EMBO J, 2003 Aug 15, 22(16), 4103 - 10 A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling; Echtay KS et al.; Oxidative stress and mitochondrial dysfunction are associated with disease and aging . Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal . Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production . We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT) . Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP) . The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice . The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT . Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production. EMBO J, 2003 Aug 15, 22(16), 4059 - 69 Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export; Pathre P et al.; The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER) . Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export . PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export . However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export . Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export . Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat. Traffic, 2003 Sep, 4(9), 631 - 41 Persistence of Golgi matrix distribution exhibits the same dependence on Sar1p activity as a Golgi glycosyltransferase; Stroud WJ et al.; We investigated the relative distributional persistence of Golgi "matrix" proteins and glycosyltransferases to an endoplasmic reticulum exit block induced by expression of a GDP-restricted Sar1p . HeLa cells were microinjected with plasmid encoding the GDP-restricted mutant (T39N) of Sar1p to block endoplasmic reticulum exit and then scored for the distribution of GM130 (Golgi matrix protein of 130 kDa), a cis located golgin; p27, a member of the p24 family of proteins; giantin, a protein that interacts indirectly with GM130; and the Golgi glycosyltransferase, N-acetylgalactosaminyltransferase-2 (GalNAcT2) . All of these proteins lost their compact, juxtanuclear distribution and displayed characteristics of endoplasmic reticulum/cytoplasmic accumulation with the same dependence on plasmid concentration . The kinetics of redistribution of GM130 and GalNAcT2 were identical . Expression of Sar1pT39N displaced the COPII coat protein Sec13p from endoplasmic reticulum exit sites consistent with disruption of these sites . This occurred without disturbing the overall distribution of endoplasmic reticulum membrane . Furthermore, the reassembly of a juxtanuclear Golgi matrix as assayed by the distribution of GM130 following washout of the Golgi disrupting drug, brefeldin A, was blocked by microinjected Sar1pT39N plasmids . We conclude that the persistence, i.e . stability and maintenance, of Golgi matrix distribution and its reassembly following drug disruption are exquisitely dependent on Sar1p activity. Biochemistry, 2003 Aug 19, 42(32), 9779 - 88 Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase; Meyer A et al.; Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase . We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase . For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates . Schizochytrium sp . was unable to convert exogenously supplied {2-(14)C}-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E . gracilis and Thraustochytrium sp . carried out this desaturation very efficiently . Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis . To clone the desaturase gene, a cDNA library of E . gracilis was subjected to mass sequencing . A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp . was isolated, and its function was verified by heterologous expression in yeast . The desaturase efficiently converted DPA to DHA . Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids . The enzyme showed strict Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate . Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position. Biochemistry, 2003 Aug 19, 42(32), 9687 - 93 Refolding of amphioxus insulin-like peptide: implications of a bifurcating evolution of the different folding behavior of insulin and insulin-like growth factor 1; Wang S et al.; Insulin and insulin-like growth factor 1 (IGF-1) share high sequence homology, but their folding behaviors are significantly different: insulin folds into one unique thermodynamically controlled structure, while IGF-1 folds into two thermodynamically controlled disulfide isomers . However, the origin of their different folding behaviors is still elusive . The amphioxus insulin-like peptide (ILP) is thought to be the common ancestor of insulin and IGF-1 . A recombinant single-chain ILP has been expressed previously, and now its folding behavior is investigated . The folding behavior of ILP shows the characteristics of both insulin and IGF-1 . On one hand, two thermodynamically controlled disulfide isomers of ILP have been identified; on the other hand, the content of isomer 1 (its disulfides are deduced identical to those of swap IGF-1) is much less than that of isomer 2 (its disulfides are deduced identical to those of native IGF-1); that is, more than 96% of ILP folds into the native structure . The present results suggest that the different folding behaviors of insulin and IGF-1 are acquired through a bifurcating evolution: the tendency of forming the thermodynamically controlled non-native disulfide isomer is diminished during evolution from ILP to insulin, while this tendency is amplified during evolution from ILP to IGF-1 . Moreover, the N-terminal Gln residue of ILP can spontaneously form a pyroglutamate residue, and its cyclization has a significant effect on the folding behavior of ILP: the percentage of isomer 1 is approximately 2-fold that of isomer 1 of the noncyclized ILP; that is, isomer 1 becomes more favored when the N-terminal residue of ILP is cyclized . So, we deduce that the N-terminal residues have a significant effect on the folding properties of insulin, IGF-1, and ILP. Biochemistry, 2003 Aug 19, 42(32), 9609 - 18 Structure, stability, and function of hDim1 investigated by NMR, circular dichroism, and mutational analysis; Zhang YZ et al.; The 142 amino acid Dim1p protein is a component of the U4/U6.U5 tri-snRNP complex required for pre-mRNA splicing and interacts with multiple splicing-associated proteins . To gain further insight into the structural basis of its function, we determined the solution structure of the reduced form of the dominant negative human hDim1 (hDim1(1)(-)(128)) using multidimensional NMR spectroscopy . This dominant negative hDim1 assumes a thioredoxin-like fold, confirming previous NMR and crystallographic results . However, in contrast to a recent crystal structure, the NMR solution structure for the reduced form of hDim1(1)(-)(128) presented here, along with thermodynamic data, indicates that the presence of a disulfide bond between Cys38 and Cys79 is structurally and functionally unimportant . Comparison of the truncated hDim1(1)(-)(128) with the full-length protein, using NMR and circular dichroism spectroscopy, indicates that the 14 C-terminal residues can undergo a local unfolding transition and assume alternative conformations, which appear to play a functional role . Other residues essential for hDim1 function are identified by using mutational and genetic approaches . The residues thus identified are not identical with those previously shown to govern Dim1 interaction with defined protein partners. J Mol Evol, 2003 Jun, 56(6), 702 - 10 A novel strategy for analysis of gene homologues and segmental genome duplications; Noskov VN et al.; Transformation-associated recombination (TAR) cloning allows selective isolation of a desired chromosomal region or gene from complex genomes . The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast Saccharomyces cerevisiae . We investigated the effect of nonhomology on the efficiency of gene capture and found that up to 15% DNA divergence did not prevent efficient gene isolation . Such tolerance to DNA divergence greatly expands the potential applications of TAR cloning for comparative genomics . In this study, we were able to use the technique to isolate nonidentical chromosomal duplications and gene homologues. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9934 - 9 Epub 2003 Aug 08. Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity; Resnick MA et al.; There are many sources of genetic diversity, ranging from programmed mutagenesis in antibody genes to random mutagenesis during species evolution or development of cancer . We propose that mutations in DNA sequence-specific transcription factors that target response elements (REs) in many genes can also provide for rapid and broad phenotypic diversity, if the mutations lead to altered binding affinities at individual REs . To test this concept, we examined the in vivo transactivation capacity of wild-type human and murine p53 and 25 partial function mutants . The p53s were expressed in yeast from a rheostatable promoter, and the transactivation capacities toward >15 promoter REs upstream of a reporter gene were measured . Surprisingly, there was wide variation in transactivation by the mutant p53s toward the various REs . This is the first study to address directly the impact of mutations in a sequence-specific transcription factor on transactivation from a wide array of REs . We propose a master gene hypothesis for phenotypic diversity where the master gene is a single transcriptional activator (or repressor) that regulates many genes through different REs . Mutations of the master gene can lead to a variety of simultaneous changes in both the selection of targets and the extent of transcriptional modulation at the individual targets, resulting in a vast number of potential phenotypes that can be created with minimal mutational changes without altering existing protein-protein interactions. J Exp Biol, 2003 Sep, 206(Pt 18), 3227 - 37 Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity; Aviezer-Hagai K et al.; V-ATPase null mutants in yeast have a distinct, conditionally lethal phenotype that can be obtained through disruption of any one of its subunits . This enables supplementation of this mutant with the relevant subunit tagged with an epitope against which an antibody is available . In this system, the effect of antibody on the activity of the enzyme can be analyzed . Towards this end we used HA to tag subunits Vma7p, Vma10p and Vma16p, which are assumed to represent, respectively, the shaft, stator and turbine of the enzyme, and used them to supplement the corresponding yeast V-ATPase null mutants . The anti-HA epitope antibody inhibited both the ATP-dependent proton uptake and the ATPase activities of the Vma16p-HA and Vma7p-HA containing complexes, in intact vacuoles and in the detergent-solubilized enzyme . Neither of these activities was inhibited by the antibody in Vma10p-HA containing enzyme . These results support the function of Vma10p as part of the stator, while the other tagged subunits are part of the rotor apparatus . The HA-tag was attached to the N terminus of Vma16p; thus the antibody inhibition points to its accessibility outside the vacuolar membrane . This assumption is supported by the supplementation of the yeast mutant by the homologues of Vma16p isolated from Arabidopsis thaliana and lemon fruit c-DNA . Contrary to yeast, which has five predicted helices, the plant subunit Vma16p has only four . Our results confirm a recent report that only four of the yeast Vma16p complexes are actually transmembrane helices. Biochem J, 2003 Nov 1, 375(Pt 3), 673 - 80 Site-directed mutagenesis of the active site of diacylglycerol kinase alpha: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms; Abe T et al.; Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover . DAGKa is activated in vitro by Ca2+ and by acidic phospholipids . The regulatory region of DAGKa includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation . DAGKa also contains tandem C1 protein kinase C homology domains . We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKa and to determine the enzymic activities of different mutant forms of pig DAGKa in vitro . Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis . Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases . Mutation of D434 (Asp434) or D650 abolished all DAGKa activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKa . Roles of homologous residues in phosphofructokinases suggested that the N-terminal half of the DAGK catalytic domain binds Mg-ATP and the C-terminal half binds diacylglycerol . A DAGKa mutant with its entire regulatory region deleted showed a much decreased activity that was not activated by Ca2+, but still exhibited PS (phosphatidylserine)-dependent activation . Moreover, mutations of aspartate residues at the catalytic domain had differential effects on activation by Ca2+ and PS . These results indicate that Ca2+ and PS stimulate DAGKa via distinct mechanisms. Nucleic Acids Res . 2003 Aug 15;31(16):e94. A sensitive transcriptome analysis method that can detect unknown transcripts; Fukumura R et al.; We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis . By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased . As a result we can determine the relationship between peaks and original transcripts unequivocally . This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks . This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species . The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression . Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs . Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes . Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available. Nucleic Acids Res, 2003 Aug 15, 31(16), 4888 - 98 Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus; Grainge I et al.; The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing . Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea . The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue . The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro . The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA . Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA . The purified Orc/Cdc6 homologue was also able to bind DNA . Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates . Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap . The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer. Genomics, 2003 Sep, 82(3), 401 - 5 The centrosomal proteins pericentrin and kendrin are encoded by alternatively spliced products of one gene; Flory MR et al.; Pericentrin, a critical centrosome component first identified in mouse, recruits factors required for assembly of the mitotic spindle apparatus . A similar yet larger human protein named kendrin was recently identified, but its relationship to pericentrin was not clear . Extensive sequence homology between the mouse chromosome 10 region encoding pericentrin and the human chromosome 21 region encoding kendrin indicates that these proteins are encoded by syntenic loci . However, comparison of the published mouse pericentrin cDNA sequence to mouse genomic DNA sequences revealed two important differences: the stop codon present in the published mouse pericentrin cDNA is not found in the mouse genomic sequence, and the 3' end of the published mouse pericentrin cDNA is a fragment from a different mouse chromosome . To resolve these discrepancies, we sequenced a mouse expressed sequence tag (EST) that corresponds to the 3' end for a 7.1-kb mouse pericentrin RNA encoded on chromosome 10 . Extensive northern blot analysis revealed that the pericentrin gene displays a complex expression pattern in both mouse and human: a 10-kb kendrin transcript is found in most tissues, whereas smaller transcripts are detected in a limited subset of tissues . These analyses demonstrate that pericentrin and kendrin are encoded by one gene, correct the previously published pericentrin cDNA sequence, and describe the complex expression pattern for a gene important for centrosome function in normal and transformed cells. Curr Biol, 2003 Aug 5, 13(15), R603 - 5 Meiosis: polo, FEAR and the art of dividing reductionally; Cohen-Fix O; Recent studies on the regulation of meiosis have uncovered new roles for old acquaintances: the polo-like kinase Cdc5 has been found to dictate proper kinetochore orientation during meiosis I, while the FEAR pathway is essential for some, but not all, aspects of meiosis I exit. Curr Biol, 2003 Aug 5, 13(15), R597 - 9 Microtubule dynamics: new surprises from an old MAP; McNally F; Dis1/XMAP215 family microtubule-binding proteins are essential for cell division in animals, plants and fungi, suggesting a conserved cell-division mechanism used by all eukaryotes . Two new studies, however, reveal that different family members can have very different effects on microtubule dynamics. Phys Rev Lett . 2003 Aug 1;91(5):058701 . Epub 2003 Aug 01. Probabilistic prediction in scale-free networks: diameter changes; Kim JH et al.; In complex systems, responses to small perturbations are too diverse to definitely predict how much they would be, and then such diverse responses can be predicted in a probabilistic way . Here we study such a problem in scale-free networks, for example, the diameter changes by the deletion of a single vertex for various in silico and real-world scale-free networks . We find that the diameter changes are indeed diverse and their distribution exhibits an algebraic decay with an exponent zeta asymptotically . Interestingly, the exponent zeta is robust as zeta approximately 2.2(1) for most scale-free networks and insensitive to the degree exponents gamma as long as 2<gamma</=3 . However, there is another type with zeta approximately 1.7(1) and its examples include the Internet and its related in silico model. Planta, 2003 Aug, 217(4), 668 - 75 Epub 2003 Mar 28. Molecular cloning of an arabidopsis homologue of GCN2, a protein kinase involved in co-ordinated response to amino acid starvation; Zhang Y et al.; DNA homologous to the yeast ( Saccharomyces cerevisiae) protein kinase gene, GCN2, was amplified from arabidopsis { Arabidopsis thaliana (L.) Heynh.} RNA and given the name AtGCN2 . The AtGCN2 peptide sequence included adjacent protein kinase and histidyl tRNA synthetase-like domains and showed 45% sequence identity with the GCN2 peptide sequence in the protein kinase domain . AtGCN2 transcripts were detectable in RNA from roots, leaves, stems, buds, flowers, siliques and seedlings . GCN2 is required for yeast cells to respond to amino acid starvation . Expression of AtGCN2 in yeast gcn2 mutants complemented the mutation, enabling growth in the presence of sulfometuron methyl, an inhibitor of branched-chain amino acid biosynthesis, and 3-aminotriazole, an inhibitor of histidine biosynthesis. Anal Bioanal Chem, 2003 Aug, 376(7), 994 - 1005 Epub 2003 Jul 09. Acetylation of the HIV-1 Tat protein: an in vitro study; Dormeyer W et al.; In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased . It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes . Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides . In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites . We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region . Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme . Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general. Nature, 2003 Aug 28, 424(6952), 1083 - 7 Epub 2003 Aug 06. Replication of a cis-syn thymine dimer at atomic resolution; Ling H et al.; Ultraviolet light damages DNA by catalysing covalent bond formation between adjacent pyrimidines, generating cis-syn cyclobutane pyrimidine dimers (CPDs) as the most common lesion . CPDs block DNA replication by high-fidelity DNA polymerases, but they can be efficiently bypassed by the Y-family DNA polymerase pol eta . Mutations in POLH encoding pol eta are implicated in nearly 20% of xeroderma pigmentosum, a human disease characterized by extreme sensitivity to sunlight and predisposition to skin cancer . Here we have determined two crystal structures of Dpo4, an archaeal pol eta homologue, complexed with CPD-containing DNA, where the 3' and 5' thymine of the CPD separately serves as a templating base . The 3' thymine of the CPD forms a Watson-Crick base pair with the incoming dideoxyATP, but the 5' thymine forms a Hoogsteen base pair with the dideoxyATP in syn conformation . Dpo4 retains a similar tertiary structure, but each unusual DNA structure is individually fitted into the active site for catalysis . A model of the pol eta-CPD complex built from the crystal structures of Saccharomyces cerevisiae apo-pol eta and the Dpo4-CPD complex suggests unique features that allow pol eta to efficiently bypass CPDs. Microbiology, 2003 Aug, 149(Pt 8), 2039 - 48 Identification and functional expression of ctaA, a P-type ATPase gene involved in copper trafficking in Trametes versicolor; Uldschmid A et al.; Here the identification and characterization of a gene encoding a copper-trafficking enzyme, ctaA (copper-transporting ATPase), from the basidiomycete Trametes versicolor are described . This P-type copper ATPase gene has two alleles, differing primarily in the length of the second, unusually long intron, and encodes a 983 aa protein with 40 % sequence identity to yeast Ccc2p . Overexpression of ctaA in yeast grown in the presence of copper led to a 15-fold increase in laccase yields, while overexpression of ctaA and tahA, a previously identified copper homeostasis gene of T . versicolor, was additive, leading to a 20-fold increase in laccase production . In T . versicolor, overexpression of ctaA and tahA led to an eightfold increase in laccase expression, and a cotransformant still expressed laccase at 3000 micro M copper when hardly any laccase activity is detected in the wild-type strain . Apparently, at low to moderate levels of copper tahA and ctaA overexpression disturbs the normal hierarchy of copper distribution, resulting in more being directed to the Golgi, while with high copper amounts that normally switch on the copper detoxification processes, tahA and ctaA gene products seem to out-compete the metallothionein copper chaperones, meaning laccase is still supplied with copper . These results may lead to a better understanding of copper trafficking and the hierarchy of copper distribution in the cell, and possibly be useful for constructing laccase-overproducing strains for biotechnological purposes. Nucleic Acids Symp Ser, 2000, (44), 259 - 60 Histone acetyltransferase (HAT) activity of ATF-2 is necessary for the CRE-dependent transcription; Kawasaki H et al.; ATF-2 is a DNA-binding protein that binds to cAMP-response elements (CREs) and forms a hetrodimer with c-Jun, via binding of the leucine zipper motif and then stimulates the CRE-dependent transcription (1,2) . Recently, we have reported that ATF-2 has an intrinsic acetyltransferase activity that is controlled by phosphorylation . Mutant form of either p300 or ATF-2 with mutations in the HAT domain failed to stimulate the CRE-dependent transcription in response to UV irradiation . Moreover, phosphorylation of ATF-2 enhanced its HAT activity and CRE-dependent transcription . Thus, these results indicate that HAT activity of ATF-2 is important for the CRE-dependent transcription. Nucleic Acids Symp Ser, 2000, (44), 245 - 6 In vitro selection by using mutated GCN4-bZIP peptides for analysis of peptide-DNA interactions; Furusawa H et al.; In vitro selection has been used as a method to determine the optimal binding site for DNA-binding proteins . We report here in vitro selection of dsDNA sequences that bind to mutated-GCN4-bZIP peptides . The GCN4-bZIP peptide mutated from alanine to histidine on a position-14 that contacts with DNA bound to different sequence from a binding site of wild type peptide. Nucleic Acids Symp Ser, 2000, (44), 13 - 4 DNA binding of a basic leucine-zipper protein with novel folding domain; Sato S et al.; DNA-binding proteins frequently utilize short alpha-helices as their critical DNA recognition elements . In this research, we have employed the structure-based design to construct a small domain that could target the specific DNA sequences recognized by the yeast transcriptional activator GCN4 . The new DNA binding motif recognizes specific DNA sequences as a dimer with high affinity and specificity under the physiological conditions. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9668 - 73 Epub 2003 Aug 05. Shrinkage-based similarity metric for cluster analysis of microarray data; Cherepinsky V et al.; The current standard correlation coefficient used in the analysis of microarray data was introduced by M . B . Eisen, P . T . Spellman, P . O . Brown, and D . Botstein {(1998) Proc . Natl . Acad . Sci . USA 95, 14863-14868} . Its formulation is rather arbitrary . We give a mathematically rigorous correlation coefficient of two data vectors based on James-Stein shrinkage estimators . We use the assumptions described by Eisen et al., also using the fact that the data can be treated as transformed into normal distributions . While Eisen et al . use zero as an estimator for the expression vector mean mu, we start with the assumption that for each gene, mu is itself a zero-mean normal random variable {with a priori distribution N(0,tau 2)}, and use Bayesian analysis to obtain a posteriori distribution of mu in terms of the data . The shrunk estimator for mu differs from the mean of the data vectors and ultimately leads to a statistically robust estimator for correlation coefficients . To evaluate the effectiveness of shrinkage, we conducted in silico experiments and also compared similarity metrics on a biological example by using the data set from Eisen et al . For the latter, we classified genes involved in the regulation of yeast cell-cycle functions by computing clusters based on various definitions of correlation coefficients and contrasting them against clusters based on the activators known in the literature . The estimated false positives and false negatives from this study indicate that using the shrinkage metric improves the accuracy of the analysis. Genome Res, 2003 Aug, 13(8), 1863 - 72 Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes; Yang E et al.; Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions . We decided to measure mRNA decay rates in two human cell lines with high-density oligonucleotide arrays that enable the measurement of decay rates simultaneously for thousands of mRNA species . Using existing annotation and the Gene Ontology hierarchy of biological processes, we assign mRNAs to functional classes at various levels of resolution and compare the decay rate statistics between these classes . The results show statistically significant organizational principles in the variation of decay rates among functional classes . In particular, transcription factor mRNAs have increased average decay rates compared with other transcripts and are enriched in "fast-decaying" mRNAs with half-lives <2 h . In contrast, we find that mRNAs for biosynthetic proteins have decreased average decay rates and are deficient in fast-decaying mRNAs . Our analysis of data from a previously published study of Saccharomyces cerevisiae mRNA decay shows the same functional organization of decay rates, implying that it is a general organizational scheme for eukaryotes . Additionally, we investigated the dependence of decay rates on sequence composition, that is, the presence or absence of short mRNA motifs in various regions of the mRNA transcript . Our analysis recovers the positive correlation of mRNA decay with known AU-rich mRNA motifs, but we also uncover further short mRNA motifs that show statistically significant correlation with decay . However, we also note that none of these motifs are strong predictors of mRNA decay rate, indicating that the regulation of mRNA decay is more complex and may involve the cooperative binding of several RNA-binding proteins at different sites. Trends Genet, 2003 Aug, 19(8), 439 - 46 Telomere maintenance and DNA replication: how closely are these two connected? Chakhparonian M, Wellinger RJ. The maintenance of the DNA at chromosome ends, the telomeres, depends on conventional semiconservative replication and on the action of telomerase, a specialized reverse transcriptase . Current research strongly suggests a regulatory interplay between this conventional semiconservative replication and telomerase, thus ensuring that no sequences are lost at the very ends of the telomeres during replication . Here, we describe recent findings on the interactions between the conventional replication machinery and telomere replication, and we discuss how DNA-integrity checkpoints might impinge on both the processing of the telomeric DNA ends and the establishment of the DNA end structure required for end protection and genome stability. Trends Genet, 2003 Aug, 19(8), 422 - 7 Genomic analysis of gene expression relationships in transcriptional regulatory networks; Yu H et al.; From merging several data sources, we created an extensive map of the transcriptional regulatory network in Saccharomyces cerevisiae, comprising 7419 interactions connecting 180 transcription factors (TFs) with their target genes . We integrated this network with gene-expression data, relating the expression profiles of TFs and target genes . We found that genes targeted by the same TF tend to be co-expressed, with the degree of co-expression increasing if genes share more than one TF . Moreover, shared targets of a TF tend to have similar cellular functions . By contrast, the expression relationships between the TFs and their targets are much more complicated, often exhibiting time-shifted or inverted behavior . Further information is available at http://bioinfo.mbb.yale.edu/regulation/TIG/ Trends Genet, 2003 Aug, 19(8), 415 - 7 Moonlighting proteins: old proteins learning new tricks; Jeffery CJ; Recently, several laboratories identifying proteins involved in the complex processes of replication, transcription and tumor suppression found that the 'new' protein they discovered had another, previously identified, function . A single protein with multiple functions might seem surprising, but there are actually many cases of proteins that 'moonlight', or have more than one role in an organism . As well as adding to the number and types of proteins that are known to moonlight, these new examples add to our understanding of the potential importance of moonlighting proteins. Biochem Biophys Res Commun, 2003 Aug 22, 308(2), 276 - 83 Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependent manner, but remain competent for mitochondrial fusion; Sesaki H et al.; The dynamin-related GTPase, Mgm1p, is critical for the fusion of the mitochondrial outer membrane, maintenance of mitochondrial DNA (mtDNA), formation of normal inner membrane structures, and inheritance of mitochondria . Although there are two forms of Mgm1p, 100 and 90 kDa, their respective functions and the mechanism by which these two forms are produced are not clear . We previously isolated ugo2 mutants in a genetic screen to identify components involved in mitochondrial fusion {J . Cell Biol . 152 (2001) 1123} . In this paper, we show that ugo2 mutants are defective in PCP1, a gene encoding a rhomboid-related serine protease . Cells lacking Pcp1p are defective in the processing of Mgm1p and produce only the larger (100 kDa) form of Mgm1p . Similar to mgm1delta cells, pcp1delta cells contain partially fragmented mitochondria, instead of the long tubular branched mitochondria of wild-type cells . In addition, pcp1delta cells, like mgm1delta cells, lack mtDNA and therefore are unable to grow on nonfermentable medium . Mutations in the catalytic domain lead to complete loss of Pcp1p function . Similar to mgm1delta cells, the fragmentation of mitochondria and loss of mtDNA of pcp1delta cells were rescued when mitochondrial division was blocked by inactivating Dnm1p, a dynamin-related GTPase . Surprisingly, in contrast to mgm1delta cells, which are completely defective in mitochondrial fusion, pcp1delta cells can fuse their mitochondria after yeast cell mating . Our study demonstrates that Pcp1p is required for the processing of Mgm1p and controls normal mitochondrial shape and mtDNA maintenance by producing the 90 kDa form of Mgm1p . However, the processing of Mgm1p is not strictly required for mitochondrial fusion, indicating that the 100 kDa form is sufficient to promote fusion. Neurobiol Dis, 2003 Aug, 13(3), 238 - 45 Presenilin-1 interacts directly with the beta-site amyloid protein precursor cleaving enzyme (BACE1); Hebert SS et al.; A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques . The major constituent of these plaques, occurring largely in brain areas important for memory and cognition, is the 40-42 amyloid residues (Abeta) . Abeta is derived from the amyloid protein precursor after cleavage by the recently identified beta-secretase (BACE1) and the putative gamma-secretase complex containing presenilin 1 (PS1) . In an attempt to develop a functional secretase enzymatic assay in yeast we demonstrate a direct binding between BACE1 and PS1 . This interaction was confirmed in vivo using coimmunoprecipitation and colocalization studies in human cultured cells . Our results show that PS1 preferably binds immature BACE1, thus possibly acting as a functional regulator of BACE1 maturation and/or activity. J Biomol Tech, 2003 Mar, 14(1), 17 - 32 A rapid method for manual or automated purification of fluorescently labeled nucleic acids for sequencing, genotyping, and microarrays; Springer AL et al.; Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids . To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors . Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation . Common methods for purifying reaction products involve several steps and require processes that are not easily automated . Prolinx, Inc . has devel oped RapXtract superparamagnetic separation technology affording rapid and easy-to-perform methods that yield high-quality product and are easily automated . The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors . These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow . RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes . RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization . The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids. J Cell Biol, 2003 Aug 4, 162(3), 413 - 23 Vps27 recruits ESCRT machinery to endosomes during MVB sorting; Katzmann DJ et al.; Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway . The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins . Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway . We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27 . ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27 . A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23 . We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin . Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes. Biochemistry, 2003 Aug 12, 42(31), 9249 - 56 Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry; Sauve AA et al.; Life span regulation and inhibition of gene silencing in yeast have been linked to nicotinamide effects on Sir2 enzymes . The Sir2 enzymes are NAD(+)-dependent protein deacetylases that influence gene expression by forming deacetylated proteins, nicotinamide and 2'-O-acetyl-ADPR . Nicotinamide is a base-exchange substrate as well as a biologically effective inhibitor . Characterization of the base-exchange reaction reveals that nicotinamide regulates sirtuins by switching between deacetylation and base exchange . Nicotinamide switching is quantitated for the Sir2s from Archeaglobus fulgidus (Sir2Af2), Saccharomyces cerevisiae (Sir2p), and mouse (Sir2alpha) . Inhibition of deacetylation was most effective for mouse Sir2 alpha, suggesting species-dependent development of this regulatory mechanism . The Sir2s are proposed to form a relatively stable covalent intermediate between ADPR and the acetyl oxygen of the acetyllysine-protein substrate . During the lifetime of this intermediate, nicotinamide occupation of the catalytic site determines the fate of the covalent complex . Saturation of the nicotinamide site for mouse, yeast, and bacterial Sir2s causes 95, 65, and 21% of the intermediate, respectively, to return to acetylated protein . The fraction of the intermediate committed to deacetylation results from competition between the nicotinamide and the neighboring 2'-hydroxyl group at the opposite stereochemical face . Nicotinamide switching supports the previously proposed Sir2 catalytic mechanism and the existence of a 1'-O-peptidyl-ADPR.Sir2 intermediate . These findings suggest a strategy for increasing Sir2 enzyme catalytic activity in vivo by inhibition of chemical exchange but not deacetylation. Recent Results Cancer Res, 2003, 164, 349 - 52 A low-calcemic vitamin D analog (Ro 25-4020) inhibits the growth of LNCaP human prostate cancer cells with increased potency by producing an active 24-oxo metabolite (Ro 29-9970); Swami S et al.; In this study, we have characterized a novel less-calcemic vitamin D analog Ro 25-4020 (1alpha, 25 dihydroxy-16-ene-5,6-trans-vitamin D3) and investigated the mechanisms underlying its enhanced growth inhibitory properties . We found that Ro 25-4020 (IC50 = 0.3 nM) exhibited greater inhibitory activity than 1,25(OH)2D3 (IC50 = 1 nM) on LNCaP human prostate cancer cell growth . However, Ro 25-4020 was tenfold less active than 1,25(OH)2D3 in receptor-binding assays, ligand-induced heterodimerization and transactivation assays using VDR . HPLC and GC-MS analyses revealed that 1,25(OH)2D3 is converted to a 24-hydroxy metabolite, which has been shown to be less potent than 1,25(OH)2D3 . In contrast, Ro 25-4020 was converted to a major 24-oxo metabolite that was more stable . Ligand-binding assays reveal that both Ro 25-4020 and its 24-oxo metabolite have similar affinity for VDR . Synthetic 24-oxo-Ro 25-4020, however, inhibited LNCaP cell proliferation as potently as 1,25(OH)2D3 and was more potent in transactivation of two out of three vitamin D target genes tested . These results suggest that conversion of Ro 25-4020 into an active and more stable 24-oxo metabolite with longer half-life contributes significantly to its potent antiproliferative actions on the LNCaP cells. J Cell Biochem, 2003 Aug 15, 89(6), 1215 - 21 alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform; Moura Lima PR et al.; The band 3 protein is the major integral protein present in the erythrocyte membrane . Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes . It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown . In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform . The assay revealed two clones containing the C-terminal region of the alpha-cardiac actin . Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay . The confocal microscopy showed band 3 in the intercalated discs . Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with alpha-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane . Nat Struct Biol, 2003 Sep, 10(9), 718 - 24 Epub 2003 Aug 03. Cooperative organization in a macromolecular complex; Seeliger MA et al.; The mechanism of assembly of multiprotein complexes and the subsequent organization of activity are not well understood . Here we report the application of biophysical tools to investigate the relationship between structure and function in protein assemblies . We used as a model system the SCF(Skp2) complex that targets p27(Kip1) for ubiquitination and subsequent degradation; this process requires an adapter protein, Cks1 . By dissecting the interactions between the different subunits we show that the properties of Cks1 are highly context dependent, and its activity is acquired only when the complex is fully assembled . The results provide insights into the central role of small adapters in macromolecular assembly and explain their high sequence conservation . Simultaneous and synergistic binding of multiple subunits in a complex provides the specificity and control required before the key cell-cycle regulator p27 is committed to degradation. Plant Cell, 2003 Aug, 15(8), 1817 - 32 Cytoplasmic N-terminal protein acetylation is required for efficient photosynthesis in Arabidopsis; Pesaresi P et al.; The Arabidopsis atmak3-1 mutant was identified on the basis of a decreased effective quantum yield of photosystem II . In atmak3-1, the synthesis of the plastome-encoded photosystem II core proteins D1 and CP47 is affected, resulting in a decrease in the abundance of thylakoid multiprotein complexes . DNA array-based mRNA analysis indicated that extraplastid functions also are altered . The mutation responsible was localized to AtMAK3, which encodes a homolog of the yeast protein Mak3p . In yeast, Mak3p, together with Mak10p and Mak31p, forms the N-terminal acetyltransferase complex C (NatC) . The cytoplasmic AtMAK3 protein can functionally replace Mak3p, Mak10p, and Mak31p in acetylating N termini of endogenous proteins and the L-A virus Gag protein . This result, together with the finding that knockout of the Arabidopsis MAK10 homolog does not result in obvious physiological effects, indicates that AtMAK3 function does not require NatC complex formation, as it does in yeast . We suggest that N-acetylation of certain chloroplast precursor protein(s) is necessary for the efficient accumulation of the mature protein(s) in chloroplasts. Mol Cell Biol, 2003 Aug, 23(16), 5928 - 38 Support for a meiotic recombination initiation complex: interactions among Rec102p, Rec104p, and Spo11p; Jiao K et al.; Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae requires at least 10 gene products . The initiation event creates double-strand breaks, which are then processed by other recombination enzymes . A variety of classical observations, such as the existence of recombination nodules, have suggested that the proteins catalyzing recombination form a complex . A variety of lines of evidence indicate that Rad50p, Mre11p, and Xrs2p interact, and genetic data suggesting interactions between Rec102p and Rec104p have been reported . It has recently been shown that Spo11p coimmunoprecipitates with Rec102p in meiosis as well . In this paper, we provide genetic and biochemical evidence that the meiosis-specific proteins Rec102p, Rec104p, and Spo11p all interact with each other in meiosis . Furthermore, we demonstrate that the interaction between Rec102p and Spo11p does not require Rec104p . Likewise, the interaction between Rec104p and Rec102p does not require Spo11p, although Spo11p may stabilize that association . The interactions suggest that Spo11p, Rec102p, and Rec104p may form a trimeric complex during the initiation of recombination. Mol Cell Biol, 2003 Aug, 23(16), 5857 - 66 Structural environment dictates the biological significance of heme-responsive motifs and the role of Hsp90 in the activation of the heme activator protein Hap1; Lee HC et al.; Heme-responsive motifs (HRMs) mediate heme regulation of diverse regulatory proteins . The heme activator protein Hap1 contains seven HRMs, but only one of them, HRM7, is essential for heme activation of Hap1 . To better understand the molecular basis underlying the biological significance of HRMs, we examined the effects of various mutations of HRM7 on Hap1 . We found that diverse mutations of HRM7 significantly diminished the extent of Hap1 activation by heme and moderately enhanced the interaction of Hap1 with Hsp90 . Furthermore, deletions of nonregulatory sequences completely abolished heme activation of Hap1 and greatly enhanced the interaction of Hap1 with Hsp90 . These results show that the biological functions of HRMs and Hsp90 are highly sensitive to structural changes . The unique role of HRM7 in heme activation stems from its specific structural environment, not its mere presence . Likewise, the role of Hsp90 in Hap1 activation is dictated by the conformational or structural state of Hap1, not by the mere strength of Hap1-Hsp90 interaction . It appears likely that HRM7 and Hsp90 act together to promote the Hap1 conformational changes that are necessary for Hap1 activation . Such fundamental mechanisms of HRM-Hsp90 cooperation may operate in diverse regulatory systems to mediate signal transduction. Mol Cell Biol, 2003 Aug, 23(16), 5572 - 80 Role of mammalian Rad54 in telomere length maintenance; Jaco I et al.; The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown . Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability . Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals . Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping . Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity. Mol Cell Biol, 2003 Aug, 23(16), 5526 - 39 p21-Activated kinase 5 (Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD; Cotteret S et al.; Pak5 is the most recently identified and least understood member of the p21-activated kinase (Pak) family . This kinase is known to promote neurite outgrowth in vitro, but its localization, substrates, and effects on cell survival have not been reported . We show here that Pak5 has unique properties that distinguish it from all other members of the Pak family . First, Pak5, unlike Pak1, cannot complement an STE20 mutation in Saccharomyces cerevisiae . Second, Pak5 binds to the GTPases Cdc42 and Rac, but these GTPases do not regulate Pak5 kinase activity, which is constitutive and stronger than any other Pak . Third, Pak5 prevents apoptosis induced by camptothecin and C2-ceramide by phosphorylating BAD on Ser-112 in a protein kinase A-independent manner and prevents the localization of BAD to mitochondria, thereby inhibiting the apoptotic cascade that leads to apoptosis . Finally, we show that Pak5 itself is constitutively localized to mitochondria, and that this localization is independent of kinase activity or Cdc42 binding . These features make Pak5 unique among the Pak family and suggest that it plays an important role in apoptosis through BAD phosphorylation. Mol Cell Biol, 2003 Aug, 23(16), 5516 - 25 Identification of a unique core domain of par-4 sufficient for selective apoptosis induction in cancer cells; El-Guendy N et al.; Recent studies indicated that the leucine zipper domain protein Par-4 induces apoptosis in certain cancer cells by activation of the Fas prodeath pathway and coparallel inhibition of NF-kappaB transcriptional activity . However, the intracellular localization or functional domains of Par-4 involved in apoptosis remained unknown . In the present study, structure-function analysis indicated that inhibition of NF-kappaB activity and apoptosis is dependent on Par-4 translocation to the nucleus via a bipartite nuclear localization sequence, NLS2 . Cancer cells that were resistant to Par-4-induced apoptosis retained Par-4 in the cytoplasm . Interestingly, a 59-amino-acid core that included NLS2 but not the C-terminal leucine zipper domain was necessary and sufficient to induce Fas pathway activation, inhibition of NF-kappaB activity, and apoptosis . Most important, this core domain had an expanded target range for induction of apoptosis, extending to previously resistant cancer cells but not to normal cells . These findings have identified a unique death-inducing domain selective for apoptosis induction in cancer cells (SAC domain) which holds promise for identifying key differences between cancer and normal cells and for molecular therapy of cancer. J Biol Chem, 2003 Oct 17, 278(42), 41083 - 92 Epub 2003 Aug 01. Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest; Yanow SK et al.; We have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts . Drf1 forms an active complex with the kinase Cdc7 . However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein . Immunodepletion of Drf1 does not prevent DNA replication in egg extracts . Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels . Nonetheless, Drf1 displays highly regulated binding to replicating chromatin . Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin . This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin . These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin . Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin . This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest . We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts . Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks. J Biol Chem, 2003 Oct 17, 278(42), 41462 - 71 Epub 2003 Aug 01. Targeting and assembly of mitochondrial tail-anchored protein Tom5 to the TOM complex depend on a signal distinct from that of tail-anchored proteins dispersed in the membrane; Horie C et al.; Mitochondrial outer membrane proteins are synthesized without a cleavable presequence but instead contain segments responsible for mitochondrial targeting and membrane integration within the molecule: the transmembrane segment (TMS) and N- or C-terminal flanking segment . We analyzed targeting and integration of Tom5, a C-tail anchor protein associated with the preprotein translocase of the outer membrane, to the yeast mitochondrial outer membrane in vivo using green fluorescent protein as the reporter and compared the signal with other signals for proteins dispersed in the membrane . The functional assembly of Tom5 into the TOM complex was assessed by blue native PAGE and complementation of temperature-sensitive deltatom5 cells . Correct targeting and assembly required (i) . an appropriate length TMS rather than hydrophobicity, (ii) . a proline residue located at correct position in the TMS and specific residues near the proline, and (iii) . that, in contrast to proteins dispersed in the outer membrane, the positive C-terminal segment was dispensable . Based on these findings, we constructed green fluorescent protein fusions with a C-terminal TMS in which the deduced sequences (minimum: Ser-Pro-Met) were inserted at an appropriate position within artificial Leu-Ala repeats . They were targeted to mitochondria and complemented the temperature-sensitive growth phenotype of deltatom5 yeast cells . The membrane-targeting mechanism of Tom5 appears to be distinct from that for proteins that are dispersed in the outer membrane. Anal Biochem, 2003 Sep 1, 320(1), 125 - 8 Ubiquinone binding protein used for determination of coenzyme Q; Hagerman RA et al.; The conventional method of assaying for the ubiquinone (CoQ) content of biological samples is to partition CoQ into an organic phase and separate it from contaminants by high-performance liquid chromatography (HPLC) . HPLC is an accurate method of quantifying CoQ content but is not ideal for routine clinical analyses . This paper describes the development of a rapid method for assaying the CoQ content of biological samples based on the binding of CoQ to a CoQ binding peptide . The 14-amino acid binding peptide was chemically synthesized, and conditions for immobilizing the peptide on microfuge tubes were established . CoQ could be selectively bound to the immobilized peptide, eluted, and determined spectrophotometrically . Limits of detection for the method were 0.25 to 5 nmol CoQ . To test biological samples, CoQ was isolated from cultures of Saccharomyces cerevisiae grown in oleic acid medium . The recovery of CoQ samples using the binding assay ranged from 99 to 102% of the values obtained with HPLC . The assay described here provides an inexpensive, rapid method for determining the CoQ content of large numbers of biological samples in a variety of laboratory settings. J Biochem Mol Biol, 2003 Jul 31, 36(4), 421 - 5 A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4; Xu K et al.; The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways . Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors . Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed . However, they require a generation of antibodies against the phosphorylated form of a specific substrate . We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phospho-threonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates . Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme. J Biochem Mol Biol, 2003 Jul 31, 36(4), 344 - 8 Identification of mutations in protein kinase CKIIbeta subunit that affect its binding to ribosomal protein L41 and homodimerization; Ahn BH et al.; Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits . The CKIIbeta subunit is thought to mediate the tetramer formation and interact with other target proteins . However, its physiological function remains obscure . In this study, point mutants of CKIIbeta that are defective for the L41 binding were isolated by using the reverse two-hybrid system . A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of CKIIbeta are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for CKIIbeta homodimerization . Two point mutants, R75 and R83, of CKIIbeta interacted with L5, topoisomerase IIbeta, and CKBBP1/SAG, but not with the wild-type CKIIbeta . This indicates that CKIIbeta homodimerization is not a prerequisite for its binding to target proteins . These CKIIbeta point mutants may be useful in exploring the biochemical physiological functions of CKIIbeta. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9873 - 7 Epub 2003 Jul 31. A SNARE required for retrograde transport to the endoplasmic reticulum; Burri L et al.; SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells . Sequence analysis of the yeast genome revealed a previously uncharacterized SNARE, SNARE-like tail-anchored protein 1 (Slt1) . Slt1 is an essential protein localized in the endoplasmic reticulum (ER) . It forms a SNARE complex with Sec22 and the ER syntaxin Ufe1 . Down-regulation of Slt1 levels leads to improper secretion of proteins normally resident in the ER . We suggest that Slt1 is a component of the SNAREpin required for retrograde traffic to the ER . Based on the previously reported association with Ufe1 and Sec22, Sec20 likely contributes the fourth SNARE to the SNAREpin. Mol Membr Biol, 2003 Jul-Sep, 20(3), 197 - 207 ER-to-Golgi transport: COP I and COP II function (Review); Duden R; COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells . These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles . COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER) . COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER . Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex. Biochem Biophys Res Commun, 2003 Aug 1, 307(3), 632 - 9 Bone morphogenetic protein signalling in NGF-stimulated PC12 cells; Althini S et al.; Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions . Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days . NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6 . PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity . Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response . Adding the inhibitory Smad7 drastically reduced this signal . In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity . A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active . Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity . However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription . The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site . Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells. Methods, 2003 Sep, 31(1), 83 - 9 Genomewide histone acetylation microarrays; Robyr D et al.; Histone acetylation and methylation are important regulators of gene activity . Chromatin immunoprecipitation (ChIP or ChrIP) has made it possible to examine not only the state of histone acetylation at a gene but also that of histone methylation and may soon be extended to other histone modifications such as phosphorylation and ubiquitination . In principle such studies are possible as long as an antibody is available to the particular histone modification . Once a target gene is identified it is instructive to see the effect of mutating putative enzymes responsible for the modification to determine how a particular enzyme is responsible for altering chromatin of that gene . Although specific target genes have been studied that contain such modifications recent technical advances have made it possible to study histone modifications genomewide . This not only allows for alternate views of particular paradigms to be investigated, but also uncovers chromosomal patterns of histone modification that would be missed in analyzing individual genes . We describe here an approach to rapidly study histone modifications genomewide by combining chromatin immunoprecipitation and DNA microarrays. Methods, 2003 Sep, 31(1), 59 - 66 In vivo assays to study histone ubiquitylation; Kao CF et al.; The importance of histone acetylation, phosphorylation, and methylation in transcription and other DNA-mediated processes is now well established . Histones are also ubiquitylated, but in contrast to the majority of ubiquitylated proteins, ubiquitylated histones are not generally targeted for degradation and may play roles similar to those of other histone modifications . Antibodies against acetylated histones have provided unique insights into the regulation, distribution, and cellular roles of these modified histones . In this report, we describe methods to identify ubiquitylated histones in budding yeast and HeLa cells . We provide protocols to detect ubiquitylated histones that are based on a combination of in vivo genetic and immunological assays . These methods should provide relatively simple and useful tools to study the global regulation of this important but poorly understood histone modification. Curr Opin Cell Biol, 2003 Aug, 15(4), 489 - 97 Peroxisome biogenesis: advances and conundrums; Lazarow PB; Investigations of peroxisome biogenesis in diverse organisms reveal new details of this unique process and its evolutionary conservation . Interactions among soluble receptors and the membrane peroxins that catalyze protein translocation are being mapped . Ubiquitination is observed . A receptor enters the organelle carrying folded cargo and recycles back to the cytosol . Tiny peroxisome remnants - vesicles and tubules - are discovered in pex3 mutants that lack the organelle . When the mutant is transfected with a good PEX3 gene, these protoperoxisomes acquire additional membrane peroxins and then import the matrix enzymes to reform peroxisomes . Thus, de novo formation need not be postulated . Dynamic imaging of yeast reveals dynamin-dependent peroxisome division and regulated actin-dependent segregation of the organelle before cell division . These results are consistent with biogenesis by growth and division of pre-existing peroxisomes. Curr Opin Cell Biol, 2003 Aug, 15(4), 446 - 55 Protein sorting into multivesicular endosomes; Raiborg C et al.; Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes . Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation . Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes . A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process . Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation . Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding. Biochem J, 2003 Oct 15, 375(Pt 2), 329 - 37 Direct interaction of beta-dystroglycan with F-actin; Chen YJ et al.; Dystroglycans are essential transmembrane adhesion receptors for laminin . Alpha-dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of beta-dystroglycan . Beta-dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton . As a part of the dystrophin-glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity . Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype . Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of beta-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells. Nature, 2003 Jul 31, 424(6948), 565 - 71 Machinery for protein sorting and assembly in the mitochondrial outer membrane; Wiedemann N et al.; Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes . It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination . Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM) . Mas37 forms a constituent of the SAM complex . The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11-15) . These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex. Nature, 2003 Jul 31, 424(6948), 549 - 52 Evolutionary capacitance as a general feature of complex gene networks; Bergman A et al.; An evolutionary capacitor buffers genotypic variation under normal conditions, thereby promoting the accumulation of hidden polymorphism . But it occasionally fails, thereby revealing this variation phenotypically . The principal example of an evolutionary capacitor is Hsp90, a molecular chaperone that targets an important set of signal transduction proteins . Experiments in Drosophila and Arabidopsis have demonstrated three key properties of Hsp90: (1) it suppresses phenotypic variation under normal conditions and releases this variation when functionally compromised; (2) its function is overwhelmed by environmental stress; and (3) it exerts pleiotropic effects on key developmental processes . But whether these properties necessarily make Hsp90 a significant and unique facilitator of adaptation is unclear . Here we use numerical simulations of complex gene networks, as well as genome-scale expression data from yeast single-gene deletion strains, to present a mechanism that extends the scope of evolutionary capacitance beyond the action of Hsp90 alone . We illustrate that most, and perhaps all, genes reveal phenotypic variation when functionally compromised, and that the availability of loss-of-function mutations accelerates adaptation to a new optimum phenotype . However, this effect does not require the mutations to be conditional on the environment . Thus, there might exist a large class of evolutionary capacitors whose effects on phenotypic variation complement the systemic, environment-induced effects of Hsp90. J Biol Chem, 2003 Oct 10, 278(41), 39517 - 26 Epub 2003 Jul 30. The mouse APG10 homologue, an E2-like enzyme for Apg12p conjugation, facilitates MAP-LC3 modification; Nemoto T et al.; Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles . The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential . In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation . The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation . The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p . Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate . Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy . Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3 . Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis . The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate. J Biol Chem, 2003 Oct 10, 278(41), 39303 - 10 Epub 2003 Jul 30. Dissection of the contribution of individual domains to the ATPase mechanism of Hsp90; Wegele H et al.; Hsp90 is a dimeric, ATP-regulated molecular chaperone . Its ATPase cycle involves the N-terminal ATP binding domain (amino acids (aa) 1-272) and, in addition, to some extent the middle domain (aa 273-528) and the C-terminal dimerization domain (aa 529-709) . To analyze the contribution of the different domains and the oligomeric state on the progression of the ATPase cycle of yeast Hsp90, we created deletion constructs lacking either the C-terminal or both the C-terminal and the middle domain . To test the effect of dimerization on the ATPase activity of the different constructs, we introduced a Cys residue at the C-terminal ends of the constructs, which allowed covalent dimerization . We show that all monomeric constructs tested exhibit reduced ATPase activity and a decreased affinity for ATP in comparison with wild type Hsp90 . The covalently linked dimers lacking only the C-terminal domain hydrolyze ATP as efficiently as the wild type protein . Furthermore, this construct is able to trap the ATP molecule similar to the full-length protein . This demonstrates that in the ATPase cycle, the C-terminal domain can be replaced by a cystine bridge . In contrast, the ATPase activity of the artificially linked N-terminal domains remains very low and bound ATP is not trapped . Taken together, we show that both the dimerization of the N-terminal domains and the association of the N-terminal with the middle domain are important for the efficiency of the ATPase cycle . These reactions are synergistic and require Hsp90 to be in the dimeric state. Biochem Biophys Res Commun, 2003 Aug 15, 308(1), 1 - 11 Composition and function of the eukaryotic N-terminal acetyltransferase subunits; Polevoda B et al.; Saccharomyces cerevisiae contains three N-terminal acetyltransferases (NATs), NatA, NatB, and NatC, composed of the following catalytic and auxiliary subunits: Ard1p and Nat1p (NatA); Nat3p and Mdm20p (NatB); and Mak3p, Mak10, and Mak31p (NatC) . The overall patterns of N-terminally acetylated proteins and NAT orthologous genes suggest that yeast and higher eukaryotes have similar systems for N-terminal acetylation . The differential expression of certain NAT subunits during development or in carcinomas of higher eukaryotes suggests that the NATs are more highly expressed in cells undergoing rapid protein synthesis . Although Mak3p is functionally the same in yeast and plants, findings with TE2 (a human Ard1p ortholog) and Tbdn100 (a mouse Nat1p ortholog) suggest that certain of the NAT subunits may have functions other than their role in NATs or that these orthologs are not functionally equivalent . Thus, the vertebrate NATs remain to be definitively identified, and, furthermore, it remains to be seen if any of the yeast NATs contribute to other functions. Mol Microbiol, 2003 Aug, 49(4), 859 - 67 Regulation of fungal gene expression via short open reading frames in the mRNA 5'untranslated region; Vilela C et al.; We review how the expression of fungal mRNAs can be controlled by ribosome interactions with short upstream open reading frames (uORFs) within the 5'untranslated region . The efficiency of uAUG recognition modulates the impact of a uORF but steps during and after translation of the uORF also influence uORF function . The post-termination behaviour of ribosomes, therefore, plays a major role in determining the expression level of these main ORFs . Translation of a uORF can produce a cis-acting peptide that causes effector molecule-dependent stalling of the ribosomes at the end of the uORF . In other cases it is the length or position, or other features of the uORF, rather than the peptide it encodes, that determine the efficiency with which ribosomes reinitiate translation downstream of it . Whether the form of the ribosome that resumes scanning after termination is the 40S subunit alone or the entire 80S ribosome is not known . Translation of the uORF can also control gene expression by affecting the stability of the mRNA . Finally, trans-acting factors may participate in the regulatory mechanisms . Future work will need not only to provide more information on the mechanisms underlying the known cases of uORF-mediated control but also to define the full complement of uORF-containing mRNAs in at least one fungal organism. Biotechnol Lett, 2003 Jun, 25(11), 891 - 3 A screening method for detecting iron reducing wood-rot fungi; Oviedo C et al.; A plate assay using the Fe(II) selective dye, ferrozine, for detecting wood-rot fungi with Fe(III) reductive abilities, was developed . The assay is fast, simple and, in most cases, more sensitive than the corresponding liquid medium test . The brown rot fungi, Gloeophyllum trabeum and Laetiporeus sulphureus, displayed higher iron reductive capabilities than white rot fungi, Trametes versicolor, Ganoderma australe and Ceriporiopsis subvermispora. J Biol Chem, 2003 Oct 10, 278(41), 39509 - 16 Epub 2003 Jul 29. Concomitant increase of histone acetyltransferase activity and degradation of p300 during retinoic acid-induced differentiation of F9 cells; Brouillard F et al.; The p300 and closely related CBP histone acetyltransferases (HAT) function as global transcriptional co-activators that play roles in many cell differentiation and signal transduction pathways . Despite their similarities, p300 and CBP have distinct functions during retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells . F9 cells constitute a well established model system for investigating the first steps of early development and retinoic acid signaling ex vivo . p300, but not CBP, was shown to be essential for F9 differentiation . In this study we have investigated the regulation of p300 during F9 differentiation . We report a dramatic decrease of p300, but not CBP protein levels, after 48 h of retinoic acid treatment . p300 is degraded via the ubiquitin-proteasome pathway . Although the large majority of p300 is degraded, its global HAT activity stays constant during F9 differentiation, which means that its specific HAT activity increases considerably . p300 is strongly phosphorylated in both undifferentiated and differentiated F9 cells; its HAT activity, however, is independent of phosphorylation before differentiation and becomes dependent on phosphorylation during differentiation . Furthermore, we show that protein kinase A affects p300 HAT activity both in vivo and in vitro as well as p300 phosphorylation in differentiated cells . Thus, we show that p300 is differentially phosphorylated in undifferentiated versus differentiated cells and that the changes in phosphorylation affect its HAT activity . Moreover, our study suggests an explanation for the functional switch of p300-mediated repression versus activation during F9 differentiation. Nucleic Acids Res, 2003 Aug 1, 31(15), 4597 - 607 The essential transcription factor Reb1p interacts with the CLB2 UAS outside of the G2/M control region; Van Slyke C et al.; Regulation of CLB2 is important both for completion of the normal vegetative cell cycle in Saccharomyces cerevisiae and for departure from the vegetative cell cycle upon nitrogen deprivation . Cell cycle-regulated transcription of CLB2 in the G2/M phase is known to be brought about by a set of proteins including Mcm1p, Fkh2/1p and Ndd1p that associate with a 35 bp G2/M-specific sequence common to a set of co-regulated genes . CLB2 transcription is regulated by additional signals, including by nitrogen levels, by positive feedback from the Clb2-Cdc28 kinase, and by osmotic stress, but the corresponding regulatory sequences and proteins have not been identified . We have found that the essential Reb1 transcription factor binds with high affinity to a sequence upstream of CLB2, within a region implicated previously by others in regulated expression, but upstream of the known G2/M-specific site . CLB2 sequence from the region around the Reb1p site blocks activation by the Gal4 protein when positioned downstream of the Gal4-binding site . Since a mutation in the Reb1p site abrogates this effect, we suggest that Reb1p is likely to occupy this site in vivo. Nucleic Acids Res, 2003 Aug 1, 31(15), 4553 - 60 The computational analysis of scientific literature to define and recognize gene expression clusters; Raychaudhuri S et al.; A limitation of many gene expression analytic approaches is that they do not incorporate comprehensive background knowledge about the genes into the analysis . We present a computational method that leverages the peer-reviewed literature in the automatic analysis of gene expression data sets . Including the literature in the analysis of gene expression data offers an opportunity to incorporate functional information about the genes when defining expression clusters . We have created a method that associates gene expression profiles with known biological functions . Our method has two steps . First, we apply hierarchical clustering to the given gene expression data set . Secondly, we use text from abstracts about genes to (i) resolve hierarchical cluster boundaries to optimize the functional coherence of the clusters and (ii) recognize those clusters that are most functionally coherent . In the case where a gene has not been investigated and therefore lacks primary literature, articles about well-studied homologous genes are added as references . We apply our method to two large gene expression data sets with different properties . The first contains measurements for a subset of well-studied Saccharomyces cerevisiae genes with multiple literature references, and the second contains newly discovered genes in Drosophila melanogaster; many have no literature references at all . In both cases, we are able to rapidly define and identify the biologically relevant gene expression profiles without manual intervention . In both cases, we identified novel clusters that were not noted by the original investigators. Nucleic Acids Res, 2003 Aug 1, 31(15), 4425 - 33 Spurious spatial periodicity of co-expression in microarray data due to printing design; Balazsi G et al.; Global transcriptome data is increasingly combined with sophisticated mathematical analyses to extract information about the functional state of a cell . Yet the extent to which the results reflect experimental bias at the expense of true biological information remains largely unknown . Here we show that the spatial arrangement of probes on microarrays and the particulars of the printing procedure significantly affect the log-ratio data of mRNA expression levels measured during the Saccharomyces cerevisiae cell cycle . We present a numerical method that filters out these technology-derived contributions from the existing transcriptome data, leading to improved functional predictions . The example presented here underlines the need to routinely search and compensate for inherent experimental bias when analyzing systematically collected, internally consistent biological data sets. Nucleic Acids Res, 2003 Aug 1, 31(15), 4391 - 400 Structural and functional homology between the RNAP(I) subunits A14/A43 and the archaeal RNAP subunits E/F; Meka H et al.; In the archaeal RNA polymerase and the eukaryotic RNA polymerase II, two subunits (E/F and RPB4/RPB7, respectively) form a heterodimer that reversibly associates with the core of the enzyme . Recently it has emerged that this heterodimer also has a counterpart in the other eukaryotic RNA polymerases: in particular two subunits of RNA polymerase I (A14 and A43) display genetic and biochemical characteristics that are similar to those of the RPB4 and RPB7 subunits, despite the fact that only A43 shows some sequence homology to RPB7 . We demonstrate that the sequence of A14 strongly suggests the presence of a HRDC domain, a motif that is found at the C-terminus of a number of helicases and RNases . The same motif is also seen in the structure of the F subunit, suggesting a structural link between A14 and the RPB4/C17/subunit F family, even in the absence of direct sequence homology . We show that it is possible to co-express and co-purify large amounts of the recombinant A14/A43 heterodimer, indicating a tight and specific interaction between the two subunits . To shed light on the function of the heterodimer, we performed gel mobility shift assays and showed that the A14/A43 heterodimer binds single-stranded RNA in a similar way to the archaeal E/F complex. Nucleic Acids Res, 2003 Aug 1, 31(15), 4326 - 31 Solution structure of the HIV-1 frameshift inducing stem-loop RNA; Staple DW et al.; The translation of reverse transcriptase and other essential viral proteins from the HIV-1 Pol mRNA requires a programmed -1 ribosomal frameshift . This frameshift is induced by two highly conserved elements within the HIV-1 mRNA: a slippery sequence comprised of a UUUUUUA heptamer, and a downstream stem-loop structure . We have determined the structure of the HIV-1 frameshift inducing RNA stem-loop, using multidimensional heteronuclear nuclear magnetic resonance (NMR) methods . The 22 nucleotide RNA solution structure {root mean squared deviation (r.m.s.d.) = 1.2 A} was determined from 475 nuclear Overhauser effect (NOE)-derived distance restrains, 20 residual dipolar couplings and direct detection of hydrogen bonds via scalar couplings . We find that the frameshift inducing stem-loop is an A-form helix capped by a structured ACAA tetraloop . The ACAA tetraloop is stabilized by an equilateral 5' and 3' stacking pattern, a sheared A-A pair and a cross-strand hydrogen bond . Unexpectedly, the ACAA tetraloop structure is nearly identical to a known tetraloop fold, previously identified in the RNase III recognition site from Saccharomyces cerevisiae. Nucleic Acids Res, 2003 Aug 1, 31(15), 4285 - 92 Mechanisms of P/CAF auto-acetylation; Santos-Rosa H et al.; P/CAF is a histone acetyltransferase enzyme which was originally identified as a CBP/p300-binding protein . In this manuscript we report that human P/CAF is acetylated in vivo . We find that P/CAF is acetylated by itself and by p300 but not by CBP . P/CAF acetylation can be an intra- or intermolecular event . The intermolecular acetylation requires the N-terminal domain of P/CAF . The intramolecular acetylation targets five lysines (416-442) at the P/CAF C-terminus, which are in the nuclear localisation signal (NLS) . Finally, we show that acetylation of P/CAF leads to an increment of its histone acetyltransferase (HAT) activity . These findings identify a new post-translation modification on P/CAF which may regulate its function. Trends Cell Biol, 2003 Aug, 13(8), 396 - 9 Cycling without CDK2? Grim JE, Clurman BE. Cyclin-dependent kinase 2 (CDK2) regulates diverse aspects of the mammalian cell cycle . Most cancer cells contain mutations in the pathways that control CDK2, and CDK2 activity has received much attention as a target for cancer therapy . However, a recent report demonstrating that some cancer cells can proliferate without CDK2 activity questions the essential role of CDK2 in cell-cycle control, as well as its suitability as a therapeutic target. Mol Cell, 2003 Jul, 12(1), 209 - 19 In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination; Sugawara N et al.; Repairing a double-strand break by homologous recombination requires binding of the strand exchange protein Rad51p to ssDNA, followed by synapsis with a homologous donor . Here we used chromatin immunoprecipitation to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HML alpha donor . Localization of Rad51p to MAT precedes its association with HML, providing evidence of the time needed for the Rad51 filament to search the genome for a homologous sequence . Rad51p binding to ssDNA requires Rad52p . The absence of Rad55p delays Rad51p binding to ssDNA and prevents strand invasion and localization of Rad51p to HML alpha . Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HML alpha; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand. Mol Cell, 2003 Jul, 12(1), 147 - 55 Involvement of actin-related proteins in ATP-dependent chromatin remodeling; Shen X et al.; Actin-related proteins (Arps) and conventional actin are enigmatic components of many chromatin-remodeling enzyme complexes . The yeast INO80 ATP-dependent chromatin-remodeling complex contains stoichiometric amounts of Arp4, Arp5, Arp8, and actin . Here we have revealed functions of Arp5 and Arp8 by analysis of mutants . arp5 Delta and arp8 Delta mutants display an ino80 Delta phenotype . Purification of INO80 complexes from arp5 Delta and arp8 Delta cells shows that protein complexes remain intact but are compromised for INO80 ATPase activity, DNA binding, and nucleosome mobilization . The INO80 (arp8 Delta) complex is strikingly deficient, not only for the Arp8 subunit, but also for Arp4 and actin, suggesting an ordered assembly of Arps . Binding of Arp8 to the INO80 complex requires an N-terminal region of Ino80 adjacent to the conserved ATPase domain . GST-Arp8 binds preferentially to histones H3 and H4 in vitro, suggesting a histone chaperone function . These findings show direct involvement of Arps in the chromatin-remodeling process. Mol Cell, 2003 Jul, 12(1), 101 - 11 Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties; Kelleher DJ et al.; Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum . Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined . The roles of specific OST subunits remained enigmatic . Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p . The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity . Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity. Mol Cell, 2003 Jul, 12(1), 51 - 62 Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state; Fulco M et al.; Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span . Prompted by the observation that the {NAD(+)}/{NADH} ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation . Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation . Conversely, cells with decreased Sir2 differentiate prematurely . To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity . The {NAD(+)}/{NADH} ratio decreases as muscle cells differentiate, while an increased {NAD(+)}/{NADH} ratio inhibits muscle gene expression . Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated {NAD(+)}/{NADH} ratio . These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor . In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression. Plant J, 2003 Aug, 35(3), 295 - 304 The soybean NRAMP homologue, GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport; Kaiser BN et al.; Iron is an important nutrient in N2-fixing legume root nodules . Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain . The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol . In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters . GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules . Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules . GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4) . The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules . Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed. J Virol, 2003 Aug, 77(16), 8695 - 701 Selection of retroviral reverse transcription primer is coordinated with tRNA biogenesis; Kelly NJ et al.; Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu . To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNA(Phe) to be supplied in trans for infectivity . Wild-type yeast tRNA(Phe) expressed in mammalian cells was transported to the cytoplasm and aminoacylated . In contrast, tRNA(Phe) without the D loop (tRNA(Phe)D(-)) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNA(Phe)D(-) was directly transfected into the cytoplasm of cells . A tRNA(Phe) mutant (tRNA(Phe)UUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNA(Phe) . Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis. J Biol Chem, 2003 Oct 10, 278(41), 39402 - 12 Epub 2003 Jul 28. The coactivator p/CIP/SRC-3 facilitates retinoic acid receptor signaling via recruitment of GCN5; Brown K et al.; p/CIP/SRC-3 is a member of a family of steroid receptor coactivators/nuclear receptor coactivators (SRC/NCoA) proteins that mediate the transcriptional effects of nuclear hormone receptors (NRs) . Using deletion analysis we have mapped the location of two distinct activation domains in p/CIP (AD1 and AD2) capable of activating transcription in mammalian cells when fused to the Gal4-DNA binding domain . In addition to AD1 being coincident with the interaction domain for CBP, we demonstrate a novel in vivo interaction between the AD1 and GCN5 . Overexpression of a Gal4-AD1 fusion protein in yeast leads to growth arrest that is relieved by mutation of genes encoding components of the SAGA complex including GCN5, ADA3, and SPT7 . In addition, the AD1 of p/CIP and the ADA3 gene are shown to be essential for retinoic acid receptor alpha-dependent transcription in yeast . Transient transfection assays in mammalian cells indicate that GCN5 cooperates with p/CIP as a coactivator of RAR alpha-dependent transcription . Down-regulation of GCN5 using small interfering RNA in mammalian cells indicates that the AD1 domain and the RAR beta promoter activity are dependent, in part, on GCN5 . Mutational analysis of AD1 has identified two helical motifs that are required for interactions with GCN5 and CBP . Taken together, these results support a model by which p/CIP functions as a ligand-dependent adapter, through specific protein-protein interactions with AD1, to recruit members from at least two distinct families of acetyltransferase proteins to NRs. J Cell Biol, 2003 Aug 4, 162(3), 403 - 12 Epub 2003 Jul 28. The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER; Morsomme P et al.; Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex . Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p . However, the t-SNARE Sed5p was not required for protein sorting upon ER exit . Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM . Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature . Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins. Dev Biol, 2003 Aug 1, 260(1), 245 - 59 MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C elegans; Yang HY et al.; In most animals, successful segregation of female meiotic chromosomes involves sequential associations of the meiosis I and meiosis II spindles with the cell cortex so that extra chromosomes can be deposited in polar bodies . The resulting reduction in chromosome number is essential to prevent the generation of polyploid embryos after fertilization . Using time-lapse imaging of living Caenorhabditis elegans oocytes containing fluorescently labeled chromosomes or microtubules, we have characterized the movements of meiotic spindles relative to the cell cortex . Spindle assembly initiated several microns from the cortex . After formation of a bipolar structure, the meiosis I spindle translocated to the cortex . When microtubules were partially depleted, translocation of the bivalent chromosomes to the cortex was blocked without affecting cell cycle timing . In oocytes depleted of the microtubule-severing enzyme, MEI-1, spindles moved to the cortex, but association with the cortex was unstable . Unlike translocation of wild-type spindles, movement of MEI-1-depleted spindles was dependent on FZY-1/CDC20, a regulator of the metaphase/anaphase transition . We observed a microtubule and FZY-1/CDC20-dependent circular cytoplasmic streaming in wild-type and mei-1 mutant embryos during meiosis . We propose that, in mei-1 mutant oocytes, this cytoplasmic streaming is sufficient to drive the spindle into the cortex . Cytoplasmic streaming is not the normal spindle translocation mechanism because translocation occurred in the absence of cytoplasmic streaming in embryos depleted of either the orbit/CLASP homolog, CLS-2, or FZY-1 . These results indicate a direct role of microtubule severing in translocation of the meiotic spindle to the cortex. FEBS Lett, 2003 Jul 31, 548(1-3), 108 - 12 Differential binding of Sin3 interacting repressor domains to the PAH2 domain of Sin3A; Pang YP et al.; The Sin3 interacting domain (SID), originally described in the Mad family of repressors, is a novel transcriptional repressor domain that binds the PAH2 domain of corepressors Sin3A and Sin3B with high affinities . The conserved SID-like domains are reportedly present in five KLF proteins . However, the KLF SIDs and the Mad SIDs can be classified into two subtypes according to sequence similarity . Here, we report the finding from computational and experimental studies that the two subtypes of SID domains bind differentially to Sin3A . This finding offers insights into a mechanism of cell growth regulation by interactions of different subtypes of SID-containing repressor proteins with Sin3 . It also provides the structural basis for developing selective modulators of Sin3. FEBS Lett, 2003 Jul 31, 548(1-3), 59 - 68 Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly; Thomas CF Jr et al.; Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV . The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity . In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature . The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis . We have identified a P . carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function . The P . carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast . Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2 . PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation . Since glucan machinery is not present in mammals, a better understanding of this pathway in P . carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall. FEBS Lett, 2003 Jul 31, 548(1-3), 33 - 6 A gene-specific effect of an internal deletion in the Bdp1 subunit of the RNA polymerase III transcription initiation factor TFIIIB; Ishiguro A et al.; The Saccharomyces cerevisiae RPR1 gene encodes the RNA subunit of its RNase P, which processes RNA polymerase (pol) III primary transcripts . RPR1, which is transcribed by pol III, has been isolated as a multicopy suppressor of a specific small internal deletion (amino acids 253-269) in the Bdp1 subunit of transcription factor TFIIIB, the core pol III transcription factor . The selective effect of this Bdp1 deletion on RPR1 transcription has been analyzed in vitro . It is shown that TFIIIC-dependent assembly of TFIIIB on the RPR1 promoter is specifically sensitive to this Bdp1 deletion, leading to gene-specifically defective single-round and multiple-round transcription. Chromosoma, 2003 Aug, 112(2), 58 - 65 Epub 2003 Jul 16. CEN plasmid segregation is destabilized by tethered determinants of Ty 5 integration specificity: a role for double-strand breaks in CEN antagonism; Fuerst PG et al.; The yeast retrotransposon Ty 5 integrates preferentially into heterochromatin at the telomeres and HM loci . Target specificity is mediated by a six amino acid sequence motif (the targeting domain, TD) of integrase that interacts with Sir4p, a structural component of heterochromatin . When tethered to CEN plasmids as part of a Gal4p DNA binding domain (GBD) fusion protein, TD destabilizes plasmid segregation in a manner similar to that observed for CEN + HM or CEN +TEL antagonism . This instability is caused by the ability of TD to nucleate components of heterochromatin on the CEN plasmid, because CEN +TD antagonism is abrogated by sir2, sir3 and sir4 mutations and by TD mutations that prevent interaction with Sir4p . In strains that acquire resistance to CEN +TD antagonism, the CEN plasmid has either recombined with a 2 mu plasmid or sustained deletions in sequences required to bind GBD-TD . CEN +TD and CEN + HM antagonism is exacerbated by mutations in components of the Ku-mediated non-homologous end-joining pathway . These observations suggest that CEN antagonism is caused by DNA breaks that result from competition between CEN - and Sir-specific segregation pathways. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 210 - 7 Epub 2003 Mar 04. Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides; Houghton-Larsen J et al.; This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides . The deduced 609-amino-acid enzyme (including signal peptide) is 63% identical to the Rhizopus oryzae raw starch-degrading glucoamylase and is the third glucoamylase reported to have the putative starch-binding domain placed N-terminally . The C-terminal catalytic domain is separated from the starch-binding domain by a serine/threonine-rich linker . An alignment of the cloned gene and cDNA sequences showed that the gene contains three introns . The transcriptional start site and the site of polyadenylation were defined by primer extension and 3'RACE, respectively . The atypical Kozak sequence is identical to the one used in R . oryzae in positions -1 to -4 . Northern slot blots revealed that glucoamylase transcription is induced during growth on starch and repressed by glucose . In silico analysis of the 1.9-kb promoter sequence cloned by inverse PCR revealed the presence of several putative regulatory elements, most notably a 19-bp sequence containing six overlapping copies of the Saccharomyces cerevisiae Nrg1p binding sequence. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 202 - 9 Epub 2003 Mar 25. cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea; Tanaka Y et al.; We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity . Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa . Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites . Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions . Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits . The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jul, 35(7), 597 - 600 Overexpression of human genes in Drosophila melanogaster by using GAL4 UAS system; Ma XZ et al.; Many human genes determined by genomic sequencing have only few information about their functions . To fill this knowledge gap, the powerful Drosophila genetics was set as a model to elucidate human gene functions effectively . By using germline transformation together with GAL4-UAS system, we studied the possibility of expressing and functionally characterization of human genes in Drosophila . Fifty-four transgenic fly lines corresponding to 10 human genes have been established . When expressed individually by crossing to an array of 6 different GAL4 driver lines, one of these genes, the translation elongation factor 1 alpha 1 (EF1 alpha-1), resulted in abnormal notum and rough eye phenotypes . This study implies the feasibility of systematically screening human gene functions by overexpression in Drosophila. Prog Nucleic Acid Res Mol Biol, 2003, 73, 221 - 50 The CCR4-NOT complex plays diverse roles in mRNA metabolism; Denis CL et al.; It is increasingly clear that the synthesis of eukaryotic mRNA involves an integrated series of events involving large multisubunit protein complexes . The evolutionarily conserved CCR4-NOT complex of proteins has been found to be involved in several aspects of mRNA formation, including repression and activation of mRNA initiation, control of mRNA elongation, and the deadenylation and subsequent degradation of mRNA . Its roles in such diverse processes make the CCR4-NOT complex central to the regulation of mRNA metabolism . In this review we describe the CCR4-NOT complex, its constituents, and its organization, discussing both the well characterized yeast proteins and their higher eukaryotic orthologs . The known biochemical roles of the individual components and of the complex are described with particular emphasis on the two known functions of the complex, repression of TFIID action and deadenylation of mRNA . Finally, the functional diversity of the CCR4-NOT complex is related to its mediating responses from a number of cellular signaling pathways. Aging Cell, 2002 Oct, 1(1), 47 - 56 Spatio-temporal analysis of gene expression during aging in Drosophila melanogaster; Seroude L et al.; The relationship between gene expression and the regulation of longevity is poorly understood . Previous studies focusing on microarray or tissue-specific changes in gene expression as a function of age have provided evidence that gene expression is a dynamic process which is regulated, even late in an organism's lifespan . Using the enhancer-trap technique, a systematic analysis of the spatio-temporal regulation of gene expression in tissues of adult Drosophila is presented . As many as 80% of enhancer traps analysed displayed (some form of) transcriptional change with age . In some cases the rate of change in expression was found to correlate with changes in longevity under various conditions, suggesting that they may be indicators of 'physiological age' and therefore valuable markers for dissecting