint kinase activation; Pike BL et al.; Forkhead-associated (FHA) domains are phosphothreonine-binding modules prevalent in proteins with important cell cycle and DNA damage response functions . The yeast checkpoint kinase Rad53 is unique in containing two FHA domains . We have generated novel recessive rad53 alleles with abolished FHA domain functions resulting from Ala substitution of the critical phosphothreonine-binding residues Arg70 and Arg605 . In asynchronous cells, inactivation of the N-terminal FHA1 domain did not impair Rad53 activation and downstream functions, whereas inactivation of the C-terminal FHA2 domain led to reduced Rad53 activation and significantly increased DNA damage sensitivity . Simultaneous inactivation of both FHA domains abolished Rad53 activation and all downstream functions and dramatically increased the sensitivity to DNA damage and replication blocks similar to kinase-defective and rad53 null alleles, but did not compromise the essential viability function of Rad53 . Interestingly, in G2/M synchronized cells, mutation of either FHA domain prevented Rad53 activation and impaired the cell cycle arrest checkpoint . Our data demonstrate that both FHA domains are required for normal Rad53 functions and indicate that the two FHA domains have differential but partially overlapping roles in Rad53 activation and downstream signaling.

Circ Res, 2003 Jul 25, 93(2), 155 - 61 Epub 2003 Jun 12.
Shear stress-mediated chromatin remodeling provides molecular basis for flow-dependent regulation of gene expression; Illi B et al.; Shear stress (SS), the tangential component of hemodynamic forces, modulates the expression of several genes in endothelial cells . However, no information is available about its effect on chromatin structure, which plays a key role in gene transcription . In this study, a link between SS and chromatin remodeling was established in human umbilical vein endothelial cells (HUVECs) . HUVECs were exposed to SS of 10 dyne/cm2 per second, in the presence or absence of the histone deacetylase inhibitor trichostatin A, and assayed for histone H3 and histone H4 modifications . SS induced histone H3 serine phosphorylation at position 10 (S10) and lysine acetylation at position 14 (K14) but required trichostatin A to induce H3 phosphoacetylation and H4 acetylation . The phosphatidylinositol 3-kinase inhibitor wortmannin and the mitogen-activated protein kinase inhibitor PD98059 decreased SS-dependent histone H3 phosphorylation, without affecting its acetylation; the p38 inhibitor SB203580 reduced both H3 phosphorylation and acetylation, whereas the protein kinase A inhibitor PKI-tide reduced histone H3 acetylation . Remarkably, the abrogation of histone acetylation inhibited SS-dependent c-fos expression . SS also activated ribosomal S6 kinase-2 and mitogen- and stress-activated kinase-1 protein kinases and promoted the formation of a cAMP-responsive element-binding protein (CREB)/CREB-binding protein complex, providing the molecular basis for the increase in histone acetyltransferase activity observed in HUVECs exposed to SS . Finally, the effect of SS on chromatin remodeling was examined . In HUVECs exposed to SS, chromatin within c-fos and c-jun promoters was specifically immunoprecipitated by an antibody against acetylated histone H3 on K14 . These results indicate that SS induces posttransduction modifications of histones; this is an early step toward the flow-dependent regulation of gene expression.

EMBO J, 2003 Jun 16, 22(12), 3188 - 97
Multiple roles of Rev3, the catalytic subunit of polzeta in maintaining genome stability in vertebrates; Sonoda E et al.; Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major postreplicational repair (PRR) pathways . The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta, which is involved in mutagenic TLS . To investigate the role of REV3 in vertebrates, we disruped the gene in chicken DT40 cells . REV3(-/-) cells are sensitive to various DNA-damaging agents, including UV, methyl methanesulphonate (MMS), cisplatin and ionizing radiation (IR), consistent with its role in TLS . Interestingly, REV3(-/-) cells showed reduced gene targeting efficiencies and significant increase in the level of chromosomal breaks in the subsequent M phase after IR in the G(2) phase, suggesting the involvement of Rev3 in HR-mediated double-strand break repair . REV3(-/-) cells showed significant increase in sister chromatid exchange events and chromosomal breaks even in the absence of exogenous genotoxic stress . Furthermore, double mutants of REV3 and RAD54, genes involved in HR, are synthetic lethal . In conclusion, Rev3 plays critical roles in PRR, which accounts for survival on naturally occurring endogenous as well as induced damages during replication.

EMBO J, 2003 Jun 16, 22(12), 3175 - 87
The nuclear actin-related proteins Arp7 and Arp9: a dimeric module that cooperates with architectural proteins for chromatin remodeling; Szerlong H et al.; Nuclear actin-related proteins (ARPs) are essential components of chromatin remodeling and modifying complexes, but their functions and relationship to actin remain elusive . The yeast SWI/SNF and RSC complexes contain Arp7 and Arp9, and are shown to form a stable heterodimer with the properties of a functional module . Arp7 and Arp9 rely on their actin-related regions for heterodimerization, and their unique C-termini cooperate for assembly into RSC . We suggest that regulated ARP-ARP (and possibly ARP-beta-actin) heterodimerization might be a conserved feature of chromatin complexes . A RSC complex lacking Arp7/9 was isolated that displays robust nucleosome remodeling activity, suggesting a separate essential role for ARPs in the regulation of chromatin structure . A screen for suppressors of arp mutations yielded the DNA bending architectural transcription factor Nhp6, which interacts with RSC complex physically and functionally and shows facilitated binding to nucleosomes by RSC . We propose that Arp7/9 dimers function with DNA bending proteins to facilitate proper chromatin architecture and complex- complex interactions.

EMBO J, 2003 Jun 16, 22(12), 3131 - 41
High-affinity DNA binding by a Mot1p-TBP complex: implications for TAF-independent transcription; Gumbs OH et al.; Yeast Mot1p, an abundant conserved member of the Snf2p-ATPase family of proteins, both dissociates TBP from DNA in vitro using the energy of ATP and represses gene transcription in vivo, yet paradoxically, loss of Mot1p function also leads to decreased transcription of certain genes . We conducted experiments utilizing fluorescently labeled DNA, TBP, fluorescence anisotropy spectroscopy and native gel electrophoresis to study Mot1p action . We have made a number of observations, the most intriguing being that a stable Mot1p-TBP complex has the ability to bind TATA DNA with high affinity, albeit with dramatically altered specificity . We propose that this altered TBP-DNA recognition is integral to Mot1p's ability to regulate transcription, and further postulate that the Mot1p-TBP complex delivers TBP to TAF-independent mRNA encoding genes.

EMBO J, 2003 Jun 16, 22(12), 3062 - 72
Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD; Baas AF et al.; The LKB1 gene encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome . Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined . Here, we report the identification and characterization of an LKB1-specific adaptor protein and substrate, STRAD (STe20 Related ADaptor) . STRAD consists of a STE20- like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity . Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners . STRAD determines the subcellular localization of wild-type, but not mutant LKB1, translocating it from nucleus to cytoplasm . One LKB1 mutation previously identified in a Peutz-Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD-dependent regulation . Removal of endogenous STRAD by siRNA abrogates the LKB1-induced G(1) arrest . Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.

Mol Biol Cell, 2003 May, 14(5), 1953 - 63 Epub 2003 Feb 06.
Constriction and Dnm1p recruitment are distinct processes in mitochondrial fission; Legesse-Miller A et al.; Mitochondria undergo cycles of fusion and fission crucial for organelle homeostasis . Fission is regulated partially by recruitment of the large GTPase Dnm1p to the outer mitochondrial membrane . Using three-dimensional time-lapse fluorescence imaging of Saccharomyces cerevisiae cells, we found that Dnm1p-EGFP appears and disappears at "hot spots" along mitochondrial tubes . It forms patches that convert rapidly into different shapes regardless of whether mitochondrial fission ensues or not . Moreover, the thickness of the mitochondrial matrix displays frequent temporal fluctuations apparently unrelated to fission or to recruitment of Dnm1p-EGFP . These results suggest that mitochondrial fission requires coordination of at least two distinct processes.

Mol Biol Cell, 2003 May, 14(5), 1852 - 67 Epub 2003 Feb 06.
Dual prenylation is required for Rab protein localization and function; Calero M et al.; The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes . In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function . Substitution of different prenyl anchors on Rab GTPases does not lead to correct function . In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4 . Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane . We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins . This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.

Mol Biol Cell, 2003 May, 14(5), 1818 - 34 Epub 2003 Feb 06.
Centrin4p, a novel mammalian centrin specifically expressed in ciliated cells; Gavet O et al.; Centriole assembly plays an important role in centrosome duplication during the cell cycle and is a prerequisite for cilia formation during the differentiation of ciliated cells . In spite of numerous investigations, the molecular machinery that governs centriole/basal body formation remains enigmatic . Recent reports suggest that the ubiquitously expressed mammalian centrins, centrin2p and centrin3p, could be involved in the centriole duplication process . To better understand the specific functions of these proteins, we performed a systematic search for novel mammalian centrins . We isolated a cDNA and the corresponding gene coding for a novel murine centrin, centrin4p, which is more closely related to centrin2p . Like centrin2p, centrin4p accumulates to centrioles and procentrioles when ectopically expressed in HeLa cells . However, centrin4p possesses two splice variants that do not localize to centrioles, suggesting a posttranscriptional regulation mechanism . We also observed that centrin4p does not share the same centriolar targeting properties with centrin2p and 3p, indicating that these proteins could recognize different centriolar partners . Centrin4 mRNA possesses a restricted expression profile and is only detected in brain, kidney, lung, and ovary . In brain, centrin4p is exclusively expressed in ependymal and choroidal ciliated cells where it is localized to basal bodies . Together, our present data suggest that centrin4p could be more specifically involved in basal bodies assembly or in a subsequent step of ciliogenesis.

Bioinformatics, 2003 Jun 12, 19(9), 1110 - 5
Novel clustering algorithm for microarray expression data in a truncated SVD space; Horn D et al.; MOTIVATION: This paper introduces the application of a novel clustering method to microarray expression data . Its first stage involves compression of dimensions that can be achieved by applying SVD to the gene-sample matrix in microarray problems . Thus the data (samples or genes) can be represented by vectors in a truncated space of low dimensionality, 4 and 5 in the examples studied here . We find it preferable to project all vectors onto the unit sphere before applying a clustering algorithm . The clustering algorithm used here is the quantum clustering method that has one free scale parameter . Although the method is not hierarchical, it can be modified to allow hierarchy in terms of this scale parameter . RESULTS: We apply our method to three data sets . The results are very promising . On cancer cell data we obtain a dendrogram that reflects correct groupings of cells . In an AML/ALL data set we obtain very good clustering of samples into four classes of the data . Finally, in clustering of genes in yeast cell cycle data we obtain four groups in a problem that is estimated to contain five families . AVAILABILITY: Software is available as Matlab programs at http://neuron.tau.ac.il/~horn/QC.htm.

Trends Genet, 2003 Jun, 19(6), 321 - 9
The diverse functions of histone acetyltransferase complexes; Carrozza MJ et al.; Although histone acetylation has historically been linked to transcription activation, recent studies indicate that this modification and the enzymes that catalyze it have much broader and diverse functions . Histone acetyltransferase complexes are involved in such diverse processes as transcription activation, gene silencing, DNA repair and cell-cycle progression . The high conservation of the acetyltransferase complexes and their functions illustrates their central role in cell growth and development.

Trends Genet, 2003 Jun, 19(6), 295 - 8
Splicing goes global; Barrass JD et al.; Transcriptomics, the analysis of the complement of mRNAs transcribed from a cell's genome, currently focuses mainly on mature, processed mRNAs . However, posttranscriptional processing of primary transcripts can significantly affect both the quantity and the structure of the mature mRNAs and therefore of the protein products . Recently, the development of an intron-specific microarray has permitted a preliminary analysis of the splicing of all intron-containing transcripts in Saccharomyces cerevisiae . Here, we discuss the findings and what might be learned from this kind of approach.

Gene, 2003 May 22, 310, 161 - 8
Identification of a larger form of the histone acetyl transferase Tip60; Legube G et al.; The histone acetyl transferase Tat interactive protein 60kD (Tip60) plays major roles in the cellular response to extra- or intra-cellular signalling . Tip60 activity appears to be tightly regulated in cells through post-translational modifications or subcellular localisation of the protein . In addition, several alternatively spliced forms of Tip60 have been described . We found here that in a significant proportion of cytoplasmic poly adenylated Tip60 mRNAs, the intron 1 was not excised . Translation of this mRNA would predictably lead to the production of a longer Tip60 protein, containing a 33 amino acids insertion four amino acids after the ATG . By transient transfection experiments, we could demonstrate that this protein was significantly produced . Thus, taken together, our results indicate the existence of a longer Tip60 protein . Whether this protein could be differentially regulated and could play different roles than the classical Tip60 protein is an intriguing possibility.

Gene, 2003 May 22, 310, 113 - 21
Characterization of a SNF1 homologue from the phytopathogenic fungus Sclerotinia sclerotiorum; Vacher S et al.; In yeast, the SNF1 gene product is essential for the release of catabolic repression . We report the isolation and characterization of an SNF1 homologue from the necrotrophic pathogen Sclerotinia sclerotiorum . Ss snf1 encodes a 765-amino-acid protein in which the catalytic domain has an overall identity with the yeast proteins varying from 55 to 76% while the C-terminal half of Ss SNF1 has a weak homology of about 20% with the yeast sequences . Reverse transcription-polymerase chain reaction showed that its transcripts were weakly and constitutively expressed in planta and in vitro regardless of the nature of the carbon sources and of the presence or absence of glucose . Expression of Ss snf1 in yeast cells allowed the snf1 mutant cells to utilize sucrose, raffinose or glycerol for growth while expression of the Ss snf1 catalytic domain did not restore growth on raffinose or glycerol . Ss SNF1 is structurally homologous to Snf1p, suggesting that the interactions between the kinase and the accessory subunits to activate the enzymatic complex are conserved in fungi.

World J Gastroenterol, 2003 Jun, 9(6), 1165 - 9
DNA polymerase zeta: new insight into eukaryotic mutagenesis and mammalian embryonic development; Zhu F et al.; Information about the mechanisms that generate mutations in eukaryotes is likely to be useful for understanding human health concerns, such as genotoxicity and cancer . Eukaryotic mutagenesis is largely the outcome of attacks by endogenous and environmental agents . Except for DNA repair, cell cycle checkpoints and DNA damage avoidance, cells have also evolved DNA damage tolerance mechanism, by which lesion-targeted mutation might occur in the genome during replication by specific DNA polymerases to bypass the lesions (translesion DNA synthesis, TLS), or mutation on undamaged DNA templates (untargeted mutation) might be induced . DNA polymerase zeta (pol zeta), which was found firstly in budding yeast Saccharomyces cerevisiae and consists of catalytic subunit scRev3 and stimulating subunit scRev7, has received more attention in recent years . Pol zeta is a member of DNA polymerase eta subfamily, which belongs to DNA polymerase B family, and exists in almost all eukaryotes . Human homolog of the scRev3 gene is located in chromosome region 6q21, and the mouse equivalent maps to chromosome 10, distal to the c-myb gene and close to the Macs gene . Alternative splicing, upstream out-of frame ATG can be found in yeast scRev3, mouse and human homologs . Furthermore, the sequence from 253-323 immediate upstream of the AUG initiator codon has the potential to form a stem-loop hairpin secondary structure in REV3 mRNA, suggesting that human REV3 protein may be expressed at low levels in human cells under normal growth conditions . The functional domain analysis showed that yeast Rev3-980 tyrosine in conserved region II is at the polymerase active site . Human REV3 amino acid residues 1 776-2 195 provide a REV7 binding domain, and REV7 amino acid residues 1-211 provide a bind domain for REV1, REV3 and REV7 itself . More interestingly, REV7 interacts with hMAD2 and therefore might function in the cell cycle control by affecting the activation of APC (anaphase promoting complex) . Currently it has been known that pol zeta is involved in most spontaneous mutation, lesion-targeted mutation via TLS, chemical carcinogen induced untargeted mutation and somatic hypermutation of antibody genes in mammalian . In TLS pathway, pol zeta acts as a "mismatch extender" with combination of other DNA polymerases, such as pol iota . Unlike in yeast, it was found that pol zeta also functioned in mouse embryonic development more recently . It was hypothesized that the roles of pol zeta in TLS and cell cycle control might contribute to mouse embryonic lethality.

Int J Cancer, 2003 Aug 20, 106(2), 244 - 51
Novel tumor antigens identified by autologous antibody screening of childhood medulloblastoma cDNA libraries; Behrends U et al.; Medulloblastoma is an embryonal childhood malignancy with poor prognosis . By screening 4 medulloblastoma cDNA expression libraries (SEREX) with autologous sera, 15 different antigens were identified . These antigens were encoded by 3 novel genes, genes of unknown function (KIAA0445, KIAA1853, KIAA0665, FLJ13942, HSPC213), a proto-oncogene (rab18), candidate tumor suppressor genes (BAP1, PRDM13) and genes encoding a motor protein (kinesin-2), a histone (H2A1.2), the ankyrin residue-rich nasopharyngeal cancer susceptibility protein (NZ16) and the transcription factor TZP, which is homologous to the tumor-associated antigens HCA58 and GLEA2 . In a consecutive analysis of serum antibody titers and tumor load, a more than 10-fold increase in serum antibodies against PRDM13 preceded the clinical diagnosis of recurrent tumor growth in a patient with aggressive large cell medulloblastoma . When sera of pediatric patients with cancer (n = 40) and healthy controls (n = 40) were tested for humoral responses against the SEREX-defined antigens, 5 antigens were exclusively recognized by sera from cancer patients . These antigens included a novel rab18 gene product translated from mRNA sequences formerly described as 3' untranslated region . Humoral responses against 2 of the remaining 10 antigens were found preferentially in cancer patients . Antibodies against these antigens were detected in 8/40 and 12/40 cancer patients, respectively, but in only 1 healthy control . The 2 antigens were characterized by a tumor-specific deletion and a tumor-specific mutation, respectively . These findings indicate that the humoral immune response against medulloblastoma is directed against diverse antigens that may be useful as diagnostic markers or targets for immunotherapy .

J Biosci, 2003 Jun, 28(4), 423 - 36
The transcriptional activator GAL4-VP16 regulates the intra-molecular interactions of the TATA-binding protein; Mishra AK et al.; Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16 . Activator also greatly suppressed the non-specific TBP-DNA complex formation . The effects were more pronounced over weaker TATA boxes . Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators . The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably . The overall stimulatory effect of the GAL4-VP16 on the TBP-TATA complex formation resembles the known effects of removal of the N-terminus of TBP on its activity, suggesting that the activator directly targets the N-terminus of TBP and facilitates its binding to the TATA box.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3157 - 65
The Upf-dependent decay of wild-type PPR1 mRNA depends on its 5'-UTR and first 92 ORF nucleotides; Kebaara B et al.; mRNAs containing premature translation termination codons (nonsense mRNAs) are targeted for deadenylation-independent degradation in a mechanism that depends on Upf1p, Upf2p and Upf3p . This decay pathway is often called nonsense- mediated mRNA decay (NMD) . Nonsense mRNAs are decapped by Dcp1p and then degraded 5' to 3' by Xrn1p . In the yeast Saccharomyces cerevisiae, a significant number of wild-type mRNAs accumulate in upf mutants . Wild-type PPR1 mRNA is one of these mRNAs . Here we show that PPR1 mRNA degradation depends on the Upf proteins, Dcp1p, Xrn1p and Hrp1p . We have mapped an Upf1p-dependent destabilizing element to a region located within the 5'-UTR and the first 92 bases of the PPR1 ORF . This element targets PPR1 mRNA for Upf-dependent decay by a novel mechanism.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3114 - 22
The SV40 T antigen modulates CBP histone acetyltransferase activity; Valls E et al.; Histone acetyltransferases (HATs) play a key role in transcription control, cell proliferation and differentiation by modulating chromatin structure; however, little is known about their own regulation . Here we show that expression of the viral oncoprotein SV40 T antigen increases histone acetylation and global cellular HAT activities . In addition, it enhances CREB-binding protein HAT activity and modulates its transcriptional activity . Finally, we show that inhibition of cellular histone deacetylases by trichostatin A increases the SV40 infectivity rate . These findings highlight the importance of histone acetylation in the regulation of the cell cycle by oncoviral proteins.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3006 - 15
Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro; Bae KH et al.; We have previously shown that replication- protein A (RPA), the heterotrimeric single-stranded DNA binding protein of eukaryotes, plays a role in Okazaki fragment processing by acting as a molecular switch between the two endonucleases, Dna2 and Fen1, to ensure the complete removal of primer RNAs in Saccharomyces cerevisiae . The stimulation of Dna2 endonuclease activity by RPA requires direct protein-protein interaction . In this report we have analyzed genetically and biochemically the interaction of Dna2 with RPA . RFA1, the gene encoding the large subunit of RPA, displayed allele-specific interactions with DNA2 that included synthetic lethality and intergenic complementation . In addition, we identified physical and functional interactions between these proteins and found that RPA binds Dna2 predominantly through its large subunit, Rpa1 . Consistent with the mapping of synthetic lethal mutations, robust interaction localizes to the C-termini of these proteins . Moreover, the N-terminal domains of Dna2 and Rpa1 appear to be important for a functional interaction because the N-terminal domain of RPA1 was required to maximally stimulate Dna2 endonuclease activity . We propose that a bimodal interaction of Dna2 with Rpa1 is important for Dna2 function both in vivo and in vitro . The relevance of each interaction with respect to the function of the Dna2 endonuclease activity is discussed.

J Cell Sci, 2003 Aug 1, 116(Pt 15), 3069 - 77 Epub 2003 Jun 10.
Relationship of DNA double-strand breaks to synapsis in Drosophila; Jang JK et al.; The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms . Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans . To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, we used an antibody that recognizes a histone modification at double-strand break sites, phosphorylation of HIS2AV (gamma-HIS2AV) . Our results support the hypothesis that double-strand break formation occurs after synapsis . Interestingly, we detected a low (10-25% of wildtype) number of gamma-HIS2AV foci in c(3)G mutants, which fail to assemble synaptonemal complex, suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks . Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged gamma-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants . There may also be an interaction between the recruitment of repair proteins and phosphorylation.

Genome Res, 2003 Jun, 13(6A), 1231 - 43
Kinase pathway database: an integrated protein-kinase and NLP-based protein-interaction resource; Koike A et al.; Protein kinases play a crucial role in the regulation of cellular functions . Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics . We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes . It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein-protein, protein-gene, and protein-compound interaction data, domain information, and structural information . It also provides an automatic pathway graphic image interface . The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group . With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables . The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/.

Genome Res, 2003 Jun, 13(6A), 1056 - 66
New evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes; Panopoulou G et al.; The 2R hypothesis predicting two genome duplications at the origin of vertebrates is highly controversial . Studies published so far include limited sequence data from organisms close to the hypothesized genome duplications . Through the comparison of a gene catalog from amphioxus, the closest living invertebrate relative of vertebrates, to 3453 single-copy genes orthologous between Caenorhabditis elegans (C), Drosophila melanogaster (D), and Saccharomyces cerevisiae (Y), and to Ciona intestinalis ESTs, mouse, and human genes, we show with a large number of genes that the gene duplication activity is significantly higher after the separation of amphioxus and the vertebrate lineages, which we estimate at 650 million years (Myr) . The majority of human orthologs of 195 CDY groups that could be dated by the molecular clock appear to be duplicated between 300 and 680 Myr with a mean at 488 million years ago (Mya) . We detected 485 duplicated chromosomal segments in the human genome containing CDY orthologs, 331 of which are found duplicated in the mouse genome and within regions syntenic between human and mouse, indicating that these were generated earlier than the human-mouse split . Model based calculations of the codon substitution rate of the human genes included in these segments agree with the molecular clock duplication time-scale prediction . Our results favor at least one large duplication event at the origin of vertebrates, followed by smaller scale duplication closer to the bird-mammalian split.

J Mol Biol, 2003 Jun 20, 329(5), 931 - 9
Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR; Schaufler LE et al.; The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif . Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance . Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions . In addition, non-specific binding by ADR1 to a random DNA sequence was measured . ADR1 binds the native sites with nanomolar affinities . Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences . The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding . The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers . Binding kinetics of two ADR1 mutants was also examined . ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity . The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate . L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type . The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions.

J Mol Graph Model, 2003 Sep, 22(1), 31 - 40
Analysis and optimization of structure-based virtual screening protocols . 2 . Examination of docked ligand orientation sampling methodology: mapping a pharmacophore for success; Good AC et al.; An important element of any structure-based virtual screening (SVS) technique is the method used to orient the ligands in the target active site . This has been a somewhat overlooked issue in recent SVS validation studies, with the assumption being made that the performance of an algorithm for a given set of orientation sampling settings will be representative for the general behavior of said technique . Here, we analyze five different SVS targets using a variety of sampling paradigms within the DOCK, GOLD and PROMETHEUS programs over a data set of approximately 10,000 noise compounds, combined with data sets containing multiple active compounds . These sets have been broken down by chemotype, with chemotype hit rate used to provide a measure of enrichment with a potentially improved relevance to real world SVS experiments . The variability in enrichment results produced by different sampling paradigms is illustrated, as is the utility of using pharmacophores to constrain sampling to regions that reflect known structural biology . The difference in results when comparing chemotype with compound hit rates is also highlighted.

Bioorg Med Chem Lett, 2003 Jul 7, 13(13), 2235 - 7
Borrelidin induces the transcription of amino acid biosynthetic enzymes via a GCN4-dependent pathway; Eastwood EL et al.; Global cellular profiling of messenger RNA levels has been used to provide insight into the effects of the angiogenesis inhibitor borrelidin on the eukaryotic model organism Saccharomyces cerevisiae . The most notable result of treatment with borrelidin is the induction of amino acid biosynthetic enzymes in a time-dependent fashion . We have ascertained that induction of this pathway involves the GCN4 transcription factor . This conclusion was determined by treating a yeast strain lacking this gene and observing the absence of increased gene transcription under Gcn4p control.

Biochem J, 2003 Sep 1, 374(Pt 2), 349 - 58
Differential expression, localization and activity of two alternatively spliced isoforms of human APC regulator CDH1; Zhou Y et al.; The timely destruction of key regulators through ubiquitin-mediated proteolysis ensures the orderly progression of the cell cycle . The APC (anaphase-promoting complex) is a major component of this degradation machinery and its activation is required for the execution of critical events . Recent studies have just begun to reveal the complex control of the APC through a regulatory network involving WD40 repeat proteins CDC20 and CDH1 . In the present paper, we report on the identification and characterization of human CDH1beta, a novel alternatively spliced isoform of CDH1 . Both CDH1alpha and CDH1beta can bind to the APC and stimulate the degradation of cyclin B1, but they are differentially expressed in human tissues and cells . CDH1alpha contains a nuclear localization signal which is absent in CDH1beta . Intracellularly, CDH1alpha appears in the nucleus whereas CDH1beta is a predominantly cytoplasmic protein . The forced overexpression of CDH1alpha in cultured cells correlates with the reduction of nuclear cyclin A, but the steady-state amount of cyclin A does not change noticeably in CDH1beta-overexpressed cells . In Xenopus embryos, ectopic overexpression of human CDH1alpha, but not of CDH1beta, induces cell-cycle arrest during the first G(1) phase at the mid-blastula transition . Taken together, our findings document the differential expression, subcellular localization and cell-cycle-regulatory activity of human CDH1 isoforms.

Nat Genet, 2003 Jul, 34(3), 326 - 9
Cadmium is a mutagen that acts by inhibiting mismatch repair; Jin YH et al.; Most errors that arise during DNA replication can be corrected by DNA polymerase proofreading or by post-replication mismatch repair (MMR) . Inactivation of both mutation-avoidance systems results in extremely high mutability that can lead to error catastrophe . High mutability and the likelihood of cancer can be caused by mutations and epigenetic changes that reduce MMR . Hypermutability can also be caused by external factors that directly inhibit MMR . Identifying such factors has important implications for understanding the role of the environment in genome stability . We found that chronic exposure of yeast to environmentally relevant concentrations of cadmium, a known human carcinogen, can result in extreme hypermutability . The mutation specificity along with responses in proofreading-deficient and MMR-deficient mutants indicate that cadmium reduces the capacity for MMR of small misalignments and base-base mismatches . In extracts of human cells, cadmium inhibited at least one step leading to mismatch removal . Together, our data show that a high level of genetic instability can result from environmental impediment of a mutation-avoidance system.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7551 - 6 Epub 2003 Jun 09.
Spreading of Sir3 protein in cells with severe histone H3 hypoacetylation; Kristjuhan A et al.; Heterochromatin formation in yeast involves deacetylation of histones, but the precise relationship between acetylation and the association of proteins such as Sir3, Sir4, and the histone deacetylase Sir2 with chromatin is still unclear . Here we show that Sir3 protein spreads to subtelomeric DNA in cells lacking the transcription-related histone acetyltransferases GCN5 and ELP3 . Spreading correlates with hypoacetylation of lysines in the histone H3 tail and results in deacetylation of lysine 16 in histone H4 . De-repression of genes situated very close to the ends of the chromosomes in gcn5 elp3 suggests that Sir3 spreads into subtelomeric DNA from the tip of the telomere . Interestingly, growth defects caused by gcn5 elp3 mutation can be suppressed by SIR deletion, suggesting that Sir proteins become detrimental for growth when chromatin is severely hypoacetylated.

Plant J, 2003 Jun, 34(6), 788 - 801
Sodium transport and HKT transporters: the rice model; Garciadeblas B et al.; Na+ uptake in the roots of K+-starved seedlings of barley, rice, and wheat was found to exhibit fast rate, low Km, and high sensitivity to K+ . Sunflower plants responded in a similar manner but the uptake was not K+ sensitive . Ba2+ inhibited Na+ uptake, but not K+ uptake in rice roots . This demonstrated that Na+ and K+ uptake are mediated by different transporters, and that K+ blocked but was not transported by the Na+ transporter . The genome of rice cv . Nipponbare contains seven HKT genes, which may encode Na+ transporters, plus two HKT pseudogenes . Yeast expressions of OsHKT1 and OsHKT4 proved that they are Na+ transporters of high and low affinity, respectively, which are sensitive to K+ and Ba2+ . Parallel experiments of K+ and Na+ uptake in yeast expressing the wheat or rice HKT1 transporters proved that they were very different; TaHKT1 transported K+ and Na+, and OsHKT1 only Na+ . Transcript expressions in shoots of the OsHKT genes were fairly constant and insensitive to changes in the K+ and Na+ concentrations of the nutrient solution . In roots, the expressions were much lower than in shoots, except for OsHKT4 and OsHKT1 in K+-starved plants . We propose that OsHKT transporters are involved in Na+ movements in rice, and that OsHKT1 specifically mediates Na+ uptake in rice roots when the plants are K+ deficient . The incidence of HKT ESTs in several plant species suggests that the rice model with many HKT genes applies to other plants.

Traffic, 2003 Jul, 4(7), 479 - 90
PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body; Shaw JD et al.; The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body . Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body . Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body . These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes . The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate {PtdIns(3,5)P2}, is necessary for proper sorting of biosynthetic cargo in the multivesicular body . Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain . Characterization of this allele revealed reduced PtdIns(3,5)P2 production, altered vacuole morphology, and biosynthetic protein sorting defects . We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants . Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane . These data show that PtdIns(3,5)P2 is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.

Biochemistry, 2003 Jun 17, 42(23), 7068 - 76
Transient protein interactions studied by NMR spectroscopy: the case of cytochrome C and adrenodoxin; Worrall JA et al.; The interaction between yeast iso-1-cytochrome c (C102T) and two forms of bovine adrenodoxin, the wild type and a truncated form comprising residues 4-108, has been investigated using a combination of one- and two-dimensional heteronuclear NMR spectroscopy . Chemical shift perturbations and line broadening of amide resonances in the {(15)N,(1)H}HSQC spectrum for both (15)N-labeled cytochrome c and adrenodoxin in the presence of the unlabeled partner protein indicate the formation of a transient complex, with a K(a) of (4 +/- 1) x 10(4) M(-)(1) and a lifetime of <3 ms . The perturbed residues map over a large surface area for both proteins . For cytochrome c, the dominating effects are located around the exposed heme edge but with other areas also affected upon formation of the complex . In the case of adrenodoxin, effects are seen in both the recognition and core domains, with the largest perturbations in the recognition domain . These results indicate that the complex has a dynamic nature, with delocalized binding of cytochrome c on adrenodoxin . A comparison with other transient complexes of redox proteins places this complex between well-defined complexes such as the cytochrome c-cytochrome c peroxidase complex and entirely dynamic complexes such as the cytochrome b(5)-myoglobin complex.

Biochemistry, 2003 Jun 17, 42(23), 7013 - 22
Substrate-induced changes in the ammonia channel for imidazole glycerol phosphate synthase; Myers RS et al.; IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-{(5'-phosphoribulosyl)-formimino}-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP) . A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate . A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR . The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues . In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism . Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away . One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein . Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction . The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein . The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state.

J Biotechnol, 2003 May 8, 102(3), 269 - 79
Cell volume changes during rapid temperature shifts; Gervais P et al.; The effect of a rapid temperature increase on the volume of different types of cells was investigated . Experiments were carried out using continuous microscopic image analysis . Volume variation of yeast cells, yeast spheroplasts and human leukaemia cells was measured during the transient phase after a thermal shift . The thermal shift was found to induce rapid increase in cell volume for cells lacking a cell wall (yeast spheroplasts and human leukaemia cells) . This increase in cell volume is assumed to be a main cause of the heat shock-induced cell death . A theoretical mechanistic model that explains the behaviour of these cells is finally proposed.

J Biol Chem, 2003 Aug 22, 278(34), 32227 - 35 Epub 2003 Jun 06.
Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways; Espinosa L et al.; Phosphorylation of Notch proteins has been indirectly correlated with Notch activation and nuclear translocation as well as cellular transformation . There is evidence that the Wnt signaling pathway, which results in glycogen synthase kinase-3 beta (GSK-3 beta) inhibition, cross-talks with the Notch pathway . In this study, we show that GSK-3 beta is able to bind and phosphorylate Notch2 in vitro and in vivo . We identify three specific phosphorylation sites in the Notch2 serine/threonine-rich domain that are dependent on GSK-3 beta activity . Phosphorylation of the serine/threonine-rich domain has been shown previously to be crucial in regulating cytokine-specific cell differentiation . Coimmunoprecipitation experiments show that full-length Notch2 binds more efficiently than intracellular Notch2 to GSK-3 beta . Nevertheless, only the processed Notch2 is a substrate for the kinase, thus suggesting that GSK-3 beta-dependent phosphorylation may be specifically regulating the activated Notch molecule . Consistent with this, GSK-3 beta inhibits the transcriptional activation of Notch target genes both in vitro and in vivo, whereas lithium chloride treatment or Wnt-1 overexpression that results in GSK-3 beta inhibition leads to the up-regulation of the Hes-1 promoter . Together, our results suggest that cross-talk between Notch and Wnt pathways may be partially mediated by specific regulation of GSK-3 beta-dependent Notch phosphorylation.

J Biol Chem, 2003 Aug 22, 278(34), 32091 - 9 Epub 2003 Jun 06.
The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans; Artal-Sanz M et al.; Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane . The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes . The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae . We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism . We demonstrate that prohibitins in C . elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans . By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad . We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells . This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.

Science, 2003 Jun 6, 300(5625), 1542 - 8
Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes; Zou L et al.; The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage . Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells . In vitro, RPA stimulates the binding of ATRIP to ssDNA . The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA . Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner . A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro . Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

J Biol Chem, 2003 Aug 8, 278(32), 29792 - 8 Epub 2003 Jun 05.
The multiple site binding of carboxypeptidase Y inhibitor (IC) to the cognate proteinase . Implications for the biological roles of the phosphatidylethanolamine-binding protein; Mima J et al.; The serine carboxypeptidase inhibitor in the cytoplasm of Saccharomyces cerevisiae, IC, specifically inhibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs) . In the presence of 1 M guanidine hydrochloride, a CPY-IC complex is formed and is almost fully activated . The reactivities of phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid, and diisopropyl fluorophosphate toward the complex are considerably increased in 1 M guanidine hydrochloride, indicating that IC contains a binding site other than its inhibitory reactive site . IC is able to form the complex with diisopropyl fluorophosphate-modified CPY . Tryptic digestion of the complex indicates that two fragments from IC are involved in complex formation with CPY . These findings demonstrate the multiple site binding of IC with CPY . Considering the fact that mouse PEBP has recently been identified as a novel thrombin inhibitor, the binding that characterizes the CPY-IC complex could be a common feature of PEBPs.

J Biol Chem, 2003 Aug 15, 278(33), 31312 - 8 Epub 2003 Jun 05.
Molecular basis for atovaquone binding to the cytochrome bc1 complex; Kessl JJ et al.; Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis . It is thought to act on these organisms by inhibiting the cytochrome bc1 complex . We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus . Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center . These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein . A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone . When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone . These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.

Eur J Pharm Sci, 2003 Jun, 19(2-3), 151 - 64
Novel high energy intermediate analogues with triazasterol-related structures as inhibitors of ergosterol biosynthesis . III . Synthesis and antifungal activity of N4-alkyl-1,6,7,11b-tetrahydro-2H-pyrimido{4,3-a}isoquinolin-4-amine salts; Gossnitzer E et al.; A series of N4-alkyl-1,6,7,11b-tetrahydro-2H-pyrimido{4,3-a}isoquinolinamine hydroiodides with triazasterol-related structures was designed and synthesized to mimic, as stable analogues, native high energy intermediates (HEI) of ergosterol biosynthesis . The title compounds can be regarded as 8,13,15-triaza-13,17-secosteroids with aromatic ring A bearing the positive charge in the guanidinium moiety . Hence, these compounds present structural similarities with corresponding carbocationic intermediates occurring during the enzyme catalyzed transformation of squalene into ergosterol . The N4-alkylaminopyrimidoisoquinolinium salts were prepared by reaction of respective S-methylthiotetrahydropyrimidoisoquinoline hydroiodides with octylamine, and appropriately methyl-branched alkyl- and alkenylamines . In order to prepare (3R)-6-isopropyl-3-methyl-6-hepten-1-amine several synthetic routes were investigated . The structures of all reported compounds were proved and completely assigned on the basis of homo- and heteronuclear correlated 1D and 2D NMR spectroscopy . The in vitro antifungal susceptibility tests of the title compounds with a standard panel of eight pathogenic fungi revealed especially against the used dermatophytes and yeasts with MICs in the range of 1-32 microg/ml moderate to good antimycotic effects . Depending on the nature of the N4-alkyl substituents structure-activity relationships were found with a maximum of antifungal efficacy of the N4-3,7-dimethyloctylaminopyrimidoisoquinolinium iodide.

Dev Cell, 2003 Jun, 4(6), 769 - 70
A mitochondrial rhomboid protease; van der Bliek AM et al.; Rhomboid proteases are integral membrane proteins, typically associated with cleavage of peptide hormones along the secretory pathway . Recent publications demonstrate that yeast mitochondria contain a rhomboid protease required for the cleavage of two mitochondrial intermembrane space proteins, suggesting that rhomboid proteases play a regulatory role in mitochondria.

Structure (Camb), 2003 Jun, 11(6), 637 - 49
Structure of the coiled-coil dimerization motif of Sir4 and its interaction with Sir3; Chang JF et al.; The yeast silent information regulators Sir2, Sir3, and Sir4 physically interact with one another to establish a transcriptionally silent state by forming repressive chromatin structures . The Sir4 protein contains binding sites for both Sir2 and Sir3, and these protein-protein interactions are required for gene silencing . Here, we report the X-ray structure of the coiled-coil dimerization motif within the C-terminus of Sir4 and show that it forms a stable 1:1 complex with a dimeric fragment of Sir3 (residues 464-978) . We have identified a cluster of residues on the surface of the Sir4 coiled coil required for specific interactions with Sir3 . The histone deacetylase Sir2 can also bind to this complex, forming a ternary complex with the truncated Sir3 and Sir4 proteins . The dual interactions of Sir4 with Sir3 and Sir2 suggest a physical basis for recruiting Sir3 to chromatin by virtue of its interactions with Sir4 and with deacetylated histones in chromatin.

Mol Microbiol, 2003 Jun, 48(6), 1693 - 709
Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans; Semighini CP et al.; The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the cellular DNA damage response . Mutations in scaANBS1, which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti-topoisomerase I drug camptothecin . We have used the scaANBS1 cDNA as a bait in a yeast two-hybrid screening and report the identification of the A . nidulans Mre11 homologue (mreA) . The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents . Septation in A . nidulans is dependent not only on the uvsBATR gene, but also on the mre11 complex . scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra-S-phase checkpoint . ScaANBS1 also participates in G2-M checkpoint control upon DNA damage caused by MMS . In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A . nidulans . Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development.

J Biol Chem, 2003 Aug 29, 278(35), 33501 - 18 Epub 2003 Jun 04.
Mice lacking phosphatidylinositol transfer protein-alpha exhibit spinocerebellar degeneration, intestinal and hepatic steatosis, and hypoglycemia; Alb JG Jr et al.; Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions . We now report that ablation of PITP alpha function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice . The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects . The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum . We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITP alpha-/- mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease . Taken together, the data suggest an unanticipated role for PITP alpha in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes.

J Biol Chem, 2003 Aug 15, 278(33), 30875 - 80 Epub 2003 Jun 04.
Mutagenesis reveals a specific role for Cox17p in copper transport to cytochrome oxidase; Punter FA et al.; The provision of copper to cytochrome oxidase is one of the requisite steps in the assembly of the holoenzyme . Several proteins are involved in this process including Cox17p, Sco1p, and Cox11p . Cox17p, an 8-kDa protein, is the only molecule thought to be involved in shuttling copper from the cytoplasm into mitochondria . Given the small size of Cox17p, we have taken a random and site-directed mutagenesis approach to studying structure-function relationships in Cox17p . Mutations have been generated in 70% of the Cox17p amino acid residues, with only a small subset leading to a detectable respiration-deficient phenotype . We have characterized the respiration-deficient cox17 mutants and found in addition to the expected cytochrome oxidase deficiency, a specific lack of Cox2p and the presence of a misassembled cytochrome oxidase in a subset of mutants . These results suggest that Cox17p is involved upstream of Sco1p in delivering copper specifically to subunit 2 of cytochrome oxidase and predict the existence of a subunit 1-specific copper chaperone.

Curr Opin Genet Dev, 2003 Jun, 13(3), 262 - 70
Nuclear genes in mitochondrial disorders; Zeviani M et al.; Nuclear genes encode hundreds of proteins involved in mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) . Nevertheless, the identification of nuclear genes responsible for OXPHOS-related disorders has proceeded at a much slower pace, compared with the discovery and characterization of mtDNA mutations . Reasons for such a gap include rarity of syndromes, genetic heterogeneity, and ignorance on this nuclear gene repertoire in humans . This scenario is changing rapidly, thanks to the discovery of several OXPHOS-related human genes, and to the identification in some of them of disease-associated mutations . In addition, new strategies - based on transcriptome and proteome analysis, and functional complementation assays - have been applied successfully to mitochondrial medicine.

Curr Opin Cell Biol, 2003 Jun, 15(3), 275 - 80
Telomerase: what are the Est proteins doing?
Taggart AK, Zakian VA.
Saccharomyces cerevisiae has proven to be a useful model organism for the study of telomerase, a specialized cellular reverse transcriptase that helps maintain genomic stability by adding telomeric DNA repeats to the ends of chromosomes . Yeast telomerase is thought to be a holoenzyme containing Est2p and TLC1 RNA, the catalytic subunit and its intrinsic template, respectively, as well as the TLC1-RNA-associated factors Est1p and Est3p . Cdc13p, a sequence-specific telomere-DNA-binding protein, is also required for action in vivo . A current model for telomerase regulation is that telomere-associated Cdc13p binds Est1p, thereby recruiting telomerase . However, recent chromatin immunoprecipitation experiments suggest an alternate role for Est1p in activating Est2p-TLC1-RNA that is already bound to the telomere . Three models for Est1p activation are presented.

Cell, 2003 May 30, 113(5), 621 - 30
Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding; Kang RS et al.; Monoubiquitination serves as a regulatory signal in a variety of cellular processes . Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs . Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination . The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin . The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation . This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling . The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch . Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin . The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins.

Cell, 2003 May 30, 113(5), 609 - 20
Mechanism of ubiquitin recognition by the CUE domain of Vps9p; Prag G et al.; Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways . The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain . The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule . The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin . The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule . Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin.

Cell, 2003 May 30, 113(5), 554 - 6
CUE'd up for Monoubiquitin; Lima CD; The first structures have been obtained for complexes between CUE domains and monoubiquitin, one by NMR (Kang et al., this issue of Cell) and one by X-ray crystallography (Prag et al., this issue of Cell), thus providing insights into ubiquitin recognition by CUE domains . Structural comparisons suggest that different CUE surfaces can interact with ubiquitin, indicating that not all CUE domains are created equal.

Genes Cells, 2003 Jun, 8(6), 559 - 71
Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone; Fujita K et al.; BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B- and T-cells . TdT synthesizes the N region in concert with many proteins including DNA-PKcs, Ku70 and Ku86 . To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact with TdT . RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT . This protein was designated as TdT interacting factor 2 (TdIF2) . The confined region of the C-terminal in TdIF2 is involved in specific interaction with the entire C-terminal in TdT . TdIF2 contains an acidic region comprised of 42 residues . TdIF2 was shown to bind specifically to a core histone by pull down assay using specific antibodies against TdIF2 . When a TdT/TdIF2 complex was applied on to a DNA-cellulose column, only TdT bound to the column while TdIF2 passed through . TdIF2 reduces the TdT activity to 46% of its maximum value in vitro assay system using activated DNA as primer . CONCLUSIONS: TdIF2 binds directly to TdT and core histone . Furthermore, TdT, TdIF2 and core histone form a ternary complex . TdIF2 liberates H2A/H2B from a core histone in correlation with PCNA . The enzymatic consequence of the TdIF2/TdT complex is the reduction of TdT activity in vitro . TdIF2 would function as a chromatin remodeling protein at the N region synthesis.

Genes Cells, 2003 Jun, 8(6), 525 - 35
Transmembrane topology of sphingoid long-chain base-1-phosphate phosphatase, Lcb3p; Kihara A et al.; BACKGROUND: Sphingoid long-chain base-1-phosphates (LCBPs) are thought to act as intracellular signalling molecules in yeast . Lcb3p is a member of the LCBPs-specific phosphatase family (SPP family) . Other yeast phosphatases, Lpp1p and Dpp1p, are members of a different lipid phosphatase family (LPP family) known to exhibit broader substrate specificities . Until now, only the membrane topology of mammalian LPP family members has been reported, whereas that of the SPP family has remained unclear . RESULTS: In our in vitro system, Lcb3p displayed major phosphatase activity against dihydrosphingosine-1-phosphate, while Dpp1p and Lpp1p also exhibited activities . Here, we determined that Lpp1p and Dpp1p exhibit the topology common to the LPP family . Moreover, we examined the transmembrane topology of Lcb3p using a C-terminal reporter approach . From our results we deduced a structural model illustrating that Lcb3p has eight membrane-spanning domains with its highly conserved phosphatase motifs positioned within the endoplasmic reticulum (ER) lumen . Consistent with this result, Lcb3p collected in low speed pellet fractions was highly resistant to exogenous proteinase K unless the membrane was disrupted . CONCLUSION: Our results suggest that the active site of Lcb3p is located in the ER lumen and, thus, the phosphate group of the LCBP is hydrolysed on the lumenal side.

Genes Dev, 2003 Jun 15, 17(12), 1524 - 39 Epub 2003 Jun 03.
Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2; Benjamin KR et al.; Meiosis is thought to require the protein kinase Ime2 early for DNA replication and the cyclin-dependent kinase Cdc28 late for chromosome segregation . To elucidate the roles of these kinases, we inhibited their activities early and late using conditional mutants that are sensitive to chemical inhibitors . Our studies reveal that both Cdc28 and Ime2 have critical roles in meiotic S phase and M phase . Early inhibition of analog-sensitive cdc28-as1 blocked DNA replication, revealing a previously undetected role for Cdc28 . Yet Cdc28 was dispensable for one of its functions in the mitotic cell cycle, degradation of Sic1 . Late addition of inhibitor to ime2-as1 revealed unexpected roles of Ime2 in the initiation and execution of chromosome segregation . The requirement of Ime2 for M phase is partially explained by its stimulation of the key meiotic transcription factor Ndt80, which is needed in turn for high Cdc28 activity . In accordance with a late role for Ime2, we observed an increase in its activity during M phase that depended on Cdc28 and Ndt80 . We speculate that several unique features of the meiotic cell division reflect a division of labor and regulatory coordination between Ime2 and Cdc28.

Hum Mol Genet, 2003 Jun 15, 12(12), 1427 - 37
The p38 subunit of the aminoacyl-tRNA synthetase complex is a Parkin substrate: linking protein biosynthesis and neurodegeneration; Corti O et al.; Parkinson's disease (PD) is a severe neurological disorder, characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the presence of Lewy bodies (LBs) . The discovery of genes responsible for familial forms of the disease has provided insights into its pathogenesis . Mutations in the parkin gene, which encodes an E3 ubiquitin-protein ligase involved in the ubiquitylation and proteasomal degradation of specific protein substrates, have been found in nearly 50% of patients with autosomal-recessive early-onset parkinsonism . The abnormal accumulation of substrates due to loss of Parkin function may be the cause of neurodegeneration in parkin-related parkinsonism . Here, we demonstrate that Parkin interacts with, ubiquitylates and promotes the degradation of p38, a key structural component of the mammalian aminoacyl-tRNA synthetase complex . We found that the ubiquitylation of p38 is abrogated by truncated variants of Parkin lacking essential functional domains, but not by the pathogenic Lys161Asn point mutant . Expression of p38 in COS7 cells resulted in the formation of aggresome-like inclusions in which Parkin was systematically sequestered . In the human dopaminergic neuroblastoma-derived SH-SY5Y cell line, Parkin promoted the formation of ubiquitylated p38-positive inclusions . Moreover, the overexpression of p38 in SH-SY5Y cells caused significant cell death against which Parkin provided protection . Analysis of p38 expression in the human adult midbrain revealed strong immunoreactivity in normal dopaminergic neurons and the labeling of LBs in idiopathic PD . This suggests that p38 plays a role in the pathogenesis of PD, opening the way for a detailed examination of its potential non-canonical role in neurodegeneration.

Hum Mol Genet, 2003 Jun 15, 12(12), 1415 - 25
IL1 receptor accessory protein like, a protein involved in X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and regulates exocytosis; Bahi N et al.; Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X-linked mental retardation . This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family . In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function . In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1alpha and beta and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL . Our results show that IL1RAPL is not a protein receptor for IL-1 . In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor . Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca(2+) binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain . The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL . Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction.

Plant Cell, 2003 Jun, 15(6), 1375 - 85
PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major; Barth I et al.; Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters . For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias . For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions . Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family . The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose . In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago . Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues . Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed.

Plant Cell, 2003 Jun, 15(6), 1347 - 59
Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis; Lois LM et al.; Post-translational modification of proteins by small polypeptides, such as ubiquitin, has emerged as a common and important mechanism for regulating protein function . Small ubiquitin-like modifier (SUMO) is a small protein that is structurally related to but functionally different from ubiquitin . We report the identification and functional analysis of AtSUMO1, AtSUMO2, and AtSCE1a as components of the SUMO conjugation (sumoylation) pathway in Arabidopsis . In yeast-two hybrid assays, AtSUMO1/2 interacts specifically with a SUMO-conjugating enzyme but not with a ubiquitin-conjugating enzyme . AtSCE1a, the Arabidopsis SUMO-conjugating enzyme ortholog, conjugates SUMO to RanGAP in vitro . AtSUMO1/2 and AtSCE1a colocalize at the nucleus, and AtSUMO1/2 are conjugated to endogenous SUMO targets in vivo . Analysis of transgenic plants showed that overexpression of AtSUMO1/2 does not have any obvious effect in general plant development, but increased sumoylation levels attenuate abscisic acid (ABA)-mediated growth inhibition and amplify the induction of ABA- and stress-responsive genes such as RD29A . Reduction of AtSCE1a expression levels accentuates ABA-mediated growth inhibition . Our results suggest a role for SUMO in the modulation of the ABA signal transduction pathway.

Biochim Biophys Acta, 2003 Jun 10, 1632(1-3), 1 - 15
Plant sphingolipids: structural diversity, biosynthesis, first genes and functions; Sperling P et al.; In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains . More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions . In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function . This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis . Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host-pathogen interactions and stress responses.

Genomics, 2003 Jun, 81(6), 570 - 8
Genomic organization and expression of mouse Tpt1 gene; Fiucci G et al.; The translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF), is encoded by a gene (Tpt1) that is highly conserved throughout phylogeny . TCTP is implicated in cell growth, acute allergic response, and apoptosis . In the present study, seven putative Tpt1 genes with different chromosomal localizations were identified in the mouse genome . In six of them, analysis of the 5' and 3' untranslated regions revealed the presence of flanking direct repeats and residual poly(A) tails typical of pseudogenes . Only three of the seven genes can produce a protein of the expected molecular weight . We isolated the genomic DNA of these three genes to analyze their sequence, genomic organization, and in vitro promoter activity . We found that mouse Tpt1 is localized on chromosome 14 with a canonical intron-exon organization, a functional promoter, and only one transcript that is ubiquitously expressed in all tissues.

Curr Biol, 2003 May 27, 13(11), R449 - 60
Captivating capture: how microtubules attach to kinetochores; Biggins S et al.; Accurate chromosome segregation is essential to ensure genomic stability because the aneuploidy that results from segregation errors leads to birth defects and contributes to the development of cancer . Chromosome segregation is directed by the kinetochore, the chromosomal site of attachment to dynamic polymers called microtubules (MTs) . Although the fidelity of chromosome segregation depends on precise interactions between kinetochores and MTs, it is still unclear how this interaction is mediated and regulated . Here we discuss current progress in determining how kinetochores assemble and attach to MTs during mitosis as well as how they correct errors.

Curr Biol, 2003 May 27, 13(11), R439 - 41
Telomere replication: an Est fest; Lundblad V; The search for subunits of the telomerase enzyme has uncovered orthologs of the budding years Est1 protein in several species, including humans . Thus, positive regulation of telomerase by Est1 appears to be a widely utilized mechanism for maintaining telomere length homeostasis.

Curr Biol, 2003 May 27, 13(11), R430 - 2
Microtubule cytoskeleton: navigating the intracellular landscape; Bloom K; Recent studies have significantly advanced our understanding of how a dividing cell asymmetrically positions the mitotic spindle--a key process in metazoan development--while maintaining a dynamic spindle state that can respond and reorient when necessary.

Biochemistry, 2003 Jun 10, 42(22), 6709 - 18
2.4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor; Holyoak T et al.; This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues . Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%) . The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases . Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor . The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine.

Nat Cell Biol, 2003 Jul, 5(7), 661 - 7
Ubp3 requires a cofactor, Bre5, to specifically de-ubiquitinate the COPII protein, Sec23; Cohen M et al.; Ubiquitination is important for a broad array of cellular functions . Although reversal of this process, de-ubiquitination, most probably represents an important regulatory step contributing to cellular homeostasis, the specificity and properties of de-ubiquitination enzymes remain poorly understood . Here, we show that the Saccharomyces cerevisiae ubiquitin protease Ubp3 requires an additional protein, Bre5, to form an active de-ubiquitination complex that cleaves ubiquitin from specific substrates . In particular, this complex rescues Sec23p, a COPII subunit essential for the transport between the endoplasmic reticulum and the Golgi apparatus, from degradation by the proteasome . This probably contributes to maintaining and adapting a Sec23 expression level that is compatible with an efficient secretion pathway, and consequently with cell growth and viability.

Biochem J, 2003 Aug 1, 373(Pt 3), 733 - 8
X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase; Erskine PT et al.; The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 A) resolution . The cyclic intermediate is bound covalently to Lys(263) with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion . The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen . The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C-C bond linking both substrates in the intermediate is formed before the C-N bond.

Plant Mol Biol, 2003 Apr, 51(6), 851 - 7
Pectate lyase gene expression and enzyme activity in ripening banana fruit; Marin-Rodriguez MC et al.; Two distinct cDNA clones showing sequence homology to higher-plant pectate lyase (Pel) genes were isolated from ripening banana fruits . The transcripts were detected only in fruit tissue and both were strongly ripening-related . Yeast transformation with the most highly expressed Pel clone produced a recombinant protein with pectate lyase activity, demonstrating that this sequence was likely to encode a pectate lyase protein in planta . An assay developed for measuring the action of the endogenous enzyme from banana pulp tissue revealed a significant increase in calcium-dependent pectate lyase activity during ripening . The enhanced levels of enzyme activity corresponded with an increase in soluble polyuronides from banana pulp.

Plant Mol Biol, 2003 Apr, 51(6), 817 - 29
Tobacco bZIP factor TGA10 is a novel member of the TGA family of transcription factors; Schiermeyer A et al.; TGA factors constitute a family of conserved plant bZ1P transcription factors that regulate transcription from as-1-like elements in response to plant signalling molecules salicylic acid (SA), methyl jasmonate (MJ) and auxin . Based on sequence similarities, two subclasses of TGA factors have been identified before in tobacco: class I factors (TGA1a and PG13) are preferentially expressed in root tip meristems, whereas class II factors (TGA2.1 and TGA2.2) are found in leaves and in roots . Here we describe a novel member of the tobacco TGA family (TGA10), which defines a distinct subclass of its own . TGA10 mRNA and TGA10 protein were found in roots but not in leaves of mature tobacco plants . TGA10 binds specifically to the as-1 element, interacts with TGA2.2, and activates transcription in yeast . When ectopically expressed in leaves, TGA10 enhanced SA-, auxin- and MJ-inducibility of target gene Nt103, which responds in the same manner to enhanced levels of TGA2.2 . This indicates that TGA10, albeit normally not present in leaves, can interact with the leaf regulatory network controlling transcription from as-1-containing promoters . However, Nt103 expression was not affected in roots of TGA10-over-expressing plants, implying the existence of root-specific mechanisms which do not allow a positive effect of increased TGA10 levels on target gene expression.

J Cell Sci, 2003 Jul 1, 116(Pt 13), 2603 - 11
Formins: signaling effectors for assembly and polarization of actin filaments; Evangelista M et al.; Eukaryotic cells require filamentous actin to maintain their shape and for movement, growth and replication . New actin filaments are formed by the cutting of existing filaments or de novo through the action of specialized nucleators . The most highly characterized nucleator is the Arp2/3 complex, which nucleates the branched actin networks in the lamellae of migrating cells . Recently, Bni1p, which is a member of the formin family of proteins, has been shown to nucleate actin filaments in vitro . Formins are implicated in the formation of actin cables in yeast, stress fibers in tissue culture cells and cytokinesis in many cell types . Formins contain two highly conserved formin-homology domains, FH1 and FH2 . The Bni1p FH2 domain is sufficient to mediate nucleation . The Bni1p FH1 domain binds profilin, an actin-monomer-binding protein that delivers actin to the growing barbed end of filaments . The Bni1p FH1-profilin interaction enhances nucleation . Formins participate in a number of signaling pathways that control the assembly of specific actin structures and bind the barbed end of actin filaments, thereby providing a cytoskeletal basis for the establishment of cell polarity.

Nature, 2003 May 29, 423(6939), 537 - 41
Mitochondrial membrane remodelling regulated by a conserved rhomboid protease; McQuibban GA et al.; Rhomboid proteins are intramembrane serine proteases that activate epidermal growth factor receptor (EGFR) signalling in Drosophila . Rhomboids are conserved throughout evolution, and even in eukaryotes their existence in species with no EGFRs implies that they must have additional roles . Here we report that Saccharomyces cerevisiae has two rhomboids, which we have named Rbd1p and Rbd2p . RBD1 deletion results in a respiratory defect; consistent with this, Rbd1p is localized in the inner mitochondrial membrane and mutant cells have disrupted mitochondria . We have identified two substrates of Rbd1p: cytochrome c peroxidase (Ccp1p); and a dynamin-like GTPase (Mgm1p), which is involved in mitochondrial membrane fusion . Rbd1p mutants are indistinguishable from Mgm1p mutants, indicating that Mgm1p is a key substrate of Rbd1p and explaining the rbd1Delta mitochondrial phenotype . Our data indicate that mitochondrial membrane remodelling is regulated by cleavage of Mgm1p and show that intramembrane proteolysis by rhomboids controls cellular processes other than signalling . In addition, mitochondrial rhomboids are conserved throughout eukaryotes and the mammalian homologue, PARL, rescues the yeast mutant, suggesting that these proteins represent a functionally conserved subclass of rhomboid proteases.

Mol Cell Biol, 2003 Jun, 23(12), 4344 - 55
The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively; Kallstrom G et al.; We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p . Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus . Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature . Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus . Since Lsg1p is cytoplasmic, its role in nuclear export is likely to be indirect . We suggest that Lsg1p is needed to recycle an export factor(s) that shuttles from the nucleus associated with the nascent 60S subunit.

EMBO J, 2003 Jun 2, 22(11), 2831 - 40
Pti1p and Ref2p found in association with the mRNA 3' end formation complex direct snoRNA maturation; Dheur S et al.; Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non-protein-coding RNAs such as snRNAs and snoRNAs . These RNAs have to be processed at their 3' ends to be functional . mRNAs are matured by cleavage and polyadenylation that require a well-characterized protein complex . Small RNAs are also subject to 3' end cleavage but are not polyadenylated . Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre-mRNA 3' end processing complex, are essential for yeast snoRNA 3' end maturation . We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p-dependent pathway for 3' end formation of non-polyadenylated transcripts.

J Gen Virol, 2003 Jun, 84(Pt 6), 1499 - 504
Seroepidemiology of the human polyomaviruses; Stolt A et al.; To assess the stability of polyomavirus antibodies in serial samples over time and the incidence and age-specific prevalence of polyomavirus infections, we established enzyme immunoassays (EIAs) using purified yeast-expressed virus-like particles (VLPs) containing the VP1 major capsid proteins of JC virus (JCV) and the AS and SB strains of BK virus (BKV) . A random subsample of 150 Finnish women who had serum samples taken during the first trimester of pregnancy and had a second pregnancy during a 5 year follow-up period was selected, grouped by age of first pregnancy . The polyomavirus antibody levels were similar in samples taken during the first and second pregnancies (correlation coefficient 0.93 for BKV SB and 0.94 for JCV) . Analysis of serum samples from 290 Swedish children aged 1-13 years, grouped by age in 2 year intervals, demonstrated that BKV seropositivity increased rapidly with increasing age of the children, reaching 98 % seroprevalence at 7-9 years of age, followed by a minor decrease . JCV seroprevalence increased only slowly with increasing age and reaching 72 % positivity among mothers >25 years of age . The age-specific seroprevalence of the human polyomaviruses measured using this VLP-based EIA was similar to previous serosurveys by other methods . The stability of the antibodies over time indicates that polyomavirus seropositivity is a valid marker of cumulative virus exposure, and polyoma VLP-based EIAs may therefore be useful for epidemiological studies of these viruses.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 6940 - 5 Epub 2003 May 27.
Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS; Gerber SA et al.; A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels . Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states . Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis . These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications . Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer . In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay . The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.

J Nutr, 2003 Jun, 133(6 Suppl 1), 2040S - 2045S
Recent advances in the understanding of amino acid regulation of gene expression; Averous J et al.; In mammals, the impact of nutrients on gene expression has become an important area of research . Because amino acids have multiple and important functions, their homeostasis has to be finely maintained . However, amino acidemia can be affected by certain nutritional conditions or various forms of stress . Consequently, mammals must adjust several of the physiological functions involved in the adaptation to amino acid availability by regulating expression of numerous genes . It has been shown that amino acids alone can modify the expression of target genes . However, understanding of amino acid-dependent control of gene expression has just started to emerge . This review focuses on recent advances in the understanding of mechanisms involved in the amino acid control of gene expression.

J Cell Sci, 2003 Jul 15, 116(Pt 14), 2875 - 83 Epub 2003 May 27.
Protein translocation across the endoplasmic reticulum membrane in cold-adapted organisms; Romisch K et al.; Secretory proteins enter the secretory pathway by translocation across the membrane of the endoplasmic reticulum (ER) via a channel formed primarily by the Sec61 protein . Protein translocation is highly temperature dependent in mesophilic organisms . We asked whether the protein translocation machinery of organisms from extremely cold habitats was adapted to function at low temperature and found that post-translational protein import into ER-derived microsomes from Antarctic yeast at low temperature was indeed more efficient than into mesophilic yeast microsomes . Analysis of the amino-acid sequences of the core component of the protein translocation channel, Sec61p, from Antarctic yeast species did not reveal amino-acid changes potentially adaptive for function in the cold, because the sequences were too divergent . We therefore analyzed Sec61alpha (vertebrate Sec61p) sequences and protein translocation into the ER of Antarctic and Arctic fishes and compared them to Sec61alpha and protein translocation into the ER of temperate-water fishes and mammals . Overall, Sec61alpha is highly conserved amongst these divergent taxa; a number of amino-acid changes specific to fishes are evident throughout the protein, and, in addition, changes specific to cold-water fishes cluster in the lumenal loop between transmembrane domains 7 and 8 of Sec61alpha, which is known to be important for protein translocation across the ER membrane . Secretory proteins translocated more efficiently into fish microsomes than into mammalian microsomes at 10 degrees C and 0 degrees C . The efficiency of protein translocation at 0 degrees C was highest for microsomes from a cold-water fish . Despite substantial differences in ER membrane lipid composition, ER membrane fluidity was identical in Antarctic fishes, mesophilic fishes and warm-blooded vertebrates, suggesting that membrane fluidity, although typically important for the function of the transmembrane proteins, is not limiting for protein translocation across the ER membrane in the cold . Collectively, our data suggest that the limited amino-acid changes in Sec61alpha from fishes may be functionally significant and represent adaptive changes that enhance channel function in the cold.

J Biol Chem, 2003 Aug 15, 278(33), 30435 - 40 Epub 2003 May 27.
Dynamic recruitment of NF-Y and histone acetyltransferases on cell-cycle promoters; Caretti G et al.; Regulation of transcription during the cell-cycle is under the control of E2 factors (E2Fs), often in cooperation with nuclear factor Y (NF-Y), a histone-like CCAAT-binding trimer . NF-Y is paradigmatic of a constitutive, ubiquitous factor that pre-sets the promoter architecture for other regulatory proteins to access it . We analyzed the recruitment of NF-Y, E2F1/4/6, histone acetyltransferases, and histone deacetylase (HDAC) 1/3/4 to several cell-cycle promoters by chromatin immunoprecipitation assays in serum-starved and restimulated NIH3T3 cells . NF-Y binding is not constitutive but timely regulated in all promoters tested, being displaced when promoters are repressed . p300 association correlates with activation, and it is never found in the absence of NF-Y, whereas PCAF/hGCN5 is often found before NF-Y association . E2F4 and E2F6, together with HDACs, are bound to repressed promoters, including the G2/M Cyclin B2 . As expected, an inverse relationship between HDACs association and histones H3/H4 acetylation is observed . Blocking cells in G1 with the cyclin-dependent kinase 2 inhibitor R-roscovitine confirms that NF-Y is bound to G1/S but not to G2/M promoters in G1 . These data indicate that following the release of E2Fs/HDACs, a hierarchy of PCAF-NF-Y-p300 interactions and H3-H4 acetylations are required for activation of cell-cycle promoters.

Insect Biochem Mol Biol, 2003 Jun, 33(6), 609 - 22
Multiple acyl-CoA desaturase-encoding transcripts in pheromone glands of Helicoverpa assulta, the oriental tobacco budworm; Jeong SE et al.; Seven desaturase cDNAs were isolated from pheromone glands of Helicoverpa assulta, a moth producing a sex pheromone blend with high Z9-16:Ald and low Z11-16:Ald, opposite to what is found in other heliothine moths such as Helicoverpa zea . Six of the seven sequences map onto recently defined lepidopteran desaturase sequence lineages and the other is orthologous to a desaturase sequence previously reported only in H . zea . The levels of desaturase-encoding transcripts in pheromone glands were determined and the three most abundant ones were functionally expressed in a desaturase-deficient mutant strain of Saccharomyces cerevisiae . The HassNPVE transcript, shown to encode a delta9 desaturase producing more Z9-18:Acid than Z9-16:Acid, was the most abundant, followed by the HassKPSE transcript, shown to encode a delta9 desaturase producing more Z9-16:Acid than Z9-18:Acid, and by the HassLPAQ transcript, shown to encode a delta11 desaturase producing only Z11-16:Acid . Thus, the relative amounts of transcripts encoding two delta9 desaturases and a single delta11 desaturase in H . assulta pheromone glands were consistent with the relative amounts of unsaturated fatty acid precursors required to produce the major and minor sex pheromone components of this species . Desaturase transcript levels in pheromone glands were also found to be as high during scotophase as during light phase, when pheromone production ceases . The other four transcripts were present at extremely low levels in H . assulta pheromone glands and the functional roles of their encoded desaturase-homologous proteins could not be determined.

Mol Cell, 2003 May, 11(5), 1379 - 87
Telomerase and ATM/Tel1p protect telomeres from nonhomologous end joining; Chan SW et al.; Telomeres protect chromosome ends from fusing to double-stranded breaks (DSBs) . Using a quantitative real-time PCR assay, we show that nonhomologous end joining between a telomere and an inducible DSB was undetectable in wild-type cells, but occurred within a few hours of DSB induction in approximately 1/2000 genomes in telomerase-deficient cells and in >1/1000 genomes in telomerase-deficient cells also lacking the ATM homolog Tel1p . The fused telomeres contained very little telomeric DNA, suggesting that catastrophic telomere shortening preceded fusion . Lengthening of telomeres did not prevent such catastrophic telomere shortening and fusion events . Telomere-DSB fusion also occurred in cells containing a catalytically inactive telomerase and in tel1 mec1 cells where telomerase cannot elongate telomeres . Thus, telomerase and Tel1p function in telomere protection as well as in telomere elongation.

Mol Cell, 2003 May, 11(5), 1323 - 36
A central role for DNA replication forks in checkpoint activation and response; Tercero JA et al.; The checkpoint proteins Rad53 and Mec1-Ddc2 regulate many aspects of cell metabolism in response to DNA damage . We have examined the relative importance of downstream checkpoint effectors on cell viability . Checkpoint regulation of mitosis, gene expression, and late origin firing make only modest contributions to viability . By contrast, the checkpoint is essential for preventing irreversible breakdown of stalled replication forks . Moreover, recruitment of Ddc2 to nuclear foci and subsequent activation of the Rad53 kinase only occur during S phase and require the assembly of replication forks . Thus, DNA replication forks are both activators and primary effectors of the checkpoint pathway in S phase.

Mol Cell, 2003 May, 11(5), 1301 - 9
Independent recruitment in vivo by Gal4 of two complexes required for transcription; Bryant GO et al.; We use a modified form of ChIP to analyze the recruitment of seven sets of proteins to the yeast GAL genes upon induction . We resolve three stages of recruitment: first SAGA, then Mediator, and finally Pol II along with four other proteins (including TBP) bind the promoter . In a strain lacking SAGA, Mediator is recruited with a time course indistinguishable from that observed in wild-type cells . Our results are consistent with the notion that a single species of activator, Gal4, separately contacts, and thereby directly recruits, SAGA and Mediator.

Mol Cell, 2003 May, 11(5), 1201 - 13
Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint; Kitagawa K et al.; The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function . The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown . We report biochemical evidence that Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast . The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression . We propose that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores.

Mol Cell, 2003 May, 11(5), 1126 - 8
mRNA decay: x (XRN1) marks the spot; Long RM et al.; Degradation of mRNA is a vital aspect of gene expression . In yeast, Dcp1p, Dcp2p, Lsm1-7p, and Xrn1p are required for mRNA decay and are localized within discrete cytoplasmic foci; in the May 2 issue of Science, Sheth and Parker provide compelling evidence that these foci represent sites for mRNA decay.

Mol Cell, 2003 May, 11(5), 1123 - 5
FEARless in meiosis; Stern BM; Degradation of mitotic cyclins is critical for exit from mitosis . Recent studies in budding yeast address the role of cyclin degradation in meiosis . Cyclin stabilization in meiosis I interferes with anaphase I spindle disassembly but, surprisingly, does not halt progression into meiosis II.

Nature, 2003 Jun 12, 423(6941), 720 - 4 Epub 2003 May 25.
Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo; Kanemaki M et al.; Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function . Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis . We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined . Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degrees C (ref . 4) . By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells . We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks . The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.

Biochem Biophys Res Commun, 2003 Jun 13, 305(4), 925 - 33
A novel murine long-chain acyl-CoA synthetase expressed in brain participates in neuronal cell proliferation; Kee HJ et al.; Refsum disease (RfD) is an autosomal recessive neurologic disorder of the lipid metabolism . We have identified a novel murine long-chain acyl-CoA synthetase (mLACS) associated with the RfD gene using yeast two-hybrid assay . Northern blot analyses revealed that mLACS was expressed mainly in the brain and testis . mLACS was highly expressed in the brain at 2 weeks after birth and maintained through adult life . Expressions of the brain-specific LACS family increased in the PC12 cells undergoing neurite outgrowth by nerve growth factor . mLACS preferentially catalyzed the formation of arachidonoyl-CoA more than palmitoyl-CoA or oleoyl-CoA in PC12 cells . Triacsin C, an inhibitor of LACS, suppressed the cell proliferation and decreased mLACS expression in parent PC12 cells, but not in stably anti-sense mLACS cDNA-transfected cells . Our results indicate that mLACS participates in neuronal cell proliferation and differentiation, and interaction of the RfD gene with brain-selective mLACS may be involved in the pathogenesis of RfD.

Biochemistry, 2003 Jun 3, 42(21), 6467 - 74
Ligand specificity in the CRAL-TRIO protein family; Panagabko C et al.; Intracellular trafficking of hydrophobic ligands is often mediated by specific binding proteins . The CRAL-TRIO motif is common to several lipid binding proteins including the cellular retinaldehyde binding protein (CRALBP), the alpha-tocopherol transfer protein (alpha-TTP), yeast phosphatidylinositol transfer protein (Sec14p), and supernatant protein factor (SPF) . To examine the ligand specificity of these proteins, we measured their affinity toward a variety of hydrophobic ligands using a competitive {(3)H}-RRR-alpha-tocopherol binding assay . Alpha-TTP preferentially bound RRR-alpha-tocopherol over all other tocols assayed, exhibiting a K(d) of 25 nM . Binding affinities of other tocols for alphaTTP closely paralleled their ability to inhibit in vitro intermembrane transfer and their potency in biological assays . All other homologous proteins studied bound alpha-tocopherol but with pronouncedly weaker (> 10-fold) affinities than alpha-TTP . Sec14p demonstrated a K(d) of 373 nM for alpha-tocopherol, similar to that for its native ligand, phosphatidylinositol (381 nM) . Human SPF had the highest affinity for phosphatidylinositol (216 nM) and gamma-tocopherol (268 nM) and significantly weaker affinity for alpha-tocopherol (K(d) 615 nM) . SPF bound {(3)H}-squalene more weakly (879 nM) than the other ligands . Our data suggest that of all known CRAL-TRIO proteins, only alphaTTP is likely to serve as the physiological mediator of alpha-tocopherol's biological activity . Further, ligand promiscuity observed within this family suggests that caution should be exercised when suggesting protein function(s) from measurements utilizing a single ligand.

Ann N Y Acad Sci, 2003 Apr, 986, 198 - 203
Function and regulation of the two major plant plasma membrane H+-ATPases; Woloszynska M et al.; Plant plasma membrane H(+)-ATPases are encoded by a family of about ten genes organized into five subfamilies . Subfamilies I and II contain the most widely and highly expressed genes . In Nicotiana plumbaginifolia, they are represented, respectively, by pma2 (plasma membrane H(+)-ATPase) and pma4 . When expressed in the yeast Saccharomyces cerevisiae, the two isoforms show different kinetics and are differently regulated by phosphorylation of the penultimate threonine residue and binding of regulatory 14-3-3 proteins . To determine if these differences also occurred in plant tissues, we developed an experimental approach allowing the characterization of a single isoform in the plant . When PMA2 bearing a 6-His tag was expressed under a strong transcription promoter in Nicotiana tabacum BY2 cells, solubilized from microsomal membranes and purified, the penultimate threonine was found to be phosphorylated, thus validating the model.

Ann N Y Acad Sci, 2003 Apr, 986, 96 - 100
Site-directed mutagenesis of amino acids in the cytoplasmic loop 6/7 of Na,K-ATPase; Xu G et al.; The loop between transmembrane helices 6 and 7 (L6/7) of P-type ATPases has been suggested to be important for the functional linkage of ion binding and enzyme phosphorylation or to be a site of initial cation binding . To investigate the role of L6/7 in Na,K-ATPase, alanine substitutions were made for charged and conserved residues in L6/7 of the human alpha1 subunit and the proteins were expressed in yeast for analysis . All mutants except the triple mutant E825A/E828A/D830A bound ouabain . Although the equilibrium dissociation constant for ouabain binding by most mutants was similar to the wild-type value, the K(d) of R837A for ouabain binding was approximately 15-fold higher than the wild-type K(d) . (18)O exchange measurements indicated that the apparent affinity of this mutant for Pi was reduced about 3-fold . The concentration dependence of KCl inhibition of ouabain binding or of NaCl inhibition of ouabain binding revealed 2-4-fold changes in the apparent affinity for cations in the E825A, E828A, and R837A mutants . The E825A and E828A mutants lost the ability to bind ouabain after extraction with 0.1% SDS or after brief heating, indicating that these mutations affected the stability of the enzyme . The ATPase activity of the other mutants was measured after extraction of crude yeast membranes with 0.1% SDS . For all mutants except R834A, R837A, and R848A, the activity was at least 50% of wild-type activity.

Ann N Y Acad Sci, 2003 Apr, 986, 90 - 5
Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase; Corre F et al.; We have found that despite a markedly low calcium affinity the D813A/D818A mutant is capable, after complexation with Cr.ATP, of occluding Ca(2+) to the same extent (1-2 Ca(2+) per ATPase monomer) as wild- type ATPase . The inherent ability of the synthetic L6-7 loop peptide to bind Ca(2+) was demonstrated with murexide and mass spectrometry . NMR analysis indicated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum with coordination by all three aspartate residues D813/D815/D818 that resulted in an altered conformation of the peptide chain . Overall our observations suggest that, in addition to mediating contact between the intramembranous Ca(2+) binding sites and the cytosolic phosphorylation site as previously suggested, the L6-7 loop, in a preceding step, participates in the formation of an entrance port important for lodging Ca(2+) at a high-affinity binding site inside the membrane.

Eur J Cancer, 2003 Jun, 39(9), 1222 - 7
Concurrent hypermethylation of gene promoters is associated with a MSI-H phenotype and diploidy in gastric carcinomas; Carvalho B et al.; Changes in the pattern of DNA methylation are among the most common alterations observed in human cancers, such as gastric carcinomas . We analysed in a series of 51 sporadic gastric carcinomas the methylation status of the promoter regions of the hMLH1, CDH1, MGMT and COX2 genes . We aimed to determine the frequency of CpG island hypermethylation and to find out whether the occurrence of concurrent hypermethylation is related to the clinicopathological features of the gastric carcinomas . Using methylation-sensitive restriction analysis/polymerase chain reaction (PCR) and methylation-specific PCR (MSP) strategies, we searched for the presence of hypermethylation on the promoter region of the 4 selected genes . All showed hypermethylation of their promoter regions with frequencies of 37, 51, 61 and 29% for hMLH1, CDH1, MGMT and COX2, respectively . Concurrent hypermethylation was more frequently observed in MSI-H (P=0.0005) and diploid (P=0.029) tumours . Hypermethylation of hMLH1 was associated with MSI-H tumours (P=0.0001), whereas hypermethylation of MGMT was associated with MSI-H (p=0.021) and diploid tumours (p=0.012) . Our results indicate that concurrent hypermethylation is a common event in gastric cancer, suggesting that global methylation changes play an important role in the development of sporadic gastric carcinoma . Moreover, inactivation of different gene promoters by hypermethylation is significantly associated with microsatellite instability (MSI-H) and diploidy: hMLH1 determines MSI-H and MGMT the diploid status of gastric carcinomas.

J Nat Prod, 2003 May, 66(5), 722 - 4
Acetylenic acids from the aerial parts of Nanodea muscosa; El-Jaber N et al.; The aerial parts of Nanodea muscosa, collected in Chile, yielded two new acetylenic acids . Their structures were elucidated by spectroscopic analyses, including 2D NMR techniques, as (13E)-octadec-13-en-11-ynoic acid (1) and (2E)-octadec-2-en-4-ynedioic acid (2) . Compound 2 constitutes the first example of a conjugated ene-yne fatty diacid isolated from a natural source . Compounds 1 and 2 did not exhibit toxicity toward a panel of DNA damage checkpoint defective yeast mutants or show affinity for the 5-HT(1A), 5-HT(2A), D(2), and H(1) receptors.

Bioinformatics, 2003 May 22, 19(8), 905 - 12
Inference of transcriptional regulation relationships from gene expression data; Kwon AT et al.; MOTIVATION: In order to find gene regulatory networks from microarray data, it is important to first find direct regulatory relationships between pairs of genes . RESULTS: We propose a new method for finding potential regulatory relationships between pairs of genes from microarray time series data and apply it to expression data for cell-cycle related genes in yeast . We compare our algorithm, dubbed the event method, with the earlier correlation method and the edge detection method by Filkov et al . When tested on known transcriptional regulation genes, all three methods are able to find similar numbers of true positives . The results indicate that our algorithm is able to identify true positive pairs that are different from those found by the two other methods . We also compare the correlation and the event methods using synthetic data and find that typically, the event method obtains better results . AVALIABILITY: software is available upon request.

J Cell Sci, 2003 Jul 1, 116(Pt 13), 2763 - 74 Epub 2003 May 20.
Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells; Santel A et al.; Mitochondrial fusion may regulate mitochondrial morphogenesis and underlie complementation between mitochondrial genomes in mammalian cells . The nuclear encoded mitochondrial proteins Mfn1 and Mfn2 are human homologues of the only known protein mediators of mitochondrial fusion, the Drosophila Fzo GTPase and Saccharomyces cerevisiae yFzo1p . Although the Mfn1 and Mfn2 genes were broadly expressed, the two genes showed different levels of mRNA expression in different tissues . Two Mfn1 transcripts were detected at similar levels in a variety of human tissues and were dramatically elevated in heart, while Mfn2 mRNA was abundantly expressed in heart and muscle tissue but present only at low levels in many other tissues . Human Mfn1 protein localized to mitochondria and participated in a high molecular weight, detergent extractable protein complex . Forced expression of Mfn1 in cultured cells caused formation of characteristic networks of mitochondria . Introduction of a point mutation in the conserved G1 region of the predicted GTPase domain (Mfn1K88T) dramatically decreased formation of mitochondrial networks upon Mfn1 overexpression, suggesting that network formation required completion of the Mfn1 GTPase cycle . Conversely, a protein variant carrying a point mutation in the G2 motif of the Mfn1 GTPase domain acted as a dominant negative: overexpression of Mfn1T109A resulted in fragmentation of mitochondria . We propose that Mfn1T109A interferes with fusion activity of endogenous Mfn1 protein, possibly by binding necessary cofactors, so to allow unopposed mitochondrial fission . Thus, Mfn1 appears to be a key player in mediating mitochondrial fusion and morphology in mammalian cells.

J Biol Chem, 2003 Aug 1, 278(31), 29288 - 97 Epub 2003 May 19.
Physical and functional interactions between PML and MDM2; Wei X et al.; The tumor suppressor protein PML and oncoprotein MDM2 have opposing effects on p53 . PML stimulates p53 activity by recruiting it to nuclear foci termed PML nuclear bodies . In contrast, MDM2 inhibits p53 by promoting its degradation . To date, neither a physical nor functional relationship between PML and MDM2 has been described . In this study, we report an in vivo and in vitro interaction between PML and MDM2 which is independent of p53 . Two separate regions of PML are recognized which can interact with MDM2 . The C-terminal half of PML, encoded by residues 300-633, can interact with the central region of MDM2 which includes the MDM2 acidic domain . In addition, PML amino acids 1-200, which encode the RING-finger and most of the B box zinc binding motifs, can interact with the C-terminal, RING-finger containing region of MDM2 . Interestingly, PML mutants in which sumoylation at lysine 160 was inhibited displayed an increased association with MDM2, suggesting that sumoylation at this site may be a determinant of PML-MDM2 binding . Coexpression with MDM2 caused a redistribution of PML from the nucleus to the cytoplasm, and this required the PML N terminus and the MDM2 RING-finger domain . These results suggest that interaction between the PML N terminus and MDM2 C terminus can promote PML nuclear exclusion . Wild-type MDM2 inhibited the ability of PML to stimulate the transcriptional activity of a GAL4-CBP fusion protein . This inhibition required the central, acidic region of MDM2, but did not require the MDM2 C terminus . Taken together, these studies demonstrate that MDM2 and PML can interact through at least two separate protein regions, and that these interactions can have specific effects on the activity and/or localization of PML.

J Mol Biol, 2003 May 30, 329(2), 207 - 15
Functional interplay between CBP and PCAF in acetylation and regulation of transcription factor KLF13 activity; Song CZ et al.; The transcriptional co-activators CBP/p300 and PCAF participate in transcriptional activation by many factors . We have shown that both CBP/p300 and PCAF stimulate the transcriptional activation by KLF13, a member of the KLF/Sp1 family, either individually or cooperatively . Here we further investigated how CBP and PCAF acetylation regulate KLF13 activity, and how these two co-activators functionally interplay in the regulation of KLF13 activity . We found that CBP and PCAF acetylated KLF13 at specific lysine residues in the zinc finger domain of KLF13 . The acetylation by CBP, however, resulted in disruption of KLF13 DNA binding . Although the acetyltransferase activity of CBP is not required for stimulating the DNA binding activity of all of the transcription factors that we have examined, the disruption of factor DNA binding by CBP acetylation is factor-specific . We further showed that PCAF and CBP act synergistically and antagonistically to regulate KLF13 DNA binding depending on the status of acetylation . PCAF blocked CBP acetylation and disruption of KLF13 DNA binding . Conversely, acetylation of KLF13 by CBP prevented PCAF stimulation of KLF13 DNA binding . PCAF blocked CBP disruption of KLF13 DNA binding by preventing CBP acetylation of KLF13 . These results demonstrate that acetylation by CBP has distinct effects on transcription factor DNA binding, and that CBP and PCAF regulate each other functionally in their regulation of transcription factor DNA binding.

Free Radic Biol Med, 2003 Jun 1, 34(11), 1458 - 72
Cloning of novel human SEC14p-like proteins: ligand binding and functional properties; Kempna P et al.; We describe the cloning and expression of two novel genes highly similar to the tocopherol-associated protein (hTAP/SEC14L2/SPF) . Immunoprecipitation of the three recombinant hTAPs and extraction of their associated lipid-soluble molecules indicates that they bind not just tocopherols, but also phosphatidylinositol, phosphatidylcholine, and phosphatidylglycerol . Ligand competition analysis by isoelectric point mobility shift assay indicates that phosphatidylcholine, tocopherols, and tocopheryl-succinate compete with phosphatidylinositol binding to hTAPs . To investigate a possible function of hTAPs on enzymes involved in phospholipids metabolism, the activity of recombinant phosphatidylinositol 3-kinase (PI3Kgamma/p110gamma) was tested . Recombinant hTAPs reduce in vitro the activity of the recombinant catalytic subunit of PI3Kgamma and stimulate it in the presence of alpha-tocopherol up to 5-fold . Immunoprecipitation of hTAP1 from cells results in co-precipitation of PI3-kinase activity, indicating a physical contact between the two proteins at a cellular level . In summary, hTAPs may modulate, in a tocopherol-sensitive manner, phosphatidylinositol-3-kinase, a central enzyme in signal transduction, cell proliferation, and apoptosis . It is possible that other phosphatidylinositol- and phosphatidylcholine-dependent signaling pathways are modulated by hTAPs and tocopherols, possibly by transporting and presenting these ligands to the corresponding enzymes.

Bioorg Med Chem, 2003 Jun 12, 11(12), 2511 - 8
Cancer preventive potential of trichothecenes from Trichothecium roseum; Konishi K et al.; Bioassay-guided separation of extracts from the culture broth and mycelium of the fungus Trichothecium roseum, aiming at the discovery for cancer preventive agents, resulted in the isolation of three new trichothecene sesquiterpenes, trichothecinols A-C (1-3) together with three known analogues, trichothecin (4), trichodermol (5) and trichothecolone (6) . Compounds 1-6 exhibited remarkably potent inhibition against Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) . Further compound 1 strongly inhibited TPA-induced tumor promotion on mouse skin initiated with 7,12-dimethylbenz{a}anthracene (DMBA) in two-stage carcinogenesis tests . These results suggest that compound 1 might be a valuable lead for further evaluation as a cancer preventive agent . In addition to their cancer preventive activity, compound 2 was found to show modest antifungal activity against Crypotcoccus albidus and Saccharomyces cerevisiae.

Cell, 2003 May 16, 113(4), 429 - 32
Tails of intrigue: phosphorylation of RNA polymerase II mediates histone methylation; Hampsey M et al.; Histone lysine methylation plays a key role in the organization of chromatin structure and the regulation of gene expression . Recent studies demonstrated that the yeast Set1 and Set2 histone methyltransferases are recruited to mRNA coding regions by the PAF transcription elongation complex in a manner dependent upon the phosphorylation state of the carboxy-terminal domain of RNA polymerase II . These studies define an unexpected link between transcription elongation and histone methylation.

Drugs R D, 2003, 4(3), 167 - 73
Anidulafungin: ECB, LY 303366, V-echinocandin, VEC, VER 002, VER-02; No major difference in inhibitory susceptibility between CYP2C9.1 and CYP2C9.3; Clinical Evaluation of Medicines and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, JapanOBJECTIVE: CYP2C9 is a polymorphic enzyme, and CYP2C9*3 is associated with decreased metabolic activity . In addition to the impaired metabolism, we investigated whether the CYP2C9*3 exhibited altered inhibitory susceptibility compared with CYP2C9*1 . METHOD: In the present study, CYP2C9.1 and CYP2C9.3 were expressed in yeast . Using typical CYP2C9 substrates (diclofenac, tolbutamide and S-warfarin) and a potent CYP2C9 inhibitor (nicardipine), the Ki values for nicardipine on the three metabolisms in CYP2C9*1 and CYP2C9*3 were determined . RESULT: The ratios of Ki(CYP2C9*3)/Ki(CYP2C9*1) on tolbutamide, diclofenac and S-warfarin metabolisms were 1.2, 3.1 and 0.8, respectively . CONCLUSION: In conclusion, there are no significant differences in the inhibitory susceptibility between the two CYP2C9 enzymes.

RNA, 2003 Jun, 9(6), 698 - 710
RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts; Peters NT et al.; We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster . In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known . Here, we address each of these questions . 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts . We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development . The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands . It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe . The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development . Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway . Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor . The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 7015 - 20 Epub 2003 May 19.
3D electron microscopy reveals the variable deposition and protein dynamics of the peripheral pyruvate dehydrogenase component about the core; Gu Y et al.; Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC) . Structures representative of PDC preparations with approximately 40% and full E1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes . The reconstructions derived from two size groups showed that the deposition of the E1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC . This study also shows that the linker of dihydrolipamide acetyltransferase (E2) that tethers E1 to the E2 core increases in length from approximately 50 to 75 A, accounting largely for the size difference of the smaller and larger structures, respectively . Extensive E1 occupancy of its 60 E2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E1 molecules . However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1 . The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 7201 - 6 Epub 2003 May 19.
BRCA2 cooperates with histone acetyltransferases in androgen receptor-mediated transcription; Shin S et al.; Germ-line mutations of the BRCA2 tumor suppressor gene greatly increase the risk of developing breast and ovarian cancers . Here, we show that wild-type BRCA2, but not a tumor-specific truncated mutant BRCA2, synergizes with the nuclear receptor coactivator p160 GRIP1 to enhance transcriptional activation by androgen receptor (AR) . BRCA2 not only associates with AR and GRIP1 but also cooperates with both the histone acetyltransferase P/CAF and BRCA1 to enhance AR- and GRIP1-mediated transactivation . As such, BRCA2 can exert its tumor suppressor function, in part, by modulating androgen signaling, which has been shown to be antiproliferative in a subset of breast cancer cells and particularly implicated in male breast tumors.

J Biol Chem, 2003 Aug 1, 278(31), 29376 - 84 Epub 2003 May 19.
BLOC-3, a protein complex containing the Hermansky-Pudlak syndrome gene products HPS1 and HPS4; Martina JA et al.; The Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles . HPS results from mutations in either one of six human genes named HPS1 to HPS6, most of which encode proteins of unknown function . Here we report that the human HPS1 and HPS4 proteins are part of a complex named BLOC-3 (for biogenesis of lysosome-related organelles complex 3) . Co-immunoprecipitation experiments demonstrated that epitope-tagged and endogenous HPS1 and HPS4 proteins assemble with each other in vivo . The HPS1.HPS4 complex is predominantly cytosolic, with a small amount being peripherally associated with membranes . Size exclusion chromatography and sedimentation velocity analyses of the cytosolic fraction indicate that HPS1 and HPS4 form a moderately asymmetric protein complex with a molecular mass of approximately 175 kDa . HPS4-deficient fibroblasts from light ear mice display normal distribution and trafficking of the lysosomal membrane protein, Lamp-2, in contrast to fibroblasts from AP-3-deficient pearl mice (HPS2), which exhibit increased trafficking of this lysosomal protein via the plasma membrane . Similarly, light ear fibroblasts display an apparently normal accumulation of Zn2+ in intracellular vesicles, unlike pearl fibroblasts, which exhibit a decreased intracellular Zn2+ storage . Taken together, these observations demonstrate that the HPS1 and HPS4 proteins are components of a cytosolic complex that is involved in the biogenesis of lysosomal-related organelles by a mechanism distinct from that operated by AP-3 complex.

J Cell Biol, 2003 May 26, 161(4), 691 - 6 Epub 2003 May 19.
The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport; Bryant NJ et al.; Protein phosphatase 1 (PP1, Glc7p) functions in the final stage of SNARE-mediated vesicle transport between docking and fusion . During this process, trans-SNARE complexes, formed between molecules in opposing membranes, convert to cis-complexes, with all participants in the same lipid bilayer . Here, we show that glc7 mutant cells accumulate SNARE complexes . These complexes are clearly different from those found in either wild-type or sec18-1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them . Given that PP1 controls fusion, the SNARE complexes that accumulate in glc7 mutants likely represent trans-SNARE complexes . Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation . These data reveal that SM proteins cycle on and off membranes in a stage-specific manner during the vesicle transport reaction, and suggest that protein phosphorylation plays a key role in the regulation of this cycle.

J Cell Biol, 2003 May 26, 161(4), 715 - 25 Epub 2003 May 19.
Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex; Nilsson I et al.; In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen . This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme . Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane . A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain . When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61alpha and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST . No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST . As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.

Biochemistry, 2003 May 27, 42(20), 5971 - 6
Structure of frataxin iron cores: an X-ray absorption spectroscopic study; Nichol H et al.; X-ray absorption spectroscopy at the iron K-edge indicates that the iron cores of human and yeast frataxin polymers assembled in vitro are identical to each other and are similar but not identical to ferritin cores . Both frataxin polymers contain ferrihydrite, a biomineral composed of ferric oxide/hydroxide octahedra . The ferrihydrite in frataxin is less ordered than iron cores of horse spleen ferritin, having fewer face-sharing Fe-Fe interactions but similar double corner-sharing interactions . The extended X-ray absorption fine structure (EXAFS) analysis agrees with previous electron microscopy data showing that frataxin cores are composed of very small ferrihydrite crystallites.

Hum Mutat, 2003 Jun, 21(6), 598 - 607
Molecular and phenotypic heterogeneity in mitochondrial trifunctional protein deficiency due to beta-subunit mutations; Spiekerkoetter U et al.; The mitochondrial trifunctional protein (TFP) is a multienzyme complex of the fatty acid beta-oxidation cycle . It is composed of four alpha-subunits (HADHA) harboring long-chain enoyl-CoA hydratase and long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) and four beta-subunits (HADHB) harboring long-chain 3-ketoacyl-CoA thiolase (LKAT) . Mutations in either subunit can result in TFP deficiency with reduced activity of all three TFP enzymes . We characterize 15 patients from 13 families with beta-subunit mutations by clinical, biochemical, and molecular features . Three clinical phenotypes are apparent: a severe neonatal presentation with cardiomyopathy, Reye-like symptoms, and early death (n=4); a hepatic form with recurrent hypoketotic hypoglycemia (n=2); and a milder later-onset neuromyopathic phenotype with episodic myoglobinuria (n=9) . Maternal HELLP syndrome occurred in two mothers independently of the fetal phenotype . Mutational analysis revealed 16 different mutations, the majority being missense mutations (n=12) . The predominance of missense mutations and the milder myopathic phenotype are consistent . Based upon homology to yeast thiolase that has been characterized structurally, the mutation localization within the protein correlates with the clinical phenotype . Outer loop mutations that are expected to alter protein stability less were only present in milder forms . The degree of reduction in thiolase antigen also correlated with the severity of clinical presentation . Although TFP deficiency is highly heterogeneous, there is genotype-phenotype correlation .

J Exp Bot, 2003 Jul, 54(388), 1701 - 9 Epub 2003 May 13.
Effect of glucose on assimilatory sulphate reduction in Arabidopsis thaliana roots; Hesse H et al.; With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5'-phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied . Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed . After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots . The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions . Treatment of roots with d-sorbitol or d-mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation . The addition of O-acetyl-l-serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity) . Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity . Contrary to nitrate reductase, APR was also increased by glucose in N-deficient plants, indicating that this effect was independent of nitrate assimilation . {35S}-sulphate feeding experiments showed that the addition of glucose to dark-treated roots resulted in an increased incorporation of {35S} into thiols and proteins, which corresponded to the increased levels of APR activity . Under N-deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins . These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co-ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation . Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergistically.

J Biol Chem, 2003 Aug 1, 278(31), 28831 - 9 Epub 2003 May 16.
Antifungal activity of amiodarone is mediated by disruption of calcium homeostasis; Gupta SS et al.; The antiarrhythmic drug amiodarone was recently demonstrated to have novel broad range fungicidal activity . We provide evidence that amiodarone toxicity is mediated by disruption of Ca2+ homeostasis in Saccharomyces cerevisiae . In mutants lacking calcineurin and various Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels (Cch1/Mid1 and Yvc1), and exchangers (Vcx1), amiodarone sensitivity correlates with cytoplasmic calcium overload . Measurements of cytosolic Ca2+ by aequorin luminescence demonstrate a biphasic response to amiodarone . An immediate and extensive calcium influx was observed that was dose-dependent and correlated with drug sensitivity . The second phase consisted of a sustained release of calcium from the vacuole via the calcium channel Yvc1 and was independent of extracellular Ca2+ entry . To uncover additional cellular pathways involved in amiodarone sensitivity, we conducted a genome-wide screen of nearly 5000 single-gene yeast deletion mutants . 36 yeast strains with amiodarone hypersensitivity were identified, including mutants in transporters (pmr1, pdr5, and vacuolar H+-ATPase), ergosterol biosynthesis (erg3, erg6, and erg24), intracellular trafficking (vps45 and rcy1), and signaling (ypk1 and ptc1) . Of three mutants examined (vps45, vma3, and rcy1), all were found to have defective calcium homeostasis, supporting a correlation with amiodarone hypersensitivity . We show that low doses of amiodarone and an azole (miconazole, fluconazole) are strongly synergistic and exhibit potent fungicidal effects in combination . Our findings point to the potentially effective application of amiodarone as a novel antimycotic, particularly in combination with conventional antifungals.

Curr Opin Plant Biol, 2003 Jun, 6(3), 231 - 5
Trehalose metabolism: a regulatory role for trehalose-6-phosphate?
Eastmond PJ, Graham IA.
Trehalose is a disaccharide that was initially thought to be rare in plants but now appears to be ubiquitous . A recent study has established that the initial step in trehalose synthesis is essential in Arabidopsis . Evidence is emerging that the precursor of trehalose (trehalose-6-phosphate) is an important regulatory molecule . In yeast, trehalose-6-phosphate regulates sugar influx into glycolysis . In plants, trehalose-6-phosphate also appears to regulate sugar metabolism, but the underlying mechanism is unresolved and may be substantially different from that in yeast.

FEBS Lett, 2003 May 22, 543(1-3), 154 - 8
The Drosophila homolog of Aut1 is essential for autophagy and development; Juhasz G et al.; The Drosophila homolog of yeast Aut1, CG6877/Draut1, is a ubiquitously expressed cytosolic protein . Draut1 loss of function was achieved by expression of an inverted repeat transgene inducing RNA interference . The effect is temperature-dependent and resembles an allelic series as described by Fortier, E . and Belote, J.M . (Genesis 26 (2000) 240-244) . Draut1 loss of function larvae are unable to induce autophagy and heterophagy in fat body cells before pupariation and die during metamorphosis . To our knowledge, this is the first report of a multicellular animal lacking the function of a gene participating in the protein conjugation systems of autophagy.

FEBS Lett, 2003 May 22, 543(1-3), 61 - 5
Interaction of Sp1 transcription factor with HIV-1 Tat protein: looking for cellular partners; Loregian A et al.; The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates HIV-1 transcription by functionally interacting with a number of cellular proteins, among which the Sp1 transcription factor . We recently demonstrated that Tat does not directly interact with Sp1 either in vitro or in vivo, and we suggested that other protein(s) could indirectly mediate Tat-Sp1 interaction . In keeping, here we showed that addition of HeLa cell nuclear extracts to purified Tat and Sp1 proteins allows the formation of a Tat/Sp1 complex in in vitro binding assays . In an attempt to identify the partner(s) that bridge Tat and Sp1, we developed a yeast multi-protein system, in which cellular proteins recently shown to play a relevant role in Tat function, namely TATA box-binding protein, cyclin T1, CDK9, and cyclin T1/CDK9 complex, were coexpressed, individually or in pair-wise combination, with Tat and Sp1 hybrids . We demonstrated that none of these candidate partners bridges Tat and Sp1 . However, our yeast multi-protein system, which allows simple and rapid detection of interactions among up to four proteins, will be most helpful to further dissect the interaction of Tat and Sp1 with other candidate partners that participate in the assembly of transcriptionally active complexes at the HIV-1 LTR.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 478 - 83 Epub 2003 May 15.
Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles; Furukawa H et al.; Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative . These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet . The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein . Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction . The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times . This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.

J Biol Chem, 2003 Jun 27, 278(26), 23686 - 90 Epub 2003 May 15.
The beta-subunit of the protein-conducting channel of the endoplasmic reticulum functions as the guanine nucleotide exchange factor for the beta-subunit of the signal recognition particle receptor; Helmers J et al.; Cotranslational protein transport to the endoplasmic reticulum is controlled by the concerted interaction of three GTPases: the SRP54 subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR) . SRbeta is related to ADP-ribosylation factor (ARF)-type GTPases, and the recently published crystal structure of SRbeta-GTP in complex with the binding domain of SRalpha suggested that SRbeta, like all ARF-type GT-Pases, requires a guanine nucleotide exchange factor (GEF) for function . Searching the sequence data base, we identified significant sequence similarity between the Sec7 domain of ARF-GEFs and the cytosolic domains of the beta-subunits of the two homologous heterotrimeric protein-conducting channels in yeast . Using a fluorescence nucleotide exchange assay, we show that the beta-subunits of the heterotrimeric protein-conducting channels function as the GEFs for SRbeta . Both the cytosolic domain of Sec61beta as well as the holo-Sec61beta, when part of the isolated trimeric Sec61p complex, function as the GEF for SRbeta, whereas the same Sec61beta, when part of the heptameric complex that facilitates posttranslational protein transport, is inactive as the GEF for SRbeta

Genetics, 2003 May, 164(1), 173 - 86
Logjam encodes a predicted EMP24/GP25 protein that is required for Drosophila oviposition behavior; Carney GE et al.; A newly characterized Drosophila melanogaster gene, logjam (loj), functions in female reproduction by modulating oviposition behavior . The locus encodes at least six overlapping transcripts with unique 5' ends . P-element mutants that express very low levels of loj transcripts are unable to oviposit mature eggs . This phenotype can be rescued by the introduction of a transgene expressing the most abundant loj transcript . As for many genes that specify behavioral outputs, loj is present in the adult central nervous system (CNS) . Interestingly, it is also observed in vitellogenic egg chambers, suggesting that there may be multiple functions for this gene in egg-laying behavior . loj encodes a predicted protein with homology to the EMP24/GP25 transmembrane components of cytoplasmic vesicles and likely functions in intracellular trafficking.

Nature, 2003 May 15, 423(6937), 309 - 12
The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments; Veaute X et al.; Homologous recombination is a ubiquitous process with key functions in meiotic and vegetative cells for the repair of DNA breaks . It is initiated by the formation of single-stranded DNA on which recombination proteins bind to form a nucleoprotein filament that is active in searching for homology, in the formation of joint molecules and in the exchange of DNA strands . This process contributes to genome stability but it is also potentially dangerous to cells if intermediates are formed that cannot be processed normally and thus are toxic or generate genomic rearrangements . Cells must therefore have developed strategies to survey recombination and to prevent the occurrence of such deleterious events . In Saccharomyces cerevisiae, genetic data have shown that the Srs2 helicase negatively modulates recombination, and later experiments suggested that it reverses intermediate recombination structures . Here we show that DNA strand exchange mediated in vitro by Rad51 is inhibited by Srs2, and that Srs2 disrupts Rad51 filaments formed on single-stranded DNA . These data provide an explanation for the anti-recombinogenic role of Srs2 in vivo and highlight a previously unknown mechanism for recombination control.

Nature, 2003 May 15, 423(6937), 305 - 9
DNA helicase Srs2 disrupts the Rad51 presynaptic filament; Krejci L et al.; Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination . Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment . Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase . Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions . We show that Srs2 acts by dislodging Rad51 from ssDNA . Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently . Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing.

Mol Cell Biol, 2003 Jun, 23(11), 3847 - 58
Identification of tetratricopeptide repeat 1 as an adaptor protein that interacts with heterotrimeric G proteins and the small GTPase Ras; Marty C et al.; The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins . Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Galpha16-binding protein . The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Galpha proteins . TPR1 was found to interact with Ha-Ras preferentially in its active form . Overexpression of TPR1 promotes accumulation of active Ras . TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras . Expression of Galpha16 strongly enhances the interaction between TPR1 and Ras . Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Galpha16 while maintaining the interaction with Galpha16 and the ability to discriminate active Ras from wild-type Ras . We have also observed that LGN, a Galphai-interacting protein with seven TPR motifs, binds Ha-Ras . Thus, TPR1 is a novel adaptor protein for Ras and selected Galpha proteins that may be involved in protein-protein interaction relating to G-protein signaling.

Mol Cell Biol, 2003 Jun, 23(11), 3837 - 46
Inhibition of p300/CBP by early B-cell factor; Zhao F et al.; Early B-cell factor (EBF) is a DNA binding protein required for early B-cell development . It activates transcription of several B-cell-specific genes, including the lambda5 gene, which encodes a protein necessary for signaling by the pre-B-cell receptor . In an effort to understand the mechanism by which EBF activates transcription, we examined its interaction with the coactivator protein p300/CBP . We found that two domains of EBF each bind the histone acetyltransferase (HAT)/CH3 domain of p300/CBP both in vitro and in vivo . Surprisingly, transcriptional activation by EBF was not sensitive to E1A, a potent p300/CBP inhibitor . In fact, overexpressed EBF mimicked E1A by severely repressing the activity of several other transcription factors, including E47, a protein that acts cooperatively with EBF to promote transcription of the lambda5 gene . This broad inhibitory profile correlated with EBF's ability to repress the HAT activity of p300/CBP in vivo and in vitro . However, such a repressed complex is not likely to form at the lambda5 promoter in vivo since (i) EBF could not bind p300/CBP and DNA simultaneously and (ii) the cooperativity imparted by E47 was sensitive to E1A . Our data reveal an intriguing inhibitory property of EBF-a property shared only by E1A, Twist, Pu.1, and the Hox family of homeodomain proteins-and suggest that E47 and EBF play distinct roles during lambda5 promoter activation.

Mol Cell Biol, 2003 Jun, 23(11), 3788 - 97
Polyphosphate loss promotes SNF/SWI- and Gcn5-dependent mitotic induction of PHO5; Neef DW et al.; Approximately 800 transcripts in Saccharomyces cerevisiae are cell cycle regulated . The oscillation of approximately 40% of these genes, including a prominent subclass involved in nutrient acquisition, is not understood . To address this problem, we focus on the mitosis-specific activation of the phosphate-responsive promoter, PHO5 . We show that the unexpected mitotic induction of the PHO5 acid phosphatase in rich medium requires the transcriptional activators Pho4 and Pho2, the cyclin-dependent kinase inhibitor Pho81, and the chromatin-associated enzymes Gcn5 and Snf2/Swi2 . PHO5 mitotic activation is repressed by addition of orthophosphate, which significantly increases cellular polyphosphate . Polyphosphate levels also fluctuate inversely with PHO5 mRNA during the cell cycle, further substantiating an antagonistic link between this phosphate polymer and PHO5 mitotic regulation . Moreover, deletion of PHM3, required for polyphosphate accumulation, leads to premature onset of PHO5 expression, as well as an increased rate, magnitude, and duration of PHO5 activation . Orthophosphate addition, however, represses mitotic PHO5 expression in a phm3delta strain . Thus, polyphosphate per se is not necessary to repress PHO transcription but, when present, replenishes cellular phosphate during nutrient depletion . These results demonstrate a dynamic mechanism of mitotic transcriptional regulation that operates mostly independently of factors that drive progression through the cell cycle.

J Biol Chem, 2003 Aug 1, 278(31), 28799 - 811 Epub 2003 May 13.
Identification of a novel Krüppel-associated box domain protein, Krim-1, that interacts with c-Myc and inhibits its oncogenic activity; Hennemann H et al.; We have used the Ras recruitment system to screen for proteins that interact with the N-terminally located transactivation domain of c-Myc . The Ras recruitment system is based on the activation of the mitogenic RAS signaling pathway in yeast by the mammalian GTPase Ha-Ras . This screen led to the identification of two novel nuclear proteins termed Krim-1A and Krim-1B that both contain an N-terminal KRAB box domain and 12 or 9 Kruppel C2H2 type zinc fingers at the C terminus, respectively . We found that sequences covering the Myc box II homology region are essential for the interaction with the Krim-1 proteins and that the second N-terminal zinc finger of Krim-1 is essential for Myc binding . Both Krim-1A and -B genes appear to be expressed ubiquitously with highest levels in spleen and lymph nodes . In particular, Krim-1B and, to a lesser extent, Krim-1A are able to decrease E-box-dependent transcriptional transactivation by c-Myc-Max complexes and also the ability of Myc to malignantly transform primary rat embryo fibroblasts, which is consistent with the functional repressive properties of their KRAB domains . The transcriptional corepressor Tif-1beta is a binding partner for Krim-1 and stabilizes the protein . Our findings suggest that Myc-mediated functions can be negatively regulated by Krim-1, potentially in a complex with Tif-1beta.

Curr Biol, 2003 May 13, 13(10), R385 - 7
Chromosome segregation: clamping down on deviant orientations; Pidoux A et al.; Chromosome segregation depends on proper orientation of sister kinetochores . The protein Csm1 is required for mono-orientation of sister kinetochores at meiosis I in budding yeast . Surprisingly, its homologue in fission yeast appears instead of clamp micro-tubule binding sites together on single mitotic kinetochores so that they all face one spindle pole.

J Basic Microbiol, 2003, 43(2), 104 - 12
An expression system for the functional analysis of pheromone genes in the tetrapolar basidiomycete Schizophyllum commune; Gola S et al.; The investigation of putative pheromone genes of basidiomycetes has been difficult since the small open reading frames are essentially annotated on the basis of a C-terminal farnesylation signal . In order to identify the functional reading frame, expression of small DNA fragments in the fungus is necessary . The expression system developed in the presented paper allows fusion to the promoter of the tef1 gene encoding the constitutively and highly expressed translation elongation factor EF1alpha . This system has been shown to be functional using an easily selectable gene, ura1 . The application to identification of functional pheromone genes has been shown with the newly detected bap2(4) gene . The Bap2(4) pheromone is the first Balpha pheromone gene activating only a single receptor specificity.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 6969 - 73 Epub 2003 May 13.
Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription; Bushnell DA et al.; The x-ray structure of complete RNA polymerase II from Saccharomyces cerevisiae has been determined, including a heterodimer of subunits Rpb4 and Rpb7 not present in previous "core" polymerase II structures . The heterodimer maintains the polymerase in the conformation of a transcribing complex, may bind RNA as it emerges from the enzyme, and is in a position to interact with general transcription factors and the Mediator of transcriptional regulation.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 6964 - 8 Epub 2003 May 13.
Architecture of initiation-competent 12-subunit RNA polymerase II; Armache KJ et al.; RNA polymerase (Pol) II consists of a 10-polypeptide catalytic core and the two-subunit Rpb4/7 complex that is required for transcription initiation . Previous structures of the Pol II core revealed a "clamp," which binds the DNA template strand via three "switch regions," and a flexible "linker" to the C-terminal repeat domain (CTD) . Here we derived a model of the complete Pol II by fitting structures of the core and Rpb4/7 to a 4.2-A crystallographic electron density map . Rpb4/7 protrudes from the polymerase "upstream face," on which initiation factors assemble for promoter DNA loading . Rpb7 forms a wedge between the clamp and the linker, restricting the clamp to a closed position . The wedge allosterically prevents entry of the promoter DNA duplex into the active center cleft and induces in two switch regions a conformation poised for template-strand binding . Interaction of Rpb4/7 with the linker explains Rpb4-mediated recruitment of the CTD phosphatase to the CTD during Pol II recycling . The core-Rpb7 interaction and some functions of Rpb4/7 are apparently conserved in all eukaryotic and archaeal RNA polymerases but not in the bacterial enzyme.

J Biol Chem, 2003 Aug 1, 278(31), 28872 - 81 Epub 2003 May 13.
Sphingolipid requirement for generation of a functional v1 component of the vacuolar ATPase; Chung JH et al.; There has been no previous indication that vacuolar ATPases (V-ATPases) require sphingolipids for function . Here we show, by using Saccharomyces cerevisiae sur4Delta and fen1Delta cells, that sphingolipids with a C26 acyl group are required for generating V1 domains with ATPase activity . Sphingolipids in sur4Delta cells contain C22 and C24 acyl groups instead of C26 acyl groups whereas about 30% of the sphingolipids in fen1Delta cells have C26 acyl groups and the rest have C22 and C24 acyl groups . sur4Delta cells have several phenotypes (vacuolar membrane ATPase, Vma-) that indicate a defect in the V-ATPase, and vacuoles purified from sur4Delta cells have little to no ATPase activity . These phenotypes are less pronounced in fen1Delta cells, consistent with the idea that the C26 acyl group in sphingolipids is necessary for V-ATPase activity . Other results show that the two V-ATPase domains, V1 and V0, are assembled and delivered to the vacuolar membrane in sur4Delta cells similar to wild-type cells . In vitro assembly studies show that V1 from sur4Delta cells associates with wild-type V0 but the complex lacks V-ATPase activity, indicating that V1 is defective . Reciprocal experiments with V0 from sur4Delta cells show that it is normal . We conclude that sphingolipids with a C26 acyl group are required for generating fully functional V1 domains.

J Biol Chem, 2003 Jul 18, 278(29), 26923 - 8 Epub 2003 May 13.
Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins; Nowotny M et al.; S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner . One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination . A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes . S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest . Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6 . We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments . Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues . Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1 . Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family . The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody . Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1 . These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.

J Med Genet, 2003 May, 40(5), 325 - 32
Disruption of the neuronal PAS3 gene in a family affected with schizophrenia; Kamnasaran D et al.; Schizophrenia and its subtypes are part of a complex brain disorder with multiple postulated aetiologies . There is evidence that this common disease is genetically heterogeneous, with many loci involved . In this report, we describe a mother and daughter affected with schizophrenia, who are carriers of a t(9;14)(q34;q13) chromosome . By mapping on flow sorted aberrant chromosomes isolated from lymphoblast cell lines, both subjects were found to have a translocation breakpoint junction between the markers D14S730 and D14S70, a 683 kb interval on chromosome 14q13 . This interval was found to contain the neuronal PAS3 gene (NPAS3), by annotating the genomic sequence for ESTs and performing RACE and cDNA library screenings . The NPAS3 gene was characterised with respect to the genomic structure, human expression profile, and protein cellular localisation to gain insight into gene function . The translocation breakpoint junction lies within the third intron of NPAS3, resulting in the disruption of the coding potential . The fact that the bHLH and PAS domains are disrupted from the remaining parts of the encoded protein suggests that the DNA binding and dimerisation functions of this protein are destroyed . The daughter (proband), who is more severely affected, has an additional microdeletion in the second intron of NPAS3 . On chromosome 9q34, the translocation breakpoint junction was defined between D9S752 and D9S972 and no genes were found to be disrupted . We propose that haploinsufficiency of NPAS3 contributes to the cause of mental illness in this family.

Mol Membr Biol, 2003 Jan-Mar, 20(1), 61 - 9
PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p; Parkes JA et al.; The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption . Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met . However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p . The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Delta 3 and Delta 19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system . The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species . Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion . These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.

J Biomol Struct Dyn, 2003 Jun, 20(6), 789 - 99
Ethidium probing of the parallel double- and four-stranded structures formed by the telomeric DNA sequences dG(GT)4G and d(GT)5; Beschetnova IA et al.; Oligonucleotides 3'-d(GT)(5)-(CH(2)CH(2)O)(3)-d(GT)(5)-3' (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) {O . F . Borisova et al . FEBS Letters 306, 140-142 (1992)} . Four d(GT)(5) strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out . The four GT repeats when flanked by guanines, 3'-dG(TG)(4)G-(CH(2)CH(2)O)(3)-dG(GT)(4)G-3' (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G.G and T.T base pairs (hp-GT ps-DNA) {A . K . Shchyolkina et al . J . Biomol . Struct . Dyn . 18, 493-503 (2001)} . In the present study the intercalator ethidium bromide (Et) was used for probing the two structures . The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared . The quantum yield (q) and the fluorescence lifetime (tau) of Et:qGT (q = 0.15 +/- 0.01 and tau = 24 +/- 1 ns) and Et:hp-GT (q = 0.10 +/- 0.01 and tau = 16.5 +/- 1 ns) indicative of intercalation mode of Et binding were determined . Et binding to qGT was found to be cooperative with corresponding coefficient omega = 3.9 +/- 0.1 and the binding constant Kappa = (6.4 +/- 0.1).10(4) M(-1) . The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of innerspaces between G-quartets (eight in our case) . The data conform to the model of Et association with GT-quadruplex suggested earlier {O . F . Borisova et al . Mol . Biol . (Russ) 35, 732-739 (2001)} . The anticooperative type of Et binding was observed in case of hp-GT ps-DNA, with the maximum number of bound Et molecules, N = 4 / 5, and the association constant Kappa = (1.5 +/- 0.1).10(5) M(-1) . Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.

J Agric Food Chem, 2003 May 21, 51(11), 3222 - 7
Determination of thiamine and its esters in beers and raw materials used for their manufacture by liquid chromatography with postcolumn derivatization; Vinas P et al.; Thiamine and its mono- and pyrophosphate esters were determined in beer and the raw materials used for its manufacture (brewer's yeast, malt, raw grain, and hops) after separation using reversed-phase liquid chromatography . The method used fluorescence detection and a new amide-based stationary phase, which avoids the need to form ion pairs, leading to narrower peaks and a simpler mobile phase . Analyses were performed by isocratic elution with a phosphate buffer mobile phase and using a postcolumn derivatization system based on the oxidation of thiamine to fluorescent thiochrome with potassium ferricyanide in alkaline solution . Only thiamine was found in the beers and raw products, especially in brewer's yeast and malt . A stability study pointed to a faster decrease in the thiamine content of samples stored at room temperature and in sunlight.

Mol Plant Microbe Interact, 2003 May, 16(5), 405 - 10
Two potato proteins, including a novel RING finger protein (HIP1), interact with the potyviral multifunctional protein HCpro; Guo D et al.; Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins . In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system . Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used . Both proteins are encoded by single-copy genes in the potato genome . Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein . The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.

Oncogene, 2003 May 8, 22(18), 2836 - 41
The adenovirus E1A oncoprotein recruits the cellular TRRAP/GCN5 histone acetyltransferase complex; Lang SE et al.; The adenovirus E1A oncoprotein stimulates cell growth and inhibits differentiation by deregulating the normal transcription program via interaction with positive and negative cellular effectors . E1A associates with transcriptional regulatory complexes containing p400 and TRRAP involved in chromatin remodeling and decondensation . TRRAP is a component of three distinct human histone acetyltransferase (HAT) complexes: the TIP60 complex and complexes containing GCN5 or PCAF . We demonstrate here that E1A binds a TRRAP complex that contains the GCN5 acetyltransferase during a normal adenovirus infection . E1A binds GCN5 and TRRAP in vivo early after virus infection . E1A is associated with significant HAT activity in vitro that is partly attributable to GCN5 . E1A represses c-Myc- and E2F-1-directed transcriptional activation in vivo by sequestering GCN5 and/or TRRAP . Our results demonstrate that E1A distinctly binds TRRAP/GCN5, p300/CBP and PCAF HAT complexes . Through interactions with multiple HAT complexes, E1A may deregulate cellular transcription programs and facilitate infection by recruiting functional HAT coactivators to viral and cellular promoter regions.

Oncogene, 2003 May 8, 22(18), 2772 - 81
Random mutagenesis of PDZ(Omi) domain and selection of mutants that specifically bind the Myc proto-oncogene and induce apoptosis; Junqueira D et al.; Omi is a mammalian serine protease that is localized in the mitochondria and released to the cytoplasm in response to apoptotic stimuli . Omi induces cell death in a caspase-dependent manner by interacting with the X-chromosome linked inhibitor of apoptosis protein, as well as in a caspase-independent way that relies on its proteolytic activity . Omi is synthesized as a precursor polypeptide and is processed to an active serine protease with a unique PDZ domain . PDZ domains recognize the extreme carboxyl terminus of target proteins . Internal peptides that are able to fold into a beta-finger are also reported to bind some PDZ domains . Using a modified yeast two-hybrid system, PDZ(Omi) mutants were isolated by their ability to bind the carboxyl terminus of human Myc oncoprotein in yeast as well as in mammalian cells . One such PDZ(m) domain (PDZ-M1), when transfected into mammalian cells, was able to bind to endogenous Myc protein and induce cell death . PDZ-M1-induced apoptosis was entirely dependent on the presence of Myc protein and was not observed when c-myc null fibroblasts were used . Our studies indicate that the PDZ domain of Omi can provide a prototype that could easily be exploited to target specifically and inactivate oncogenes by binding to their unique carboxyl terminus.

J Biol Chem, 2003 Aug 15, 278(33), 30896 - 904 Epub 2003 May 12.
Ybp1 is required for the hydrogen peroxide-induced oxidation of the Yap1 transcription factor; Veal EA et al.; We describe the characterization of Ybp1, a novel protein, in Saccharomyces cerevisiae, that is required for the oxidative stress response to peroxides . Ybp1 is required for H2O2-induced expression of the antioxidant encoding gene TRX2 . Our data indicate that the effects of Ybp1 are mediated through the Yap1 transcription factor . Indeed, Ybp1 forms a stress-induced complex with Yap1 in vivo and stimulates the nuclear accumulation of Yap1 in response to H2O2 but not in response to the thiol-oxidizing agent diamide . The H2O2-induced nuclear accumulation of Yap1 is regulated by the oxidation of specific cysteine residues and is dependent on the thiol peroxidase Gpx3 . Our data suggest that Ybp1 is required for the H2O2-induced oxidation of Yap1 and acts in the same pathway as Gpx3 . Consequently, Ybp1 represents a novel class of stress regulator of Yap1 . These data have important implications for the regulation of protein oxidation and stress responses in eukaryotes.

EMBO J, 2003 May 15, 22(10), 2526 - 35
Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication; Hashimoto Y et al.; Fission yeast Cut5/Rad4 and its budding yeast homolog Dpb11 are required for both DNA replication and the S-phase checkpoint . Here, we have investigated the role of the Xenopus homolog of Cut5 in the initiation of DNA replication using Xenopus egg extracts . Xenopus Cut5, which shows sequence similarity to DmMus101 and HsTopBP1, is essential for DNA replication in the egg extracts . It is required for the chromatin binding of Cdc45 and DNA polymerases, but not for the formation of pre-replicative complexes or the elongation stage of DNA replication . The chromatin binding of Cut5 consists of two distinct modes . S-phase cyclin-dependent kinase (S-CDK)-independent binding is sufficient for DNA replication while S-CDK-dependent binding is dispensable . Further, S-CDK acts after the chromatin binding of Cut5 and before the binding of Cdc45 . These results demonstrate that the chromatin binding of Cut5 is required for the action of S-CDK, which in turn triggers the formation of pre-initiation complexes of DNA replication.

EMBO J, 2003 May 15, 22(10), 2433 - 42
Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II; Alepuz PM et al.; In budding yeast, the mitogen-activated protein kinase (MAPK) Hog1 coordinates the transcriptional program required for cell survival upon osmostress . The Hot1 transcription factor acts downstream of the MAPK and regulates a subset of Hog1-responsive genes . In response to high osmolarity, Hot1 targets Hog1 to specific osmostress-responsive promoters . Here, we show that assembly of the general transcription machinery at Hot1-dependent promoters depends on the presence of Hot1 and active Hog1 MAPK . Unexpectedly, recruitment of RNA polymerase (Pol) II complex to target promoters does not depend on the phosphorylation of the Hot1 activator by the MAPK . Hog1 interacts with the RNA Pol II and with general components of the transcription machinery . More over, when tethered to a promoter as a LexA fusion protein, Hog1 activates transcription in a stress- regulated manner . Thus, anchoring of active Hog1 to promoters by the Hot1 activator is essential for recruitment and activation of RNA Pol II . The mammalian p38 also interacts with the RNA Pol II, which might suggest a conserved mechanism for regulation of gene expression by SAPKs among eukaryotic cells.

EMBO J, 2003 May 15, 22(10), 2380 - 6
Tom40, the import channel of the mitochondrial outer membrane, plays an active role in sorting imported proteins; Gabriel K et al.; The Translocase of the Outer Mitochondrial membrane (TOM complex) is centred on a channel, created by Tom40, serving as the only means of entry for proteins into the mitochondrion . Proteins destined for internal mitochondrial compartments interact subsequently with one of the two distinct protein Translocases of the Inner Mitochondrial membrane (TIM23 and TIM54 complexes) or follow specialized paths into the intermembrane space . To investigate the sorting of precursor proteins to these various sub-mitochondrial compartments, we created a library of tom40 mutants and screened for alleles selectively corrupt in protein sorting . One of the tom40 mutants, tom40-97, carries a single point mutation (W(243)R) resulting in an ineffective transfer of precursors to the TIM23 complex . There is no defect on transfer of precursors to the TIM54 complex or insertion of proteins into the outer membrane . The Tom40 channel is not a passive pore, but plays an active role in protein sorting for all sub-mitochondrial locations.

Trends Cell Biol, 2003 May, 13(5), 221 - 8
Never say never . The NIMA-related protein kinases in mitotic control; O'Connell MJ et al.; Mitosis sees a massive reorganization of cellular architecture . The microtubule cytoskeleton is reorganized to form a bipolar spindle between duplicated microtubule organizing centers, the chromosomes are condensed, attached to the spindle at their kinetochores, and, through the action of multiple molecular motors, the chromosomes are segregated into two daughter cells . Mitosis also sees a substantial wave of protein phosphorylation, controlling signaling events that coordinate mitotic processes and ensure accurate chromosome segregation . The key switch for the onset of mitosis is the archetypal cyclin-dependent kinase, Cdc2 . Under the direction of Cdc2 is an executive of protein serine/threonine kinases that fall into three families: the Polo kinases, Aurora kinases and the NIMA-related kinases (Nrk) . The latter family has proven the most enigmatic in function, although recent advances from several sources are beginning to reveal a common functional theme.

Biochemistry, 2003 May 20, 42(19), 5600 - 8
High-resolution crystal structures and spectroscopy of native and compound I cytochrome c peroxidase; Bonagura CA et al.; Cytochrome c peroxidase (CCP) is a 32.5 kDa mitochondrial intermembrane space heme peroxidase from Saccharomyces cerevisiae that reduces H(2)O(2) to 2H(2)O by oxidizing two molecules of cytochrome c (cyt c) . Here we compare the 1.2 A native structure (CCP) with the 1.3 A structure of its stable oxidized reaction intermediate, Compound I (CCP1) . In addition, crystals were analyzed by UV-vis absorption and electron paramagnetic resonance spectroscopies before and after data collection to determine the state of the Fe(IV) center and the cationic Trp191 radical formed in Compound I . The results show that X-ray exposure does not lead to reduction of Fe(IV) and only partial reduction of the Trp radical . A comparison of the two structures reveals subtle but important conformational changes that aid in the stabilization of the Trp191 cationic radical in Compound I . The higher-resolution data also enable a more accurate determination of changes in heme parameters . Most importantly, when one goes from resting state Fe(III) to Compound I, the His-Fe bond distance increases, the iron moves into the porphyrin plane leading to shorter pyrrole N-Fe bonds, and the Fe(IV)-O bond distance is 1.87 A, suggesting a single Fe(IV)-O bond and not the generally accepted double bond.

J Cell Biol, 2003 Apr 28, 161(2), 393 - 402
FAK induces expression of Prx1 to promote tenascin-C-dependent fibroblast migration; McKean DM et al.; Fibroblast migration depends, in part, on activation of FAK and cellular interactions with tenascin-C (TN-C) . Consistent with the idea that FAK regulates TN-C, migration-defective FAK-null cells expressed reduced levels of TN-C . Furthermore, expression of FAK in FAK-null fibroblasts induced TN-C, whereas inhibition of FAK activity in FAK-wild-type cells had the opposite effect . Paired-related homeobox 1 (Prx1) encodes a homeobox transcription factor that induces TN-C by interacting with a binding site within the TN-C promoter, and it also promotes fibroblast migration . Therefore, we hypothesized that FAK regulates TN-C by controlling the DNA-binding activity of Prx1 and/or by inducing Prx1 expression . Prx1-homeodomain binding site complex formation observed with FAK-wild-type fibroblasts failed to occur in FAK-null fibroblasts, yet expression of Prx1 in these cells induced TN-C promoter activity . Thus, FAK is not essential for Prx1 DNA-binding activity . However, activated FAK was essential for Prx1 expression . Functionally, Prx1 expression in FAK-null fibroblasts restored their ability to migrate toward fibronectin, in a manner that depends on TN-C . These results appear to be relevant in vivo because Prx1 and TN-C expression levels were reduced in FAK-null embryos . This paper suggests a model whereby FAK induces Prx1, and subsequently the formation of a TN-C-enriched ECM that contributes to fibroblast migration.

Nat Biotechnol, 2003 Jun, 21(6), 697 - 700 Epub 2003 May 12.
Global protein function prediction from protein-protein interaction networks; Vazquez A et al.; Determining protein function is one of the most challenging problems of the post-genomic era . The availability of entire genome sequences and of high-throughput capabilities to determine gene coexpression patterns has shifted the research focus from the study of single proteins or small complexes to that of the entire proteome . In this context, the search for reliable methods for assigning protein function is of primary importance . There are various approaches available for deducing the function of proteins of unknown function using information derived from sequence similarity or clustering patterns of co-regulated genes, phylogenetic profiles, protein-protein interactions (refs . 5-8 and Samanta, M.P . and Liang, S., unpublished data), and protein complexes . Here we propose the assignment of proteins to functional classes on the basis of their network of physical interactions as determined by minimizing the number of protein interactions among different functional categories . Function assignment is proteome-wide and is determined by the global connectivity pattern of the protein network . The approach results in multiple functional assignments, a consequence of the existence of multiple equivalent solutions . We apply the method to analyze the yeast Saccharomyces cerevisiae protein-protein interaction network . The robustness of the approach is tested in a system containing a high percentage of unclassified proteins and also in cases of deletion and insertion of specific protein interactions.

Nat Genet, 2003 Jun, 34(2), 166 - 76
Module networks: identifying regulatory modules and their condition-specific regulators from gene expression data; Segal E et al.; Much of a cell's activity is organized as a network of interacting modules: sets of genes coregulated to respond to different conditions . We present a probabilistic method for identifying regulatory modules from gene expression data . Our procedure identifies modules of coregulated genes, their regulators and the conditions under which regulation occurs, generating testable hypotheses in the form 'regulator X regulates module Y under conditions W' . We applied the method to a Saccharomyces cerevisiae expression data set, showing its ability to identify functionally coherent modules and their correct regulators . We present microarray experiments supporting three novel predictions, suggesting regulatory roles for previously uncharacterized proteins.

J Biol Chem, 2003 Jul 25, 278(30), 28246 - 51 Epub 2003 May 09.
Arabidopsis histone acetyltransferase AtGCN5 regulates the floral meristem activity through the WUSCHEL/AGAMOUS pathway; Bertrand C et al.; Histone acetyltransferases, which are able to acetylate histone and non-histone proteins, play important roles in gene regulation . Many histone acetyltransferases are related to yeast Gcn5, a component of two transcription regulatory complexes SAGA and ADA . In this work, by characterizing a mutation in the Arabidopsis GCN5 gene (AtGCN5) we studied the regulatory function of this gene in controlling floral meristem activity . We show that in addition to pleiotropic effects on plant development, this mutation also leads to the production of terminal flowers . The flowers show homeotic transformations of petals into stamens and sepals into filamentous structures and produce ectopic carpels . The phenotypes correlate to an expansion of the expression domains within floral meristems of the key regulatory genes WUSCHEL (WUS) and AGAMOUS (AG) . These results suggest that AtGCN5 is required to regulate the floral meristem activity through the WUS/AG pathway . This study brings new elements on the elucidation of specific developmental pathways regulated by AtGCN5 and on the control mechanism of meristem regulatory gene expression.

J Biol Chem, 2003 Aug 8, 278(32), 30328 - 38 Epub 2003 May 09.
Roles for sphingolipid biosynthesis in mediation of specific programs of the heat stress response determined through gene expression profiling; Cowart LA et al.; Previous studies have demonstrated roles for de novo production of sphingolipids in Saccharomyces cerevisiae in the regulation of the transient cell cycle arrest and nutrient permease degradation associated with the heat stress response, suggesting multiple functions for yeast sphingolipids in this response . We, therefore, sought to determine the generalized involvement of sphingolipids in the heat stress response by using microarray hybridization of RNA isolated from heat-stressed cultures of the mutant strain lcb1-100, which is unable to produce sphingolipids in response to heat . Approximately 70 genes showed differential regulation during the first 15 min of heat stress in the lcb1-100 strain compared with the wild type strain, indicating a requirement for de novo sphingolipid biosynthesis for proper regulation of these genes during heat stress . Grouping these genes into functional categories revealed several pathways, including some in which sphingolipids were previously suspected to play a role, such as stress response pathways and cell cycle regulation . Hierarchical clustering analysis revealed sphingolipid involvement in regulation of tRNA synthesis and metabolic genes and transporters . Additionally, the microarray results demonstrated novel sphingolipid involvement in transcriptional regulation of pathways of translation and cell wall organization and biogenesis . Our results demonstrate a broad-reaching effect of sphingolipids in the yeast heat stress response and suggest that the mechanism of sphingolipid involvement in several cellular pathways occurs via sphingolipid-mediated regulation of message levels.

BMC Genomics . 2003 May 09;4(1):19.
Development and validation of a T7 based linear amplification for genomic DNA; Liu CL et al.; BACKGROUND: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure . These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation . To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified . Current protocols use PCR-based approaches to amplify in exponential fashion . However, exponential amplification protocols are highly susceptible to bias . Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis . These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail . RESULTS: We have developed a novel linear amplification protocol for genomic DNA . Terminal transferase is used to add polyT tails to the ends of DNA fragments . Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP . Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT) . Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material . In contrast, we find that PCR amplification is biased towards species of greater size . Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach . CONCLUSION: We present a T7-based linear amplification protocol for genomic DNA . Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

Planta, 2003 Aug, 217(4), 547 - 58 Epub 2003 May 09.
Molecular and biochemical characterization of an aminoalcoholphosphotransferase (AAPT1) from Brassica napus: effects of low temperature and abscisic acid treatments on AAPT expression in Arabidopsis plants and effects of over-expression of BnAAPT1 in transgenic Arabidopsis; Qi Q et al.; Aminoalcoholphosphotransferases (AAPT, EC 2.7.8.1 and EC 2.7.8.2) catalyze the transfer of CDP-aminoalcohols to sn-1, 2 diacylglycerol (DAG) to form phosphatidylaminoalcohols with the release of CMP . The Brassica napus L . AAPT1 gene (designated BnAAPT1) was identified from cDNA libraries of seedlings and developing seeds . Functional characterization was accomplished by heterologous expression of BnAAPT1 in a yeast strain deficient in AAPT activities . BnAAPT1 exhibited a greater preference for utilizing CDP-choline as a substrate with Vmax of 35 {14C}phosphatidylcholine nmol h(-1) mg(-1) protein and apparent Km of 32 microM while CDP-ethanolamine had a Vmax of 13 {14C}phosphatidylethanolamine nmol h(-1) mg(-1)protein and an apparent Km of 127 microM . The enzyme was activated by Mg2+, Mn2+ and phospholipid mixtures, and inhibited by Ca2+ . A CDP-alcohol phosphotransferase motif, Asp99-Gly100-(X2)-Ala103-Arg104-(X8)-Gly113-(X3)-Asp117-(X3)-Asp121, was completely conserved in BnAAPT1 and its catalytic role was confirmed by scanning alanine mutagenesis . Over-expression of BnAAPT1 under the control of the double 35S promoter in transgenic Arabidopsis thaliana (L.) Heynh . plants led to elevated levels of the corresponding transcript and enzyme activity . In four of the high over-expression transgenic lines, phospholipid and fatty acid composition analyses revealed that chloroplastidic and extrachloroplastidic membranes isolated from transgenic leaves had about a 25% increase in phosphatidylcholine and in the proportions of polyunsaturated fatty acids {18:2+18:3}, relative to the control . There were also consistent, but small differences observed in the proportions of 18:3 in transgenic green siliques and in 20:1 in mature transgenic seeds of these lines . Induction of Arabidopsis AAPT transcription in response to (+)-abscisic acid and low-temperature treatments, and the cold tolerance in BnAAPT1 transgenic seedlings implies that AAPT may play a role in resistance to damage at low growth temperatures.

J Biol Chem, 2003 Jul 11, 278(28), 25408 - 16 Epub 2003 May 07.
Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1; Fitch MJ et al.; The Saccharomyces cerevisiae Mcm7 protein is a subunit of the presumed heteromeric MCM helicase that melts origin DNA and unwinds replication forks . Previous work showed that Mcm1 binds constitutively to the MCM7 promoter and regulates MCM7 expression . Here, we identify Mcm7 as a novel cofactor of Mcm1 in the regulation of MCM7 expression . Transcription of MCM7 is increased in the mcm7-1 mutant and decreased in the mcm1-1 mutant, suggesting that Mcm7 modulates its own expression in conjunction with Mcm1 . Indeed, Mcm7 stimulates Mcm1 binding to the early cell cycle box upstream of the promoters of MCM7 as well as CDC6 and MCM5 . Whereas Mcm1 binds these promoters constitutively, Mcm7 is recruited during late M phase, consistent with Mcm7 playing a direct role in modulating the periodic expression of early cell cycle genes . The multiple roles of Mcm7 in replication initiation, replication elongation, and autoregulation parallel those of the oncoprotein, the large T-antigen of the SV40 virus.

Dev Cell, 2003 May, 4(5), 727 - 39
Division of the nucleolus and its release of CDC14 during anaphase of meiosis I depends on separase, SPO12, and SLK19; Buonomo SB et al.; Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together . It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules . Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata . The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres . We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle . Separase is also required but surprisingly not its protease activity . It has two mechanistically different roles during meiosis I . Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12delta and slk19delta mutants.

Dev Cell, 2003 May, 4(5), 711 - 26
The Cdc14 phosphatase and the FEAR network control meiotic spindle disassembly and chromosome segregation; Marston AL et al.; During meiosis, DNA replication is followed by two consecutive rounds of chromosome segregation . Cells lacking the protein phosphatase CDC14 or its regulators, SPO12 and SLK19, undergo only a single meiotic division, with some chromosomes segregating reductionally and others equationally . We find that this abnormal chromosome behavior is due to an uncoupling of meiotic events . Anaphase I spindle disassembly is delayed in cdc14-1, slk19Delta, or spo12Delta mutants, but the chromosome segregation cycle continues, so that both meiotic chromosome segregation phases take place on the persisting meiosis I spindle . Our results show that Cdc14, Slk19, and Spo12 are not only required for meiosis I spindle disassembly but also play a pivotal role in establishing two consecutive chromosome segregation phases, a key feature of the meiotic cell cycle.

Biochem J, 2003 Aug 15, 374(Pt 1), 165 - 73
The acetyltransferase 60 kDa trans-acting regulatory protein of HIV type 1-interacting protein (Tip60) interacts with the translocation E26 transforming-specific leukaemia gene (TEL) and functions as a transcriptional co-repressor; Nordentoft I et al.; The translocation E26 transforming-specific (ETS) leukaemia (TEL), alias the ETS variant (ETV6), gene is expressed in most human tissues and encodes a transcriptional repressor . The TEL gene is involved in more than 40 different chromosomal translocations associated with haematological malignancies . As little is known about the function of intact TEL, we searched for TEL-interacting proteins by yeast two-hybrid screening . Among the interacting partners, we identified the histone acetyltransferase protein Tip60 {60 kDa trans-acting regulatory protein of HIV type 1 (Tat)-interacting protein} . The interaction was reproduced in vitro, and in mammalian cells we mapped the interaction regions in TEL to the ETS domain and those in Tip60 to the MYST ('MOZ, Ybf2/Sas3, SAS2 and Tip60', where MOZ stands for male absent on the first, SAS for something about silencing and Ybf2 for identical with SAS2) region . Detailed analysis of the Tip60 MYST domain by introduction of point mutations revealed that an N-terminal C2HC zinc finger was essential for interaction with TEL . Finally, we showed that Tip60 functions in a reporter system as a co-repressor in TEL-mediated transcription repression.

Cell Res, 2003 Apr, 13(2), 69 - 81
Function and regulation of Aurora/Ipl1p kinase family in cell division; Ke YW et al.; During mitosis, the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation, a complex movements orchestrated by mitotic kinases and its effector proteins . Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell . Defects in these processes can lead to aneuploidy or polyploidy . Aurora/Ipl1p family, a class of conserved serine/threonine kinases, plays key roles in chromosome segregation and cytokinesis . This article highlights the function and regulation of Aurora/Ipl1p family in mitosis and provides potential links between aberrant regulation of Aurora/Ipl1p kinases and pathogenesis of human cancer.

Nucleic Acids Res . 2003 May 15;31(10):e56.
Real-time PCR-based method for the estimation of genome sizes; Wilhelm J et al.; The fast and reliable estimation of the genome sizes of various species would allow for a systematic analysis of many organisms and could reveal insights into evolutionary processes . Many methods for the estimation of genome sizes have already been described . The classical methods are based on the determination of the phosphate content in the DNA backbone of total DNA isolated from a defined number of cells or on reassociation kinetics of high molecular weight genomic DNA (c(0)t assay) . More recent techniques employ DNA-specific fluorescent dyes in flow cytometry analysis, image analysis or absorption cytometry after Feulgen staining . The method presented here is based on the absolute quantification of genetic elements in a known amount (mass) of genomic DNA by real-time quantitative PCR . The method was evaluated on three different eukaryotic species, Saccharomyces cerevisiae (12.1 Mb), Xiphophorus maculatus (550 Mb) and Homo sapiens sapiens (2.9 Gb), and found to be fast, highly accurate and reliable.

Nucleic Acids Res, 2003 May 15, 31(10), 2534 - 43
Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array; Ji SJ et al.; Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule . To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver . We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes . cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases . Twenty-nine cDNAs, including a putative vacuolar (H+)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a . fiber cells when compared to that in 0 d.p.a . ovules . Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period . This report constitutes the first systematic analysis of genes involved in cotton fiber development . Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.

Nucleic Acids Res, 2003 May 15, 31(10), 2475 - 82
The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation; Schaft D et al.; Existing evidence indicates that SET2, the histone 3 lysine 36 methyltransferase of Saccharomyces cerevisiae, is a transcriptional repressor . Here we show by five main lines of evidence that SET2 is involved in transcriptional elongation . First, most, if not all, subunits of the RNAP II holoenzyme co-purify with SET2 . Second, all of the co-purifying RNAP II subunit, RPO21, was phosphorylated at serines 5 and 2 of the C-terminal domain (CTD) tail, indicating that the SET2 association is specific to either the elongating or SSN3 repressed forms (or both) of RNAP II . Third, the association of SET2 with CTD phosphorylated RPO21 remained in the absence of ssn3 . Fourth, in the absence of ssn3, mRNA production from gal1 required SET2 . Fifth, SET2 was detected on gal1 by in vivo crosslinking after, but not before, the induction of transcription . Similarly, SET2 physically associated with the transcribed region of pdr5 but was not detected on gal1 or pdr5 promoter regions . Since SET2 is also a histone methyltransferase, these results suggest a role for histone 3 lysine 36 methylation in transcriptional elongation.

J Biol Chem, 2003 Jul 25, 278(30), 28324 - 34 Epub 2003 May 07.
Mutagenesis suggests several roles of Snu114p in pre-mRNA splicing; Bartels C et al.; Snu114p, a yeast U5 small nuclear ribonucleoprotein (snRNP) homologous to the ribosomal GTPase EF-2, was recently found to play a part in the dissociation of U4 small nuclear RNA (snRNA) from U6 snRNA . Here, we show that purified Snu114p binds GTP specifically . To test the possibility that binding and hydrolysis of GTP by Snu114p are required to stimulate the unwinding of U4 from U6, we produced several mutations of Snu114p . Residues whose mutations led to lethal phenotypes were all clustered in the P loop and in the guanine-ring binding sequence (NKXD) of the G domain, which in elongation factor-G is required for the binding and hydrolysis of GTP . An arginine residue in domain II, which in EF-G forms a salt bridge with a residue of the G domain, when mutated in Snu114p (R487E), led to a temperature-sensitive phenotype . The substitution D271N in the NKXD sequence is predicted to bind XTP instead of GTP . Spliceosomes containing this mutant, isolated by affinity chromatography after heat treatment, retained U4 snRNA paired with the U6 snRNA . U4 snRNA was released efficiently only when these arrested spliceosomes were reactivated by lowering the temperature in the presence of a mixture of ATP and XTP . Because non-hydrolyzable XTP analogues did not consent the release of U4, we conclude that the release requires hydrolysis of XTP . This suggests that Snu114p needs GTP to influence, directly or indirectly, the unwinding of U4 from U6 . An additional role for Snu114p is also demonstrated: after growth of the D271N and R487E strains at high temperatures, we observed decreased levels of the U5 and the U4/U6.U5 snRNPs . This indicates that, before splicing, Snu114p plays a part in the assembly of both particles.

Eur J Cancer, 2003 May, 39(8), 1165 - 75
Histone acetylation-mediated regulation of genes in leukaemic cells; Chambers AE et al.; Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression . Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays . Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR) . A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression . A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer . The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader-Willi syndrome.

Yeast, 2003 May, 20(7), 645 - 52
Gene regulation in response to protein disulphide isomerase deficiency; Norgaard P et al.; We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency . Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue, revealed a cis-acting element responding to deficiency of protein disulphide isomerase activity (designated CERP) . The presence of the sequence element is necessary and sufficient for the upregulation in response to disulphide isomerase deficiency, as measured by a minimal promoter containing the CERP element . The sequence (GACACG) does not resemble the unfolded protein response element . It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes .

Yeast, 2003 May, 20(7), 625 - 32
Guanidine reduces stop codon read-through caused by missense mutations in SUP35 or SUP45; Bradley ME et al.; Sup35 and Sup45 are essential protein components of the Saccharomyces cerevisiae translation termination factor . Yeast cells harbouring the {PSI(+)} prion form of Sup35 have impaired stop codon recognition (nonsense suppression) . It has long been known that the {PSI(+)} prion is not stably transmitted to daughter cells when yeast are grown in the presence of mM concentrations of guanidine hydrochloride (GuHCl) . In this paper, Mendelian suppressor mutations whose phenotypes are likewise hidden during growth in the presence of millimolar GuHCl are described . Such GuHCl-remedial Mendelian suppressors were selected under conditions where {PSI(+)} appearance was limiting, and were caused by missense mutations in SUP35 or SUP45 . Clearly, anti-suppression caused by growth in the presence of GuHCl is not sufficient to distinguish missense mutations in SUP35 or SUP45, from {PSI(+)} . However, the Mendelian and prion suppressors can be distinguished by subsequent growth in the absence of GuHCl, where only the nonsense suppression caused by the {PSI(+)} prion remains cured . Recent reports indicate that GuHCl blocks the inheritance of {PSI(+)} by directly inhibiting the activity of the protein remodelling factor Hsp104, which is required for the transmission of {PSI(+)} from mother to daughter cells . However, the nonsense suppressor activity caused by the GuHCl-remedial sup35 or sup45 suppressors does not require Hsp104 . Thus, GuHCl must anti-suppress the sup35 and sup45 mutations via an in vivo target distinct from Hsp104 .

Acta Neuropathol (Berl), 2003 Jun, 105(6), 593 - 602 Epub 2003 Mar 05.
Cytokines in sural nerve biopsies from inflammatory and non-inflammatory neuropathies; Lindenlaub T et al.; Proinflammatory cytokines are supposed to play a major role in the pathophysiology of vasculitis and in the development of neuropathic pain . Here we studied the cytokine expression in sural nerve biopsy specimens from patients with vasculitic and other inflammatory and non-inflammatory neuropathies, and investigated whether an increased cytokine expression was correlated with the presence of neuropathic pain . We used immunohistochemistry including double labeling and morphometry to localize and quantify the expression of interleukin-1 beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF) in sural nerve biopsy samples of 41 patients with vasculitic neuropathy (VANP), chronic inflammatory demyelinating neuropathy (CIDP), non-inflammatory chronic axonal neuropathy (CANP), and 3 controls . Overall cytokine immunoreactivity was highest in VANP, less strong in CIDP and lowest in CANP . Cytokine immunoreactivity was directly correlated with the degree of axonal degeneration, endoneurial macrophages and epineurial T cells . In VANP and CANP, a higher cytokine content was associated with neuropathic pain.

Cell Cycle, 2003 May-Jun, 2(3), 238 - 45
Loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development; Gascoyne DM et al.; Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a multiple step process characterized by distinctive proliferative stages . Chromosomal aberrations are identified early in this tumorigenic pathway, however, the mechanism that originates them is unknown . Using a functional assay, we investigated the status of the mitotic spindle cell cycle checkpoint (MSCCC) that regulates the metaphase to anaphase transition, in cells representing different stages of fibrosarcoma progression . Loss of MSCCC activity was apparent in mild fibromatosis and completely abolished in aggressive fibromatosis and fibrosarcoma lesions . This altered MSCCC status was confirmed biochemically by deregulated expression of Cks1 protein and unscheduled cyclin B metabolism . Immunoprecipitation and sequencing analyses indicated that mutation of p53 was not required for the abrogation of the MSCCC . These results demonstrate that loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development . To our knowledge, these studies constitute the first report of a transition in MSCCC activity in a tumorigenesis model.

J Cell Sci, 2003 Jun 15, 116(Pt 12), 2431 - 42 Epub 2003 May 06.
Increased ploidy and KAR3 and SIR3 disruption alter the dynamics of meiotic chromosomes and telomeres; Trelles-Sticken E et al.; We investigated the sequence of chromosomal events during meiotic prophase in haploid, diploid and autotetraploid SK1 strains of Saccharomyces cerevisiae . Using molecular cytology, we found that meiosis-specific nuclear topology (i.e . dissolution of centromere clustering, bouquet formation and meiotic divisions) are significantly delayed in polyploid SK1 meiosis . Thus, and in contrast to the situation in plants, an increase in ploidy extends prophase I in budding yeast . Moreover, we found that bouquet formation also occurs in haploid and diploid SK1 meiosis deficient in the telomeric heterochromatin protein Sir3p . Diploid sir3Delta SK1 meiosis showed pleiotropic defects such as delayed centromere cluster resolution in a proportion of cells and impeded downstream events (i.e . bouquet formation, homologue pairing and meiotic divisions) . Meiotic telomere clustering occurred in diploid and haploid sir3Delta strains . Using the haploid system, we further show that a bouquet forms at the kar3Delta SPB . Comparison of the expression of meiosis-specific Ndj1p-HA and Zip1p in haploid control and kar3Delta time courses revealed that fewer cells enter the meiotic cycle in absence of Kar3p . Elevated frequencies of bouquets in kar3Delta haploid meiosis suggest a role for Kar3p in regulation of telomere dynamics.

J Biol Chem, 2003 Jul 25, 278(30), 27804 - 10 Epub 2003 May 06.
Giardia lamblia RNA polymerase II: amanitin-resistant transcription; Seshadri V et al.; Giardia lamblia is an early branching eukaryote, and although distinctly eukaryotic in its cell and molecular biology, transcription and translation in G . lamblia demonstrate important differences from these processes in higher eukaryotes . The cyclic octapeptide amanitin is a relatively selective inhibitor of eukaryotic RNA polymerase II (RNAP II) and is commonly used to study RNAP II transcription . Therefore, we measured the sensitivity of G . lamblia RNAP II transcription to alpha-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin . In contrast, 50 microg/ml amanitin inhibits 85% of RNAP III transcription activity using leucyl-tRNA as a template . To better understand transcription in G . lamblia, we identified 10 of the 12 known eukaryotic rpb subunits, including all 10 subunits that are required for viability in Saccharomyces cerevisiae . The amanitin motif (amanitin binding site) of Rpb1 from G . lamblia has amino acid substitutions at six highly conserved sites that have been associated with amanitin resistance in other organisms . These observations of amanitin resistance of Giardia RNA polymerase II support previous proposals of the mechanism of amanitin resistance in other organisms and provide a molecular framework for the development of novel drugs with selective activity against G . lamblia.

Genome Biol . 2003;4(5):R34 . Epub 2003 Apr 25.
Clustering gene-expression data with repeated measurements; Yeung KY et al.; Clustering is a common methodology for the analysis of array data, and many research laboratories are generating array data with repeated measurements . We evaluated several clustering algorithms that incorporate repeated measurements, and show that algorithms that take advantage of repeated measurements yield more accurate and more stable clusters . In particular, we show that the infinite mixture model-based approach with a built-in error model produces superior results.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5760 - 5 Epub 2003 May 05.
The structure of nonvertebrate actin: implications for the ATP hydrolytic mechanism; Vorobiev S et al.; The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 A . These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively . The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies . This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization . The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5688 - 93 Epub 2003 May 05.
NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1; Nguyen BD et al.; FCP1 {transcription factor IIF (TFIIF)-associated carboxyl-terminal domain (CTD) phosphatase} is the only identified phosphatase specific for the phosphorylated CTD of RNA polymerase II (RNAP II) . The phosphatase activity of FCP1 is enhanced in the presence of the large subunit of TFIIF (RAP74 in humans) . It has been demonstrated that the CTD of RAP74 (cterRAP74; residues 436-517) directly interacts with the highly acidic CTD of FCP1 (cterFCP; residues 879-961 in human) . In this manuscript, we have determined a high-resolution solution structure of a cterRAP74cterFCP complex by NMR spectroscopy . Interestingly, the cterFCP protein is completely disordered in the unbound state, but forms an alpha-helix (H1'; E945-M961) in the complex . The cterRAP74cterFCP binding interface relies extensively on van der Waals contacts between hydrophobic residues from the H2 and H3 helices of cterRAP74 and hydrophobic residues from the H1' helix of cterFCP . The binding interface also contains two critical electrostatic interactions involving aspartic acid residues from H1' of cterFCP and lysine residues from both H2 and H3 of cterRAP74 . There are also three additional polar interactions involving highly conserved acidic residues from the H1' helix . The cterRAP74cterFCP complex is the first high-resolution structure between an acidic residue-rich domain from a holoenzyme-associated regulatory protein and a general transcription factor . The structure defines a clear role for both hydrophobic and acidic residues in proteinprotein complexes involving acidic residue-rich domains in transcription regulatory proteins.

J Cell Biol, 2003 May 12, 161(3), 535 - 45 Epub 2003 May 05.
A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase; Gromley A et al.; Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function . They also participate in cytokinesis and cell cycle progression in ways that are poorly understood . Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression . Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges . Most cells continue to cycle, reenter mitosis, and form multicellular syncytia . Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis . At later times, viable cells arrest in G1/G0 . The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis . The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway . We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.

Cell, 2003 May 2, 113(3), 395 - 404
Program-specific distribution of a transcription factor dependent on partner transcription factor and MAPK signaling; Zeitlinger J et al.; Specialized gene expression programs are induced by signaling pathways that act on transcription factors . Whether these transcription factors can function in multiple developmental programs through a global switch in promoter selection is not known . We have used genome-wide location analysis to show that the yeast Ste12 transcription factor, which regulates mating and filamentous growth, is bound to distinct program-specific target genes dependent on the developmental condition . This condition-dependent distribution of Ste12 requires concurrent binding of the transcription factor Tec1 during filamentation and is differentially regulated by the MAP kinases Fus3 and Kss1 . Program-specific distribution across the genome may be a general mechanism by which transcription factors regulate distinct gene expression programs in response to signaling.

Am J Hum Genet, 2003 Jun, 72(6), 1359 - 69 Epub 2003 May 02.
Cohen syndrome is caused by mutations in a novel gene, COH1, encoding a transmembrane protein with a presumed role in vesicle-mediated sorting and intracellular protein transport; Kolehmainen J et al.; Cohen syndrome is an uncommon autosomal recessive disorder whose diagnosis is based on the clinical picture of nonprogressive psychomotor retardation and microcephaly, characteristic facial features, retinal dystrophy, and intermittent neutropenia . We have refined the critical region on chromosome 8q22 by haplotype analysis, and we report the characterization of a novel gene, COH1, that is mutated in patients with Cohen syndrome . The longest transcript (14,093 bp) is widely expressed and is transcribed from 62 exons that span a genomic region of approximately 864 kb . COH1 encodes a putative transmembrane protein of 4,022 amino acids, with a complex domain structure . Homology to the Saccharomyces cerevisiae VPS13 protein suggests a role for COH1 in vesicle-mediated sorting and transport of proteins within the cell.

Science, 2003 May 2, 300(5620), 805 - 8
Decapping and decay of messenger RNA occur in cytoplasmic processing bodies; Sheth U et al.; A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay . We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies) . First, proteins that activate or catalyze decapping are concentrated in P bodies . Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies . Finally, mRNA degradation intermediates are localized to P bodies . These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.

J Biol Chem, 2003 Jul 11, 278(28), 25628 - 36 Epub 2003 May 01.
Physical and genetic interactions of cytosolic malate dehydrogenase with other gluconeogenic enzymes; Gibson N et al.; A truncated form (deltanMDH2) of yeast cytosolic malate dehydrogenase (MDH2) lacking 12 residues on the amino terminus was found to be inadequate for gluconeogenic function in vivo because the mutant enzyme fails to restore growth of a Deltamdh2 strain on minimal medium with ethanol or acetate as the carbon source . The DeltanMDH2 enzyme was also previously found to be refractory to the rapid glucose-induced inactivation and degradation observed for authentic MDH2 . In contrast, kinetic properties measured for purified forms of MDH2 and deltanMDH2 enzymes are very similar . Yeast two-hybrid assays indicate weak interactions between MDH2 and yeast phosphoenolpyruvate carboxykinase (PCK1) and between MDH2 and fructose-1,6-bisphosphatase (FBP1) . These interactions are not observed for deltanMDH2, suggesting that differences in cellular function between authentic and truncated forms of MDH2 may be related to their ability to interact with other gluconeogenic enzymes . Additional evidence was obtained for interaction of MDH2 with PCK1 using Hummel-Dreyer gel filtration chromatography, and for interactions of MDH2 with PCK1 and with FBP1 using surface plasmon resonance . Experiments with the latter technique demonstrated a much lower affinity for interaction of deltanMDH2 with PCK1 and no interaction between deltanMDH2 and FBP1 . These results suggest that the interactions of MDH2 with other gluconeogenic enzymes are dependent on the amino terminus of the enzyme, and that these interactions are important for gluconeogenic function in vivo.

J Biol Chem, 2003 Jul 18, 278(29), 26526 - 32 Epub 2003 Apr 30.
Nonpolar thymine isosteres in the Ty3 polypurine tract DNA template modulate processing and provide a model for its recognition by Ty3 reverse transcriptase; Lener D et al.; Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (+) U3 RNA and DNA by their cognate reverse transcriptases (RTs) . In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (+)-RNA and failure to release (+)-DNA from the primer . Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (-)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F) . We observed a consistent spatial correlation between the site of T --> F substitution and enhanced ribonuclease H (RNase H) activity approximately 12-13 bp downstream . In the most pronounced case, dual T --> F substitution at PPT positions -1/-2 redirects RNase H cleavage almost exclusively to the novel site . The structural features of this unusual base suggest that its insertion into the Ty3 PPT (-)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT approximately 12-13 bp from its RNase H catalytic center . A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex . The spatial relationship derived from T --> F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5' vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper.

J Biol Chem, 2003 Jul 11, 278(28), 25331 - 40 Epub 2003 May 01.
Nuclear accumulation of the small GTPase Gsp1p depends on nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the acetyl-CoA carboxylase Acc1p; Gao H et al.; The small GTPase Ran/Gsp1p plays an essential role in nuclear trafficking of macromolecules, as Ran/Gsp1p regulates many transport processes across the nuclear pore complex (NPC) . To determine the role of nucleoporins in the generation of the nucleocytoplasmic Gsp1p concentration gradient, mutations in various nucleoporin genes were analyzed in the yeast Saccharomyces cerevisiae . We show that the nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the enzyme acetyl-CoA carboxylase (MTR7) control the distribution and cellular concentration of Gsp1p . At the restrictive temperature the reporter protein GFP-Gsp1p, which is too large to diffuse across the nuclear envelope, fails to concentrate in nuclei of nup133delta, rat2-1, nup85delta, nic96deltaC, and mtr7-1 cells, demonstrating that GFP-Gsp1p nuclear import is deficient . In addition, the concentration of Gsp1p is severely reduced in mutants nup133Delta and mtr7-1 under these conditions . We have now identified the molecular mechanisms that contribute to the dissipation of the Gsp1p concentration gradient in these mutants . Loss of the Gsp1p gradient in nup133delta and rat2-1 can be explained by reduced binding of the Gsp1p nuclear carrier Ntf2p to NPCs . Likewise, nup85delta cells that mislocalize GFP-Gsp1p at the permissive as well as non-permissive temperature have a diminished association of Ntf2p-GFP with nuclear envelopes under both conditions . Moreover, under restrictive conditions Prp20p, the guanine nucleotide exchange factor for Gsp1p, mislocalizes to the cytoplasm in nup85delta, nic96deltaC, and mtr7-1 cells, thereby contributing to a collapse of the Gsp1p gradient . Taken together, components of the NPC subcomplex containing Rat2p/Nup120p, Nup133p, and Nup85p, in addition to proteins Nic96p and Mtr7p, are shown to be crucial for the formation of a nucleocytoplasmic Gsp1p gradient.

J Biol Chem, 2003 Aug 8, 278(32), 29400 - 9 Epub 2003 May 02.
Sigma 1- and mu 1-Adaptin homologues of Leishmania mexicana are required for parasite survival in the infected host; Gokool S; The sorting of membrane-bound proteins from the trans-Golgi network to lysosomal/endosomal compartments is achieved by preferential inclusion into clathrin-coated vesicles . Contained within the cytoplasmic domains of such proteins, specific sequence motifs have been identified (tyrosine-based and/or di-leucine-based) that are essential for targeting and are recognized by a family of heterotetrameric adaptor complexes, which then recruit clathrin . These cytosolic protein complexes, which have been found in a wide variety of higher eukaryotic organisms, are essential for the development of multicellular organisms . In trypanosomatids, the adaptin-mediated sorting of proteins is largely uncharacterized . In order to identify components of the adaptor-complex machinery, this study reports the cloning and characterization of sigma 1- and mu 1-adaptin gene homologues from the eukaryotic protozoan parasite, Leishmania mexicana . Generation of sigma 1- and mu 1-adaptin gene deletion mutants shows that these promastigote parasites are viable in culture, but are unable to establish infection of macrophages or mice, indicating that adaptin function is crucial for pathogenesis in these unicellular organisms.

J Biol Chem, 2003 Jul 11, 278(28), 25688 - 99 Epub 2003 May 02.
The Vid vesicle to vacuole trafficking event requires components of the SNARE membrane fusion machinery; Brown CR et al.; The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is targeted to Vid vesicles when glucose-starved cells are replenished with glucose . Vid vesicles then deliver FBPase to the vacuole for degradation . A modified alkaline phosphatase assay was developed to study the trafficking of Vid vesicles to the vacuole . For this assay, FBPase was fused with a truncated form of alkaline phosphatase . Under in vivo conditions, FBPase-delta60Pho8p was targeted to the vacuole via Vid vesicles, and it exhibited Pep4p- and Vid24p-dependent alkaline phosphatase activation . Vid vesicle-vacuole targeting was reconstituted using Vid vesicles that contained FBPase-delta60Pho8p . These vesicles were incubated with vacuoles in the presence of cytosol and an ATP-regenerating system . Under in vitro conditions, alkaline phosphatase was also activated in a Pep4p- and Vid24p-dependent manner . The GTPase Ypt7p was identified as an essential component in Vid vesicle-vacuole trafficking . Likewise, a number of v-SNAREs (Ykt6p, Nyv1p, Vti1p) and homotypic fusion vacuole protein sorting complex family members (Vps39p and Vps41p) were required for the proper function of Vid vesicles . In contrast, the t-SNARE Vam3p was a necessary vacuolar component . Vid vesicle-vacuole trafficking exhibits characteristics similar to heterotypic membrane fusion events.

Genes Dev, 2003 May 1, 17(9), 1141 - 52
A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication; Kubota Y et al.; We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins . These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts . The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK . The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5 . On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery . Electron microscopy further reveals that the complex has a ring-like structure . These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.

FEBS Lett, 2003 May 8, 542(1-3), 125 - 31
Mining gene expression data using a novel approach based on hidden Markov models; Ji X et al.; In this work we have developed a new framework for microarray gene expression data analysis . This framework is based on hidden Markov models . We have benchmarked the performance of this probability model-based clustering algorithm on several gene expression datasets for which external evaluation criteria were available . The results showed that this approach could produce clusters of quality comparable to two prevalent clustering algorithms, but with the major advantage of determining the number of clusters . We have also applied this algorithm to analyze published data of yeast cell cycle gene expression and found it able to successfully dig out biologically meaningful gene groups . In addition, this algorithm can also find correlation between different functional groups and distinguish between function genes and regulation genes, which is helpful to construct a network describing particular biological associations . Currently, this method is limited to time series data . Supplementary materials are available at http://www.bioinfo.tsinghua.edu.cn/~rich/hmmgep_supp/.

FEBS Lett, 2003 May 8, 542(1-3), 119 - 24
Solution structure and p43 binding of the p38 leucine zipper motif: coiled-coil interactions mediate the association between p38 and p43; Ahn HC et al.; p38, which has been suggested to be a scaffold protein for the assembly of a macromolecular tRNA synthetase complex, contains a leucine zipper-like motif . To understand the importance of the leucine zipper-like motif of p38 (p38LZ) in macromolecular assembly, the p38LZ solution structure was investigated by circular dichroism and nuclear magnetic resonance spectroscopy . The solution structure of p38LZ showed an amphipathic alpha-helical structure and characteristics similar to a coiled-coil motif . The protein-protein interaction mediated by p38LZ was examined by an in vitro binding assay . The p43 protein, another non-synthetase component of the complex, could bind to p38LZ via its N-terminal domain, which is also predicted to have a potential coiled-coil motif . Thus, we propose that the p38-p43 complex would be formed by coiled-coil interactions, and the formation of the binary complex would facilitate the macromolecular assembly of aminoacyl-tRNA synthetases.

FEBS Lett, 2003 May 8, 542(1-3), 100 - 4
Identification of primula fatty acid delta 6-desaturases with n-3 substrate preferences; Sayanova OV et al.; Fatty acid Delta(6)-desaturation, the first committed step in C(20) polyunsaturated fatty acid biosynthesis, is generally considered not to discriminate between n-3 and n-6 substrates . We previously identified higher plant species that showed preferential Delta(6)-desaturation of n-3 C(18) fatty acid substrates . A polymerase chain reaction-based approach was used to isolate 'front-end' cytochrome b(5) fusion desaturases from Primula vialii Franchet and Primula farinosa L . Functional analysis in Saccharomyces cerevisiae identified fatty acid Delta(6)-desaturases with a strong specificity for the n-3 substrate alpha-linolenic acid (18:3 Delta(9,12,15)) . These results indicate that the accumulation of octadecatetraenoic acid (18:4 Delta(6,9,12,15)) in planta is due to the activity of a novel n-3-specific fatty acid Delta(6)-desaturase.

Arch Biochem Biophys, 2003 May 15, 413(2), 262 - 70
Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase; Jennewein S et al.; The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group . The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases . The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene . One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene . Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM . Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures . Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.

Bioorg Chem, 2003 Apr, 31(2), 109 - 21
Synthesis and biological evaluation of the disulfide form of the glutathione analogue gamma-(L-glutamyl)-L-cysteinyl-L-aspartyl-L-cysteine; Cacciatore I et al.; By using the chain to chain mode of cyclization the title glutathione analogue (4), containing the 11-membered disulfide ring replacing the native -Cys-Gly fragment, has been synthesized and characterized together with its reduced dithiol form gamma-Glu-Cys-Asp-Cys (5) . The activity of (4) with gamma-glutamyl-transferase and glutathione reductase has been evaluated and compared with those of the two conformationally restricted glutathione analogues (2) and (3) previously reported.

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 362 - 8 Epub 2003 May 01.
Differential response to low temperature of two Delta6 fatty acid desaturases from Mucor circinelloides; Michinaka Y et al.; A recently identified Delta6 fatty acid desaturase in Mucor rouxii shows a low sequence homology (approximately 24% at the amino acid level) to that isolated from Mortierella alpina, but is phylogenetically closer to a plant enzyme, suggesting the occurrence of Delta6 desaturase isozymes in Mucorales molds . In the present study, two types of Delta6 desaturases, mcD6-1 ( Mo . alpina type) and mcD6-2 ( M . rouxii type), were cloned from Mucor circinelloides . When the cloned genes were expressed in the yeast Saccharomyces cerevisiae in the presence of a linoleic acid substrate (C18:2Delta9, 12), a newly generated gamma-linolenic acid (C18:3Delta6, 9, 12) was detected in the cells, which confirmed the suspected enzymatic function of the recombinant protein . This is the first report of Delta6 desaturase isozymes present in one organism . Northern analysis demonstrated that the amount of mcD6-2 mRNA was less than half of that of mcD6-1 mRNA in cells grown at 28 degrees C . However, upon cultivation of the cells at 15 degrees C for 0.5-1 h, mcD6-2 mRNA rapidly increased by up to 1.5-fold and then gradually decreased . By contrast, mcD6-1 transcripts levels did not fluctuate significantly for 1 h after the temperature shift, but declined by 75% over the next 2 h . The gamma-linolenic acid content in total fatty acid from M . circinelloides decreased at 28 degrees C, but was maintained at approximately 30% at 15 degrees C . These data suggest that Delta6 desaturase isozymes play physiologically distinct roles in the maintenance of cellular lipids and adaptation to low temperature.

Nat Rev Genet, 2003 May, 4(5), 391 - 7
Mitochondrial gene history and mRNA localization: is there a correlation?
Karlberg EO, Andersson SG.
Phylogenetic studies of the yeast mitochondrial proteome have shown a complex evolutionary scenario, in which proteins of bacterial origin form complexes with proteins of eukaryotic origin . Exciting new results from whole-genome microarray studies of subcellular mRNA localizations have shown that mRNAs that are of putative bacterial origin are mainly translated on polysomes that are associated with the mitochondrion, whereas those of eukaryotic origin are generally translated on free cytosolic polysomes . Understanding these newly discovered relationships promises insights into old questions about organelle origins and mRNA localization in the eukaryotic cell.

Genome Res, 2003 May, 13(5), 883 - 95
Discovering novel cis-regulatory motifs using functional networks; Ettwiller LM et al.; We combined functional information such as protein-protein interactions or metabolic networks with genome information in Saccharomyces cerevisiae to predict cis-regulatory motifs in the upstream region of genes . We developed a new scoring metric combining these two information sources and used this metric in motif discovery . To estimate the statistical significance of this metric, we used brute-force randomization, which shows a consistent well-behaved trend . In contrast, real data showed complex nonrandom behavior . With conservative parameters we were able to find 42 degenerate motifs (that touch 40% of yeast genes) based on 647 original patterns, five of which are well known . Some of these motifs also show limited spatial position in the promoter, indicative of a true motif . We also tested the metric on other known motifs and show that this metric is a good discriminator of real motifs . As well as a pragmatic motif discovery method, with many applications beyond this work, these results also show that interacting proteins are often coordinated at the level of transcription, even in the absence of obvious coregulation in gene expression data sets.

EMBO J, 2003 May 1, 22(9), 2274 - 83
Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells; Heidenreich E et al.; Resting cells experience mutations without apparent external mutagenic influences . Such DNA replication-independent mutations are suspected to be a consequence of processing of spontaneous DNA lesions . Using experimental systems based on reversions of frameshift alleles in Saccharomyces cerevisiae, we evaluated the impact of defects in DNA double-strand break (DSB) repair on the frequency of replication-independent mutations . The deletion of the genes coding for Ku70 or DNA ligase IV, which are both obligatory constituents of the non-homologous end joining (NHEJ) pathway, each resulted in a 50% reduction of replication-independent mutation frequency in haploid cells . Sequencing indicated that typical NHEJ-dependent reversion events are small deletions within mononucleotide repeats, with a remarkable resemblance to DNA polymerase slippage errors . Experiments with diploid and RAD52- or RAD54-deficient strains confirmed that among DSB repair pathways only NHEJ accounts for a considerable fraction of replication-independent frameshift mutations in haploid and diploid NHEJ non-repressed cells . Thus our results provide evidence that G(0) cells with unrepressed NHEJ capacity pay for a large-scale chromosomal stability with an increased frequency of small-scale mutations, a finding of potential relevance for carcinogenesis.

EMBO J, 2003 May 1, 22(9), 2264 - 73
CTG repeat instability and size variation timing in DNA repair-deficient mice; Savouret C et al.; Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene . We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA-PKcs (non-homologous end-joining) . Genes of the non-homologous end-joining and homologous recombination pathways did not seem to affect repeat instability . Only lack of Rad52 led to a slight decrease in expansion range . Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model . Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations . Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability . We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.

EMBO J, 2003 May 1, 22(9), 2178 - 87
Regions of GAL4 critical for binding to a promoter in vivo revealed by a visual DNA-binding analysis; Mizutani A et al.; Binding of transcriptional activators to specific sites on DNA, mediated by their DNA-binding domain, is a key regulatory point in transcriptional regulation . With a GFP-based microscopic assay, we investigated how the prototypical activator GAL4 effectively binds to a promoter in living yeast cells . We show that GAL4 relies on a previously unrevealed mechanism involving 'DNA-binding enhancers' (DBEs), the regions of GAL4 that assist DNA-binding domain association with DNA . GAL4 contains two DBEs, one, but not the other, physically overlapping the principal transcriptional activation domain . Either of the DBEs, however, can function independently of transcriptional activation, indicating a discrete mechanism responsible for DNA-binding enhancement . The effect of DBEs, while not limited to natural target promoters, is still not universal and can be profoundly affected by the binding-site context . The GAL4 DBEs can also enhance promoter binding of an unrelated DNA-binding domain, and possibly represent a new modular functional unit responsible for effective binding of diverse regulatory factors to DNA in vivo.

EMBO J, 2003 May 1, 22(9), 2025 - 35
A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP; Loewen CJ et al.; Intracellular lipid traffic is mediated both by membrane vesicles and by a number of non-vesicular pathways facilitated by cytoplasmic lipid binding proteins . For these proteins to act effectively they must be targeted accurately to specific membranes . Here we identify a novel short conserved determinant called the FFAT motif that is shared by several seemingly unrelated lipid binding proteins and is also found in Opi1p, a transcriptional regulator of phospholipid synthesis in yeast . FFAT motifs act as membrane- targeting determinants by their direct interaction with homologues of VAMP-associated protein (VAP), a conserved endoplasmic reticulum (ER) protein . In budding yeast, all four proteins with FFAT motifs interact with Scs2p, a homologue of VAP, to target the ER to some extent . The precise intracellular distribution of each of these proteins depends on the integration of the FFAT-Scs2p interaction with other targeting determinants, and the interaction is functionally significant . We conclude that binding to a VAP homologue is a common mechanism by which proteins with FFAT motifs, most of which are involved in lipid metabolism, target ER membranes.

Curr Opin Struct Biol, 2003 Apr, 13(2), 249 - 55
Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures; Hopfner KP et al.; ATP-binding cassette (ABC)-type ATPases are chemo-mechanical engines for diverse biological pathways . ABC ATPase domains act not only in ABC transporters but also in DNA mismatch, nucleotide excision and double-strand break repair enzymes, as well as in chromosome segregation . Atomic-resolution crystal structures suggest molecular mechanisms for ABC ATPases and reveal surprisingly significant mechanistic and architectural conservation . This emerging unified structural biochemistry provides general medical and biological insights into how ABC proteins function as chemo-mechanical devices . ATP binding by the signature and Q-loop motifs drives the conformations of substrate-specific domains to accomplish diverse functions in transmembrane transport and DNA repair.

Fitoterapia, 2003 Apr, 74(3), 231 - 6
Analgesic, antipyretic and anti-inflammatory effects of essential oil of Lippia multiflora; Abena AA et al.; The essential oil of Lippia multiflora Moldenke (Verbenaceae) produced by conventional hydrodistillation was analyzed and studied for its analgesic, antipyretic and anti-inflammatory activities in rats and mice . At the doses used (2, 4 and 8 ml/kg o.s.) the essential oil of L . multiflora showed significant and dose-dependent analgesic effect on acetic acid-induced writhing in mice . Only the dose of 8 ml/kg of essential oil, antagonized hyperexia induced by brewer's yeast . No effect on granuloma formation was observed.

Ageing Res Rev, 2003 Jul, 2(3), 287 - 97
Hyper nuclear acetylation (HNA) in proliferation, differentiation and apoptosis; Kawahara K et al.; Coactivators such as cyclic AMP-response-element binding protein (CREB)-binding protein (CBP) and p300/CBP associated factor (P/CAF) play a crucial role in coordinating and cointegrating eukaryotic transcription . One of the recent paradigms in the eukaryotic transcription field is the finding of molecular basis of coactivator function . The well characterized coactivators such as CBP and P/CAF have been proposed to coactivate/cointegrate gene expression with many transcription activators through two mechanisms . One is complex formation with the components with basal transcriptional machinery . Another is its intrinsic and associated enzymatic activity, which transfers an acetyl-base to the epsilon ( epsilon ) portion of lysine-residues in histones and certain nuclear proteins (factor acetyltransferases; FATs), such as p53, lymphoid enhancer-binding factor (LEF), and transcription factor IIE (TFIIE), which often results in increased transcriptional activity . Recently, the status of hyper nuclear acetylation (HNA) has been thought to influence proliferation, differentiation and apoptosis . Furthermore, recent reports showed that histone acetyltransferase (HAT) activity is increased in human disease, such as cancer and atherosclerosis, and studies have shown associations between nuclear acetylation/deacetylation and cell proliferation/differentiation.

Curr Biol, 2003 Apr 29, 13(9), R360 - 2
Dispatch . GTPase signalling: new functions for Diaphanous-related formins; Olson MF; The Diaphanous-related formins are effectors for Rho GTPases . Two recent reports have unveiled previously unappreciated activities of Drf3 (mDia2), eliciting cytoskeletal rearrangements at the behest of Cdc42, and of DRF2 (hDia2C), regulating endosome dynamics for RhoD.

Curr Biol, 2003 Apr 29, 13(9), 715 - 24
The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function; Wesche S et al.; BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis . In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function . The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization . RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins . She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain . In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches . In vitro, She4p stimulates binding of Myo5p to filamentous actin . Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip . This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap . Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p . CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.

Plant Cell, 2003 May, 15(5), 1131 - 42
Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance; Delhaize E et al.; The yeast Saccharomyces cerevisiae expressing a cDNA library prepared from Stylosanthes hamata was screened for enhanced Mn(2+) tolerance . From this screen, we identified four related cDNAs that encode membrane-bound proteins of the cation diffusion facilitator (CDF) family . One of these cDNAs (ShMTP1) was investigated in detail and found to confer Mn(2+) tolerance to yeast by internal sequestration rather than by efflux of Mn(2+) . Expression of ShMTP1 in a range of yeast mutants suggested that it functions as a proton:Mn(2+) antiporter on the membrane of an internal organelle . Similarly, when expressed in Arabidopsis, ShMTP1 conferred Mn(2+) tolerance through internal sequestration . The ShMTP1 protein fused to green fluorescent protein was localized to the tonoplast of Arabidopsis cells but appeared to localize to the endoplasmic reticulum of yeast . We suggest that the ShMTP1 proteins are members of the CDF family involved in conferring Mn(2+) tolerance and that at least one of these proteins (ShMTP1) confers tolerance by sequestering Mn(2+) into internal organelles.

Mol Cell Biol, 2003 May, 23(10), 3527 - 35
Functional relation among RecQ family helicases RecQL1, RecQL5, and BLM in cell growth and sister chromatid exchange formation; Wang W et al.; Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes . Cells derived from individuals suffering from these syndromes show significant levels of genomic instability . However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5 . We generated here RECQL1(-/-), RECQL5(-/-), RECQL1(-/-)/RECQL5(-/-), RECQL1(-/-)/BLM(-/-), and RECQL5(-/-)/BLM(-/-) cells from chicken B-lymphocyte line DT40 cells . Although BLM(-/-) DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an approximately 10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1(-/-), RECQL5(-/-), and RECQL1(-/-)/RECQL5(-/-) cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE . However, both RECQL1(-/-)/BLM(-/-) and RECQL5(-/-)/BLM(-/-) cells grew more slowly than BLM(-/-) cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition . Surprisingly, RECQL5(-/-)/BLM(-/-) cells showed a higher frequency of SCE than BLM(-/-) cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.

Mol Cell Biol, 2003 May, 23(10), 3497 - 505
Two ubiquitin-conjugating enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, have distinct functions for ubiquitination of mitotic cyclin; Seino H et al.; Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases . Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins . The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase . In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C . Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition . Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11 . Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13 . They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4+ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11+ cannot be suppressed by high expression of UbcP1/Ubc4 . In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells . Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.

Mol Cell Biol, 2003 May, 23(10), 3487 - 96
The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10; Bastin-Shanower SA et al.; Mus81-Mms4 and Rad1-Rad10 are homologous structure-specific endonucleases that cleave 3' branches from distinct substrates and are required for replication fork stability and nucleotide excision repair, respectively, in the yeast Saccharomyces cerevisiae . We explored the basis of this biochemical and genetic specificity . The Mus81-Mms4 cleavage site, a nick 5 nucleotides (nt) 5' of the flap, is determined not by the branch point, like Rad1-Rad10, but by the 5' end of the DNA strand at the flap junction . As a result, the endonucleases show inverse substrate specificity; substrates lacking a 5' end within 4 nt of the flap are cleaved poorly by Mus81-Mms4 but are cleaved well by Rad1-10 . Genetically, we show that both mus81 and sgs1 mutants are sensitive to camptothecin-induced DNA damage . Further, mus81 sgs1 synthetic lethality requires homologous recombination, as does suppression of mutant phenotypes by RusA expression . These data are most easily explained by a model in which the in vivo substrate of Mus81-Mms4 and Sgs1-Top3 is a 3' flap recombination intermediate downstream of replication fork collapse.

Mol Cell Biol, 2003 May, 23(10), 3468 - 76
Multiple mechanistically distinct functions of SAGA at the PHO5 promoter; Barbaric S et al.; Our previous studies have shown that the rate of chromatin remodeling and consequently the rate of PHO5 activation are strongly decreased in the absence of Gcn5 histone acetyltransferase activity . Using chromatin immunoprecipitation, we demonstrate that SAGA is physically recruited to the PHO5 promoter . Recruitment is dependent on the specific activator Pho4 and occurs only under inducing conditions . Spt3, another subunit of SAGA, also plays a role in PHO5 activation but has a function that is completely different from that of Gcn5 . An SPT3 deletion severely compromises the PHO5 promoter and reduces the extent of transcriptional activation by diminishing the binding of the TATA binding protein to the promoter without, however, affecting the rate or the extent of chromatin remodeling . A gcn5 spt3 double mutant shows a synthetic phenotype almost as severe as that observed for an spt7 or spt20 mutant . The latter two mutations are known to prevent the assembly of the complex and consequently lead to the loss of all SAGA functions . The absence of the Ada2 subunit causes a strong delay in chromatin remodeling and promoter activation that closely resembles the delay observed in the absence of Gcn5 . A deletion of only the Ada2 SANT domain has exactly the same effect, strongly suggesting that Ada2 controls Gcn5 activity by virtue of its SANT domain . Finally, the Gcn5 bromodomain also contributes to but is not essential for Gcn5 function at the PHO5 promoter . Taken together, the results provide a detailed and differentiated description of the role of SAGA as a coactivator at the PHO5 promoter.

Mol Cell Biol, 2003 May, 23(10), 3456 - 67
Identification and characterization of three new components of the mSin3A corepressor complex; Fleischer TC et al.; The mSin3A corepressor complex contains 7 to 10 tightly associated polypeptides and is utilized by many transcriptional repressors . Much of the corepressor function of mSin3A derives from associations with the histone deacetylases HDAC1 and HDAC2; however, the contributions of the other mSin3A-associated polypeptides remain largely unknown . We have purified an mSin3A complex from K562 erythroleukemia cells and identified three new mSin3A-associated proteins (SAP): SAP180, SAP130, and SAP45 . SAP180 is 40% identical to a previously identified mSin3A-associated protein, RBP1 . SAP45 is identical to mSDS3, the human ortholog of the SDS3p component of the Saccharomyces cerevisiae Sin3p-Rpd3p corepressor complex . SAP130 does not have detectable homology to other proteins . Coimmunoprecipitation and gel filtration data suggest that the new SAPs are, at the very least, components of the same mSin3A complex . Each new SAP repressed transcription when tethered to DNA . Furthermore, repression correlated with mSin3A binding, suggesting that the new SAPs are components of functional mSin3A corepressor complexes . SAP180 has two repression domains: a C-terminal domain, which interacts with the mSin3A-HDAC complex, and an N-terminal domain, which functions independently of mSin3A-HDAC . SAP130 has a repression domain at its C terminus that interacts with the mSin3A-HDAC complex and an N-terminal domain that probably mediates an interaction with a transcriptional activator . Together, our data suggest that these novel SAPs function in the assembly and/or enzymatic activity of the mSin3A complex or in mediating interactions between the mSin3A complex and other regulatory complexes . Finally, all three SAPs bind to the HDAC-interaction domain (HID) of mSin3A, suggesting that the HID functions as the assembly interface for the mSin3A corepressor complex.

J Cell Sci, 2003 Jun 15, 116(Pt 12), 2409 - 19 Epub 2003 Apr 30.
Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway; Thomas F et al.; The production of ribosomes constitutes a major biosynthetic task for cells . Eukaryotic small and large ribosomal subunits are assembled in the nucleolus and independently exported to the cytoplasm . Most nuclear export pathways require RanGTP-binding export receptors . We analyzed the role of CRM1, the export receptor for leucine-rich nuclear export signals (NES), in the biogenesis of ribosomal subunits in vertebrate cells . Inhibition of the CRM1 export pathway led to a defect in nuclear export of both 40S and 60S subunits in HeLa cells . Moreover, the export of newly made ribosomal subunits in Xenopus oocytes was efficiently and specifically competed by BSA-NES conjugates . The CRM1 dependence of 60S subunit export suggested a conserved function for NMD3, a factor proposed to be a 60S subunit export adaptor in yeast . Indeed, we observed that nuclear export of human NMD3 (hNMD3) is sensitive to leptomycin B (LMB), which inactivates CRM1 . It had, however, not yet been demonstrated that Nmd3 can interact with CRM1 . Using purified recombinant proteins we have shown here that hNMD3 binds to CRM1 directly, in a RanGTP-dependent manner, by way of a C-terminal NES sequence . Our results suggest that the functions of CRM1 and NMD3 in ribosomal subunit export are conserved from yeast to higher eukaryotes.

Bioinformatics, 2003 May 1, 19(7), 842 - 50
Reconstructing the temporal ordering of biological samples using microarray data; Magwene PM et al.; MOTIVATION: Accurate time series for biological processes are difficult to estimate due to problems of synchronization, temporal sampling and rate heterogeneity . Methods are needed that can utilize multi-dimensional data, such as those resulting from DNA microarray experiments, in order to reconstruct time series from unordered or poorly ordered sets of observations . RESULTS: We present a set of algorithms for estimating temporal orderings from unordered sets of sample elements . The techniques we describe are based on modifications of a minimum-spanning tree calculated from a weighted, undirected graph . We demonstrate the efficacy of our approach by applying these techniques to an artificial data set as well as several gene expression data sets derived from DNA microarray experiments . In addition to estimating orderings, the techniques we describe also provide useful heuristics for assessing relevant properties of sample datasets such as noise and sampling intensity, and we show how a data structure called a PQ-tree can be used to represent uncertainty in a reconstructed ordering . AVAILABILITY: Academic implementations of the ordering algorithms are available as source code (in the programming language Python) on our web site, along with documentation on their use . The artificial 'jelly roll' data set upon which the algorithm was tested is also available from this web site . The publicly available gene expression data may be found at and http://caulobacter.stanford.edu/CellCycle/.

Tsitologiia, 2003, 45(2), 196 - 215
{The role of tyrosine phosphorylation in the regulation of cell proliferation and differentiation in lower eukaryotes}; Shemarova IV; The review summarizes current data on signaling transduction mechanisms in some unicellular eukaryotes, based on sequential translation of phosphotyrosine signals from the cell surface to the nucleus.

J Androl, 2003 May-Jun, 24(3), 332 - 42
Evidence for a role of glycogen synthase kinase-3 beta in rodent spermatogenesis; Guo TB et al.; Glycogen synthase kinase-3 beta (GSK-3 beta) regulates cell metabolism, cell cycle, and cell fate through the phosphorylation of a diverse array of substrates . Herein, we provide evidence that supports a role for GSK-3 in mammalian meiosis and spermatogenesis . Immunostaining of testis sections showed that while GSK-3 alpha was ubiquitous in the seminiferous tubules, GSK-3 beta was expressed in premeiotic type B spermatogonia, in both meiotic preleptotene and leptotene spermatocytes, as well as in Sertoli cells in both the mouse and rat . Thus, GSK-3 beta is expressed in germ cells entering meiosis . In addition, intense immunoreactivity was detected in rat step 6 though 11 spermatids . In situ hybridization (ISH) in rat testis confirmed the immunostaining pattern in leptotene and spermatids and showed a GSK-3 beta messenger RNA (mRNA) signal in some pachytene spermatocytes . The restricted pattern of expression suggests cell-specific regulation of Gsk-3 beta mRNA . To determine whether GSK-3 is required for meiosis entry, rat stage VIIa seminiferous tubule segments were cultured with selective small-molecule GSK-3 inhibitors . These compounds markedly and dose-dependently suppressed meiotic synthesis (S)-phase DNA . Since a yeast GSK-3 homolog, Rim11p (regulator of inducer of meiosis), is pivotal to meiosis entry, we tested whether GSK-3 beta complements Rim11p function in meiosis . Rim11p phosphorylates transcription factors Ume6p (unscheduled meiotic gene expression) and Ime1p (inducer of meiosis) to induce meiosis entry . Overexpression of murine GSK-3 beta in a rim11 mutant yeast failed to rescue the sporulation defect . Our finding that GSK-3 beta interacted only with Ume6p but not with IME1 in a yeast 2-hybrid assay suggests that noncomplementation reflects partial divergence in substrate specificity . This work provides the basis for future studies of GSK-3 beta signaling in mammalian meiosis and spermatogenesis.

BMC Bioinformatics . 2003 Apr 29;4(1):15.
DNA microarray data and contextual analysis of correlation graphs; Rougemont J et al.; BACKGROUND: DNA microarrays are used to produce large sets of expression measurements from which specific biological information is sought . Their analysis requires efficient and reliable algorithms for dimensional reduction, classification and annotation . RESULTS: We study networks of co-expressed genes obtained from DNA microarray experiments . The mathematical concept of curvature on graphs is used to group genes or samples into clusters to which relevant gene or sample annotations are automatically assigned . Application to publicly available yeast and human lymphoma data demonstrates the reliability of the method in spite of its simplicity, especially with respect to the small number of parameters involved . CONCLUSIONS: We provide a method for automatically determining relevant gene clusters among the many genes monitored with microarrays . The automatic annotations and the graphical interface improve the readability of the data . A C++ implementation, called Trixy, is available from http://tagc.univ-mrs.fr/bioinformatics/trixy.html.

J Cell Biol, 2003 Apr 28, 161(2), 333 - 47
LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway; Chen EJ et al.; LST8, a Saccharomyces cerevisiae gene encoding a 34-kD WD-repeat protein, was identified by mutations that caused defects in sorting Gap1p to the plasma membrane . Here, we report that the Gap1p sorting defect in the lst8-1 mutant results from derepression of Rtg1/3p activity and the subsequent accumulation of high levels of intracellular amino acids, which signal Gap1p sorting to the vacuole . To identify the essential function of Lst8p, we isolated lst8 mutants that are temperature-sensitive for growth . These mutants show hypersensitivity to rapamycin and derepressed Gln3p activity like cells with compromised TOR pathway activity . Like tor2 mutants, lst8 mutants also have cell wall integrity defects . Confirming a role for Lst8p in the TOR pathway, we find that Lst8p associates with both Tor1p and Tor2p and is a peripheral membrane protein that localizes to endosomal or Golgi membranes and cofractionates with Tor1p . Further, we show that a sublethal concentration of rapamycin mimics the Gap1p sorting defect of an lst8 mutant . Finally, the different effects of lst8 alleles on the activation of either the Rtg1/3p or Gln3p transcription factors reveal that these two pathways constitute distinct, genetically separable outputs of the Tor-Lst8 regulatory complex.

Hum Mol Genet, 2003 May 15, 12(10), 1145 - 54
A very long-chain acyl-CoA synthetase-deficient mouse and its relevance to X-linked adrenoleukodystrophy; Heinzer AK et al.; X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative and endocrine disorder resulting from mutations in ABCD1 which encodes a peroxisomal membrane protein in the ATP binding cassette superfamily . The biochemical signature of X-ALD is increased levels of saturated very long-chain fatty acids (VLCFA; carbon chains of 22 or more) in tissues and plasma that has been associated with decreased peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity and decreased peroxisomal VLCFA beta-oxidation . It has been hypothesized that ABCD1, which has no demonstrable VLCS activity itself, has an indirect effect on peroxisomal VLCS activity and VLCFA beta-oxidation by transporting fatty acid substrates, VLCS protein or some required co-factor into peroxisomes . Here we report the characterization of a Vlcs knockout mouse that exhibits decreased peroxisomal VLCS activity and VLCFA beta-oxidation but does not accumulate VLCFA . The XALD/Vlcs double knockout mouse has the biochemical abnormalities observed in the individual knockout mice but does not display a more severe X-ALD phenotype . These data lead us to conclude that (1) VLCFA levels are independent of peroxisomal fatty acid beta-oxidation, (2) there is no ABCD1/VLCS interaction and (3) the common severe forms of X-ALD cannot be modeled by decreasing peroxisomal VLCS activity in the XALD mouse.

Mol Cell, 2003 Apr, 11(4), 853 - 64
Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores; Harel A et al.; The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins . Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export . However, these altered pores all still possessed the bulk of pore structure . Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components . The resulting reconstituted nuclei are severely defective for NLS import and DNA replication . Strikingly, they show a profound defect for every tested nucleoporin . Even the integral membrane proteins POM121 and gp210 are absent or unorganized . Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores . We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.

Nat Cell Biol, 2003 May, 5(5), 480 - 5
Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I; Clyne RK et al.; During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors . Three innovations in chromosome behaviour during meiosis I accomplish this unique division . First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together . Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated . Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed . The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5 . We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers . They also fail to properly localize the Lrs4 (ref . 3) and Mam1 (ref . 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores . Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.

J Biol Chem, 2003 Jul 11, 278(28), 25970 - 6 Epub 2003 Apr 25.
Sti1 is a novel activator of the Ssa proteins; Wegele H et al.; The molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes . Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop . In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction . With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins . Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro . The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90 . Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system . Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1 . Thus, Sti1 is a potent novel effector for the Hsp70 ATPase.

J Biol Chem, 2003 Jul 11, 278(28), 25958 - 63 Epub 2003 Apr 25.
Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter; Kamiyama S et al.; Nucleotide sulfate, namely 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation . Although a specific PAPS transporter is present in Golgi membrane, no study has reported the corresponding gene . We have identified a novel human gene encoding a PAPS transporter, which we have named PAPST1, and the Drosophila melanogaster ortholog, slalom (sll) . The amino acid sequence of PAPST1 (432 amino acids) exhibited 48.1% identity with SLL (465 amino acids), and hydropathy analysis predicted the two to be type III transmembrane proteins . The transient expression of PAPST1 in SW480 cells showed a subcellular localization in Golgi membrane . The expression of PAPST1 and SLL in yeast Saccharomyces cerevisiae significantly increased the transport of PAPS into the Golgi membrane fraction . In human tissues, PAPST1 is highly expressed in the placenta and pancreas and present at lower levels in the colon and heart . An RNA interference fly of sll produced with a GAL4-UAS system revealed that the PAPS transporter is essential for viability . It is well known that mutations of some genes related to PAPS synthesis are responsible for human inherited disorders . Our findings provide insights into the significance of PAPS transport and post-translational sulfation.

J Biol Chem, 2003 Aug 1, 278(31), 28961 - 7 Epub 2003 Apr 26.
Structure-expression relationship of tumor necrosis factor receptor mutants that increase expression; Schweickhardt RL et al.; The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-alpha (TNF-alpha) . The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds . Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor . Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express several-fold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-alpha . The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds . Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation.

Plant J, 2003 May, 34(3), 293 - 306
Three SAC1-like genes show overlapping patterns of expression in Arabidopsis but are remarkably silent during embryo development; Despres B et al.; In Saccharomyces cerevisiae, the SAC1 gene encodes a polyphosphoinositide phosphatase (PPIPase) that modulates the levels of phosphoinositides, which are key regulators of a number of signal transduction processes . SAC1p has been implicated in multiple cellular functions: actin cytoskeleton organization, secretory functions, inositol metabolism, ATP transport, and multiple-drug sensitivity . Here, we describe the characterization of three genes in Arabidopsis thaliana, AtSAC1a, AtSAC1b, and AtSAC1c, encoding proteins similar to those of yeast SAC1p . We demonstrated that the three AtSAC1 proteins are functional homologs of the yeast SAC1p because they can rescue the cold-sensitive and inositol auxotroph yeast sac1-null mutant strain . The fact that Arabidopsis and yeast SAC1 genes derived from a common ancestor suggests that this plant multigenic family is involved in the phosphoinositide pathway and in a range of cellular functions similar to those in yeast . Using GFP fusion experiments, we demonstrate that the three AtSAC1 proteins are targeted to the endoplasmic reticulum . Their expression patterns are overlapping, with at least two members expressed in each organ . Remarkably, AtSAC1 genes are not expressed during seed development, and therefore additional phosphatases are required to control phosphoinositide levels in seeds.

Biochem J, 2003 Jul 15, 373(Pt 2), 381 - 91
Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins; Larsson M et al.; Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family . In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions . Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin . PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction . The two proteins were found to be co-localized in these cells by confocal microscopy . Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95 . We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95 . The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102 . We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin . The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97 . The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily . One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain . Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.

Nucleic Acids Res, 2003 May 1, 31(9), 2333 - 43
Dependence of pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions; Chawla G et al.; Saccharomyces cerevisiae PRP17 (CDC40) encodes a second-step pre-mRNA splicing factor with a role in cell division . The functions of Prp17 in specific cell cycle transitions were examined using temperature-sensitive alleles in arrest/release experiments . We find that G(1)/S and G(2)/M transitions depend on Prp17 . G(1)-synchronized prp17::LEU2 cells arrest at non-permissive temperatures as unbudded haploid cells with low levels of CLN1, CLB5 and RNR1 transcripts . This indicates a Prp17 execution point at or prior to Start . Reduced levels of alpha-tubulin protein, a mitotic spindle component, underlie the benomyl sensitivity of prp17 mutants and possibly their G(2)/M arrest . Splicing of TUB1 and TUB3 transcripts, which encode alpha-tubulin, was analyzed in prp17 and other second-step factor mutants . TUB1 splicing is inefficient in prp17, prp16 and prp22, and marginally affected in prp18, slu7-1 and psf1-1 . TUB3 splicing is similarly affected . In vitro splicing with TUB3 pre-mRNA demonstrates a compromised second step in prp17::LEU2 extracts, implicating a direct role for Prp17 in its efficient splicing . Genomic replacement of an intronless TUB1 gene relieves the benomyl sensitivity of prp17 mutants; however, they remain temperature sensitive, implying multiple limiting factors for mitosis . The data suggest that integration of splicing with the cell cycle is important for G(1)/S and G(2)/M transitions.

Nucleic Acids Res, 2003 May 1, 31(9), 2279 - 88
Molecular determinants of the cell-cycle regulated Mcm1p-Fkh2p transcription factor complex; Boros J et al.; The MADS-box transcription factor Mcm1p and forkhead (FKH) transcription factor Fkh2p act in a DNA-bound complex to regulate cell-cycle dependent expression of the CLB2 cluster in Saccharomyces cerevisiae . Binding of Fkh2p requires prior binding by Mcm1p . Here we have investigated the molecular determinants governing the formation of the Mcm1p- Fkh2p complex . Fkh2p exhibits cooperativity in complex formation with Mcm1p and we have mapped a small region of Fkh2p located immediately upstream of the FKH DNA binding domain that is required for this cooperativity . This region is lacking in the related protein Fkh1p that cannot form ternary complexes with Mcm1p . A second region is identified that inhibits Mcm1p-independent DNA binding by Fkh2p . The spacing between the Mcm1p and Fkh2p binding sites is also a critical determinant for complex formation . We also show that Fkh2p can form ternary complexes with the human counterpart of Mcm1p, serum response factor (SRF) . Mutations at analogous positions in Mcm1p, which are known to affect SRF interaction with its partner protein Elk-1, abrogate complex formation with Fkh2p, demonstrating evolutionary conservation of coregulatory protein binding surfaces . Our data therefore provide molecular insights into the mechanisms of Mcm1p- Fkh2p complex formation and more generally aid our understanding of MADS-box protein function.

Anal Biochem, 2003 May 15, 316(2), 171 - 4
A high-throughput system for two-hybrid screening based on growth curve analysis in microtiter plates; Diaz-Camino C et al.; The yeast two-hybrid system is a powerful tool for identifying novel protein-protein interactions . In general, biochemical marker genes such as lacZ are exploited for indirect quantification of the interaction, and commonly involve the conduct of rather laborious beta-galactosidase assays . This paper describes a simple alternative method based on growth curve analysis of yeast cultures that is amenable to microtiter plate format, and therefore allows the quantification of large numbers of yeast two-hybrid combinations . The analyzed results of yeast cultures grown in microtiter plates were compared with those obtained from the classical beta-galactosidase assay . We conclude that the method presented here is reproducible, of equal or greater sensitivity than the beta-galactosidase assay, and can be further adapted for application to the conduct of large-scale, automated yeast two-hybrid experiments.

Fish Shellfish Immunol, 2003 May, 14(5), 423 - 34
Chemiluminescence response of beta-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax; Vazzana M et al.; The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with beta-glucan . The CL response by P.C . and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood . This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros . Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros . Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction) . Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O-2 dismutation to H(2)O(2), supported the correlation between CL and respiratory burst . Treatment with ouabain and DNP suggested that in this response, Ca(++) pump channels and calmodulin are involved in a metabolic energy-dependent pathway.

Trends Genet, 2003 May, 19(5), 238 - 42
Predicting gene function by conserved co-expression; van Noort V et al.; We show that gene co-expression, which generally provides only a very weak signal for the prediction of functional interactions, can provide a reliable signal by exploiting evolutionary conservation . The encoded proteins of conserved co-expressed gene pairs are highly likely to be part of the same pathway not only after speciation (98%), but also after parallel gene duplication (97%) . Conserved co-expression combined with homology data enables us to predict specific gene functions . The use of conservation between parallel duplicated gene pairs to predict function is especially promising given that gene duplication is common in eukaryotes, and that data from only a single organism can be used.

Lung Cancer, 2003 May, 40(2), 157 - 64
DNA repair rate and etoposide (VP16) resistance of tumor cell subpopulations derived from a single human small cell lung cancer; Hansen LT et al.; Two human small cell lung cancer (SCLC) subpopulations, CPH 54A, and CPH 54B, established from the same patient tumor by in vitro cloning, were investigated . The tumor was classified as intermediate-type SCLC . The cellular sensitivity to ionizing radiation (IR) was previously determined in the two sublines both in vivo and in vitro . Here we measured the etoposide (VP16) sensitivity together with the induction and repair of VP16- and IR-induced DNA double-strand breaks (DSBs) . The two subpopulations were found to differ significantly in sensitivity to VP16, with the radioresistant 54B subline also being VP16 resistant . In order to explain the VP16 resistant phenotype several mechanisms where considered . The p53 status, P-glycoprotein, MRP, topoisomerase IIalpha, and Mre11 protein levels, as well as growth kinetics, provided no explanations of the observed VP16 resistance . In contrast, a significant difference in repair of both VP16- and IR-induced DSBs, together with a difference in the levels of the DSB repair proteins DNA-dependent protein kinase (DNA-PK(cs)) and RAD51 was observed . The VP16- and radioresistant 54B subline exhibited a pronounced higher repair rate of DSBs and higher protein levels of both DNA-PK(cs) and RAD51 compared with the sensitive 54A subline . We suggest, that different DSB repair rates among tumor cell subpopulations of individual SCLC tumors may be a major determinant for the variation in clinical treatment effect observed in human SCLC tumors of identical histological subtype.

J Steroid Biochem Mol Biol, 2003 Feb, 84(2-3), 259 - 68
Comparison of hormonal activity (estrogen, androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy; Beck V et al.; Extracts from red clover (Trifolium pratense), soybean (Glycine max.) and black cohosh (Cimicifuga racemosa) are frequently used as alternative compounds for hormone replacement therapy (HRT) to treat menopausal disorders . Fifteen commercially available products made either from red clover, soybean or black cohosh were tested in in vitro assays in this study . The main polycyclic phenolic compounds of soy and red clover products were biochanin A, genistein, daidzein, formononetin, and glycitein . In red clover products glycitein was not abundant . All the compounds showed clear estrogenic activity through estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) and affinity to progesterone receptor (PR) and androgen receptor (AR), whereas the compounds from black cohosh did not . This was corroborated by synthetic isoflavones such as biochanin A, daidzein, genistein and formononetin . They exerted affinity to PR and AR in the range of 0.39-110 mM . Statistical analysis applying principal component analysis (PCA) revealed that all red clover and soy products are grouped in different clusters . Red clover products showed a higher affinity to AR and PR than soy products, which is explained by the higher amount of isoflavones present . In vitro assays and chemical analysis showed that theoretical estrogenic activity expressed as equivalent E2 concentration is in the same range as recommended for synthetic estrogens . Broader spectrum of action and hypothesized lower side effects by action through ERbeta make them suitable for alternative hormone replacement therapy.

Biochem J, 2003 Jul 15, 373(Pt 2), 571 - 81
Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence; Kirwan AF et al.; The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity . Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence {Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J . Biol . Chem . 273, 8940-8945} . This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity . To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions {GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)} . GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays . These results suggested that a region within C1/V2 directly inhibits catalytic domain activity . Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner . This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester . Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate . Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity . Taken together, these observations support strongly the existence of multiple regions within the PKCalpha-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

Nucleosides Nucleotides Nucleic Acids, 2003, 22(1), 13 - 9
Microwave-assisted synthesis of O'-adamantylated uracil-derived nucleosides; Gorska A et al.; Microwave-induced synthesis of O'-adamantyl derivatives of AZT, thymidine, 2'-deoxyuridine and uridine was investigated . Contrary to heterocyclus adamantylation of uracil and uridine in trifluoroacetic acid, the microwave-induced reaction provided sugar-substituted compounds.

Cancer Sci, 2003 Feb, 94(2), 148 - 52
Polo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancers; Takahashi T et al.; PLK (polo-like kinase), the human counterpart of polo in Drosophila melanogaster and of CDC5 in Saccharomyces cerevisiae, belongs to a family of serine/threonine kinases . It is intimately involved in spindle formation and chromosome segregation during mitosis . The purpose of this study was to determine whether PLK1 is overexpressed in primary colorectal cancer specimens as compared with normal colon mucosa and to assess its relation to other kinases as a potential new tumor marker . In the present study, immunohistochemical analyses were performed of PLK1 expression in 78 primary colorectal cancers as well as 15 normal colorectal specimens . Furthermore, we examined the relationship between other kinases, Aurora-A and Aurora-C, and PLK1 expression . In normal colon mucosa, some crypt cells showed weakly positive staining for PLK1 in 13 out of 15 cases, the remaining cases being negative . Elevated expression of PLK1 was observed in 57 (73.1%) of the colorectal cancers, statistically significant associations being evident with pT (primary tumor invasion) (P=0.0006, Mann-Whitney U test), pN (regional lymph nodes) (P=0.008, chi2 test) and the Dukes' classification (P=0.0005, Mann-Whitney U test) . Mean proliferating cell nuclear antigen-labeling index was 52.3%, with a range of 24.1% to 77.3% . Values for lesions with high and low PLK1 expression were 54.7+/-10.3% (mean+/-SD) and 45.9+/-11.9% (P=0.002, Student's t test) . PLK1 was significantly associated with Aurora-A, but PLK1 staining was more diffuse and extensive than for Aurora-A or Aurora-C . Interestingly, PLK1 overexpression was significantly associated with p53 accumulation in colorectal cancers . Our results suggest overexpression of PLK1 might be of pathogenic, prognostic and proliferative importance, so that this kinase might have potential as a new tumor marker for colorectal cancers.

Electrophoresis, 2003 Apr, 24(7-8), 1137 - 44
Simple circuit to improve electric field homogeneity in contour-clamped homogeneous electric field chambers; Herrera JA et al.; We redesigned contour-clamped homogeneous electric field (CHEF) circuitry to eliminate crossover distortion, to set identical potentials at electrodes of each equipotential pair and to drive pairs with transistors in emitter follower stages . An equipotential pair comprised the two electrodes set at the same potential to provide electric field homogeneity inside of the hexagonal array . The new circuitry consisted of two identical circuits, each having a resistor ladder, diodes and transistors . Both circuits were interconnected by diodes that controlled the current flow to electrodes when the array was energized in the 'A' or 'B' direction of the electric field . The total number of transistors was two-thirds of the total number of electrodes . Average voltage deviation from potentials expected at electrodes to achieve a homogeneous electric field was 0.06 V, whereas 0.44 V was obtained with another circuit that used transistors in push-pull stages . The new voltage clamp unit is cheap, generated homogeneous electric field, and gave reproducible and undistorted DNA band patterns.

J Biol Chem, 2003 Jul 25, 278(30), 27781 - 8 Epub 2003 Apr 21.
Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA; Herlan M et al.; The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes . Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA . Mgm1 and its human ortholog OPA1, associated with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation (Wong, E . D., Wagner, J . A., Gorsich, S . W., McCaffery, J . M., Shaw, J . M., and Nunnari, J . (2000) J . Cell Biol . 151, 341-352; Wong, E . D., Wagner, J . A., Scott, S . V., Okreglak, V., Holewinske, T . J., Cassidy-Stone, A., and Nunnari, J . (2003) J . Cell Biol . 160, 303-311; Sesaki, H., Southard, S . M., Yaffe, M . P., and Jensen, R . E . (2003) Mol . Biol . Cell, in press) . Mgm1 and its orthologs exist in two forms of different lengths . To obtain new insights into their biogenesis and function, we have characterized these isoforms . The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1) . The large isoform is an integral inner membrane protein facing the intermembrane space . Furthermore, the conversion of l-Mgm1 into s-Mgm1 was found to be dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphology . Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila melanogaster, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1 . Expression of s-Mgm1 can partially complement the Deltapcp1 phenotype . Expression of both isoforms but not of either isoform alone was able to partially complement the Deltamgm1 phenotype . Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphology and maintenance of mitochondrial DNA.

J Biol Chem, 2003 Jun 27, 278(26), 23502 - 7 Epub 2003 Apr 21.
Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan; Burchmore RJ et al.; While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa . We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily . The gene was expressed in both the procyclic and bloodstream forms of the organism . Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine . A transporter with an indistinguishable kinetic profile was identified in T . b . brucei procyclics and designated H4 . RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90% . Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

J Mol Biol, 2003 May 2, 328(3), 721 - 36
Computer modeling of the membrane interaction of FYVE domains; Diraviyam K et al.; FYVE domains are membrane targeting domains that are found in proteins involved in endosomal trafficking and signal transduction pathways . Most FYVE domains bind specifically to phosphatidylinositol 3-phosphate (PI(3)P), a lipid that resides mainly in endosomal membranes . Though the specific interactions between FYVE domains and the headgroup of PI(3)P have been well characterized, principally through structural studies, the available experimental structures suggest several different models for FYVE/membrane association . Thus, the manner in which FYVE domains adsorb to the membrane surface remains to be elucidated . Towards this end, recent experiments have shown that FYVE domains bind PI(3)P in the context of phospholipid bilayers and that hydrophobic residues on a conserved loop are able to penetrate the membrane interface in a PI(3)P-dependent manner.Here, the finite difference Poisson-Boltzmann (FDPB) method has been used to calculate the energetic interactions of FYVE domains with phospholipid membranes . Based on the computational analysis, it is found that (1) recruitment to membranes is facilitated by non-specific electrostatic interactions that occur between basic residues on the domains and acidic phospholipids in the membrane, (2) the energetic analysis can quantitatively differentiate among the modes of membrane association proposed by the experimentally determined structures, (3) FDPB calculations predict energetically feasible models for the membrane-associated states of FYVE domains, (4) these models are consistent with the observation that conserved hydrophobic residues insert into the membrane interface, and (5) the calculations provide a molecular model for the hydrophobic partitioning: binding of PI(3)P significantly neutralizes positive potential in the region of the hydrophobic residues, which acts as an "electrostatic switch" by reducing the energetic barrier for membrane penetration . Finally, the computational results are extended to FYVE domains of unknown structure through the construction of high quality homology models for human FYVE sequences.

Bioorg Med Chem, 2003 Apr 3, 11(7), 1521 - 30
Non-thiol farnesyltransferase inhibitors: N-(4-acylamino-3-benzoylphenyl)-3-{5-(4-nitrophenyl)-2-furyl}acrylic acid amides; Kettler K et al.; We have designed the nitrophenylfurylacryl-substituted benzophenone 4f as a non-thiol farnesyltransferase inhibitor utilizing a novel aryl binding site of farnesyltransferase . Variation of the 2-acylamino substituent at the benzophenone core structure of our initial lead 4f yielded several non-thiol farnesyltransferase inhibitors with improved activity . These compounds display activity in the low nanomolar range.

J Biol Chem, 2003 Jun 27, 278(26), 23460 - 71 Epub 2003 Apr 18.
Feedback inhibition on cell wall integrity signaling by Zds1 involves Gsk3 phosphorylation of a cAMP-dependent protein kinase regulatory subunit; Griffioen G et al.; We report here that budding yeast cAMP-dependent protein kinase (cAPK) is controlled by heat stress . A rise in temperature from 30 to 37 degrees C was found to result in both a higher expression and an increased cytoplasmic localization of its regulatory subunit Bcy1 . Both of these effects required phosphorylation of serines located in its localization domain . Surprisingly, classic cAPK-controlled processes were found to be independent of Bcy1 phosphorylation, indicating that these modifications do not affect cAPK activity as such . Alternatively, phosphorylation may recruit cAPK to, and thereby control, a specific subset of (perhaps novel) cAPK targets that are presumably localized extranuclearly . Zds1 and Zds2 may play a role in this process, since these were found required to retain hyperphosphorylated Bcy1 in the cytoplasm at 37 degrees C . Mck1, a homologue of mammalian glycogen synthase kinase 3 and a downstream component of the heat-activated Pkc1-Slt2/Mpk1 cell wall integrity pathway, is partly responsible for hyperphosphorylations of Bcy1 . Remarkably, Zds1 appears to act as a negative regulator of cell wall integrity signaling, and this activity is dependent in part on the phosphorylation status of Bcy1 . Thus, Mck1 phosphorylation of Bcy1 and Zds1 may constitute an unprecedented negative feedback control on the cell wall integrity-signaling pathway.

Infect Immun, 2003 May, 71(5), 2819 - 26
Deletion of the Aspergillus fumigatus gene encoding the Ras-related protein RhbA reduces virulence in a model of Invasive pulmonary aspergillosis; Panepinto JC et al.; Aspergillus fumigatus is the predominant mold pathogen in patients who lack functional innate immunity . The A . fumigatus rhbA gene was first identified as a transcript that was upregulated when the organism was grown in the presence of mammalian cells . To gain insight into the function of rhbA in the growth and pathogenesis of A . fumigatus, we constructed a strain that lacks a functional rhbA gene . The Delta rhbA mutant showed a significant reduction in virulence compared to the virulence of the wild type in a mouse model of invasive aspergillosis . Complementation of the deletion with the wild-type gene restored full virulence . Although the Delta rhbA mutant grew as well as the wild type on solid medium containing the rich nitrogen source ammonium, the growth of the mutant was impaired on medium containing poor nitrogen sources . Like the Saccharomyces cerevisiae rhb1 mutant, the Delta rhbA mutant exhibited increased uptake of arginine . In addition, the Delta rhbA strain underwent asexual development in submerged cultures, even under ammonium-excess conditions . Growth of the mutant with poor nitrogen sources eliminated both the arginine uptake and submerged asexual development phenotypes . The mutant showed enhanced sensitivity to the TOR kinase inhibitor rapamycin . These findings establish the importance of rhbA for A . fumigatus virulence and suggest a role for rhbA in nutrient sensing.

Genes Dev, 2003 Apr 15, 17(8), 1055 - 67
NQK1/NtMEK1 is a MAPKK that acts in the NPK1 MAPKKK-mediated MAPK cascade and is required for plant cytokinesis; Soyano T et al.; The tobacco protein kinase NPK1 is a MAPKKK that regulates formation of the cell plate during cytokinesis . In the present study, we have identified tobacco NQK1/NtMEK1 and NRK1 as a MAPKK and a MAPK, respectively, downstream of NPK1 . NQK1/NtMEK1 complements the mutation in the PBS2 MAPKK gene of yeast in a manner that depends on both NPK1 and its activator, NACK1, a kinesin-like protein . Active NPK1 and NQK1/NtMEK1 phosphorylate and activate NQK1/NtMEK1 and NRK1, respectively . Both NQK1/NtMEK1 and NRK1, as well as NPK1, are activated at the late M phase of the cell cycle in tobacco cells, and they are rapidly inactivated by depolymerization of phragmoplast microtubules . These results suggest the existence of a MAPK cascade that consists of NPK1, NQK1/NtMEK1, and NRK1 and functions in a process related to the architecture of phragmoplasts at the late M phase of the cell cycle . Overexpression of kinase-negative NQK1/NtMEK1 in tobacco cells generates multinucleate cells with incomplete cross-walls . Arabidopsis plants with a mutation in the ANQ1 gene, an ortholog of NQK1/NtMEK1, display a dwarf phenotype, with unusually large cells that contain multiple nuclei and cell-wall stubs in various organs . In addition, anq1 homozygotes set fewer flowers and produce large and malformed pollen grains with a tetrad structure . Thus, NQK1/NtMEK1 (ANQ1) MAPKK appears to be a positive regulator of plant cytokinesis during meiosis as well as mitosis.

RNA, 2003 May, 9(5), 533 - 42
Structure of the U6 RNA intramolecular stem-loop harboring an S(P)-phosphorothioate modification; Reiter NJ et al.; Phosphorothioate-substitution experiments are often used to elucidate functionally important metal ion-binding sites on RNA . All previous experiments with S(P)-phosphorothioate-substituted RNAs have been done in the absence of structural information for this particular diastereomer . Yeast U6 RNA contains a metal ion-binding site that is essential for spliceosome function and includes the pro-S(P) oxygen 5' of U(80) . S(P)-phosphorothioate substitution at this location creates spliceosomes dependent on thiophilic ions for the first step of splicing . We have determined the solution structure of the U(80) S(P)-phosphorothioate-substituted U6 intramolecular stem-loop (ISL), and also report the refined NMR structure of the unmodified U6 ISL . Both structures were determined with inclusion of (1)H-(13)C residual dipolar couplings . The precision of the structures with and without phosphorothioate (RMSD = 1.05 and 0.79 A, respectively) allows comparison of the local and long-range structural effect of the modification . We find that the U6-ISL structure is unperturbed by the phosphorothioate . Additionally, the thermodynamic stability of the U6 ISL is dependent on the protonation state of the A(79)-C(67) wobble pair and is not affected by the adjacent phosphorothioate . These results indicate that a single S(P)-phosphorothioate substitution can be structurally benign, and further validate the metal ion rescue experiments used to identify the essential metal-binding site(s) in the spliceosome.

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