Virology, 2002 Dec 20, 304(2), 167 - 75 The domain order of mammalian capping enzyme can be inverted and baculovirus phosphatase can function in cap formation in vivo; Martins A et al.; The bifunctional mammalian mRNA capping enzyme (Mce1) consists of an N-terminal triphosphatase domain Mce1(1-210) fused to a C-terminal guanylyltransferase domain Mce1(211-597) . The physical domain order H(2)N-triphosphatase-guanylyltransferase-COOH mimics the temporal order of the capping reactions . To determine if the physical domain order is functionally important in vivo, we engineered an "inverted" mammalian capping enzyme InvMce1 {H(2)N-Mce1(211-597)-(1-210)-COOH} . We found that InvMce1 complemented the growth of Saccharomyces cerevisiae cet1delta and ceg1delta strains in which the endogenous yeast triphosphatase and guanylyltransferase genes were deleted . By testing truncated versions of InvMce1, we determined that Mce1(1-178) comprises a minimal functional triphosphatase domain . Baculovirus phosphatase (BVP) is a monofunctional single-domain protein with RNA triphosphatase and RNA diphosphatase activities and an undefined role in viral RNA metabolism . Here we demonstrated that BVP can function as an RNA triphosphatase for cap formation in vivo when fused to the C-terminus of Mce1(211-597) . By characterizing a series of InvMce1-BVP derivatives with amino acid substitutions in the phosphate-binding loop of BVP, we showed that the in vivo activity of the mutant chimeras in cap formation is contingent upon in vitro phosphohydrolase activity of the respective BVP proteins . BVP catalysis in vitro was not limited to 5'-phosphorylated RNA or nucleotide substrates, but also embraced tripolyphosphatase and pyrophosphatase activities . BVP-specific activities with nucleotide and inorganic substrates were as follows: ATP (14 min(-1)), ADP (31 min(-1)), PPP(i) (3.7 min(-1)), and PP(i) (1 min(-1)) . BVP did not hydrolyze AMP . We surmise that BVP has adapted the cysteinyl phosphatase fold to the hydrolysis of phosphoanhydrides . Biochem Biophys Res Commun, 2003 Jan 10, 300(2), 297 - 304 Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a; Kaneko C et al.; UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor . UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4 . We have isolated and characterized the mouse gene (Ube4b) for UFD2a . A full-length (approximately 5700 bp) Ube4b cDNA was isolated and the corresponding gene spans >100 kb, comprising 27 exons . Luciferase reporter gene analysis of the 5(') flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity . This functional sequence contains two putative Sp1 binding sites but not a TATA box . Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues . We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding . These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway. Mol Cell, 2002 Dec, 10(6), 1511 - 8 NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases; Liu J et al.; Cullin proteins assemble a large number of RING E3 ubiquitin ligases and regulate various physiological processes . Covalent modification of cullins by the ubiquitin-like protein NEDD8 activates cullin ligases through an as yet undefined mechanism . We show here that p120(CAND1) selectively binds to unneddylated CUL1 and is dissociated by CUL1 neddylation . CAND1 formed a ternary complex with CUL1 and ROC1 . CAND1 dissociated SKP1 from CUL1 and inhibited SCF ligase activity in vitro . Suppression of CAND1 in vivo increased the level of the CUL1-SKP1 complex . We suggest that by restricting SKP1-CUL1 interaction, CAND1 regulated the assembly of productive SCF ubiquitin ligases, allowing a common CUL1-ROC core to be utilized by a large number of SKP1-F box-substrate subcomplexes. Mol Cell, 2002 Dec, 10(6), 1489 - 94 Maf1 is an essential mediator of diverse signals that repress RNA polymerase III transcription; Upadhya R et al.; Maf1 is a putative repressor of RNA polymerase (pol) III transcription that is conserved from yeast to humans . Here we show that Maf1 is a common component of multiple signaling pathways in S . cerevesiae that sense changes in the cellular environment and repress pol III transcription . Signaling pathways activated in response to rapamycin-induced nutrient limitation, DNA damage, and secretory pathway defects all require Maf1 in order to affect pol III transcriptional repression . In addition, Maf1 was required for repression of pol III transcription during the normal yeast growth cycle . Biochemical studies identified the initiation factor TFIIIB as a target of Maf1-dependent repression and revealed a defect in TFIIIB-DNA complex assembly under repressing conditions. Mol Cell, 2002 Dec, 10(6), 1429 - 39 The RNA polymerase II CTD kinase CTDK-I affects pre-mRNA 3' cleavage/polyadenylation through the processing component Pti1p; Skaar DA et al.; There are several kinases in Saccharomyces cerevisiae that phosphorylate the CTD of RNA polymerase II, but specific and distinct functions of the phospho-CTDs generated by the different kinases are not well understood . A genetic screen for suppressors of loss of yeast CTD kinase I (CTDK-I) function (by deletion of the catalytic subunit gene CTK1) identified PTI1, a potential 3' cleavage/polyadenylation factor . Genetic and physical interactions connect Pti1p to components of CF IA and CF II/CPF, and mutations of PTI1 or CTK1 affect 3' cleavage site choice and transcript abundance of particular genes . Therefore, one important function of the CTDK-I-generated phospho-CTD appears to be the coupling of transcription to 3' processing of pre-mRNAs by a Pti1p-containing complex. Mol Cell, 2002 Dec, 10(6), 1417 - 27 Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes; Makeyev EV et al.; Recent genetic data suggest that proteins homologous to a plant RNA-dependent RNA polymerase (RdRP) play a central role in posttranscriptional gene silencing (PTGS) in many organisms . We show here that purified recombinant protein QDE-1, a genetic component of PTGS ("quelling") in the fungus Neurospora crassa, possesses RNA polymerase activity in vitro . The full-length enzyme and its enzymatically active C-terminal fragment perform two different reactions on single-stranded RNA templates, synthesizing either extensive RNA chains that form template-length duplexes or approximately 9-21-mer complementary RNA oligonucleotides scattered along the entire template . QDE-1 supports both de novo and primer-dependent initiation mechanisms . These results suggest that several distinct activities of cell-encoded RdRPs can be employed for efficient PTGS in vivo. Mol Cell, 2002 Dec, 10(6), 1391 - 401 Transcriptional activators stimulate DNA repair; Frit P et al.; To counteract the deleterious effects of genotoxic injury, cells have set up a sophisticated network of DNA repair pathways . We show that Gal4-VP16 and RAR transcriptional activators stimulate nucleotide excision repair (NER) . This DNA repair activation is not coupled to transcription since it occurs in Cockayne syndrome cells (which are transcription-coupled repair deficient) and is observed in vitro in the presence of alpha-amanitin and in the absence of the basal transcription factors . Using a reconstituted dual incision assay, we also show that binding of activators to their cognate sequences induces a local chromatin remodeling mediated by ATP-driven chromatin remodeling and acetyltransferase activities to facilitate DNA repair. Mol Cell, 2002 Dec, 10(6), 1307 - 18 Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1; Panaretou B et al.; Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined . A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system . In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90 . The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle. Mol Cell, 2002 Dec, 10(6), 1295 - 305 Regulation of subtelomeric silencing during stress response; Ai W et al.; Sir proteins play a critical role in silent chromatin domains . While mutations can cause derepression of heterochromatin, it remains unclear whether silencing is actively involved in transcriptional control under changing environmental conditions . We find that TOR inhibits Sir3 phosphorylation . Rapamycin or stress induced by chlorpromazine leads to activation of MAP kinase Mpk1/Slt2, which phosphorylates Sir3 . Sir3 hyperphosphorylation is correlated with reduced subtelomeric silencing, increased subtelomeric cell wall gene expression, and stress resistance to chlorpromazine, but does not affect the silent HML and rDNA loci . Based on these observations, we propose that regulation of silencing may be used to control gene expression at specific silent chromatin domains in response to stress and possibly other environmental changes. Mol Cell, 2002 Dec, 10(6), 1257 - 8 Hitchhikers guide to the vacuole-mechanisms of cargo sequestration in the Cvt and autophagic pathways; Thumm M; In the yeast Saccharomyces cerevisiae, precursor aminopeptidase I and alpha-mannosidase are selectively targeted via the Cvt and the autophagic pathways to the vacuole . Two studies now uncover the molecular basis of the selective sequestration of these two enzymes. Mol Cell, 2002 Dec, 10(6), 1255 - 6 Aha, another regulator for hsp90 chaperones; Mayer MP et al.; A large number of key regulators controlling homeostasis and cell fate are chaperoned by the Hsp90 folding machine . In this issue of Molecular Cell, report the discovery of a new stress-regulated cochaperone, Aha1, which accelerates the dynamics of this machine. Biotechniques, 2002 Dec, 33(6), 1349 - 53 Small-scale telomere repeat sequence content assay using pyrophosphorolysis coupled with ATP detection; Learish RD et al.; Studies of telomere length have been carried out in diverse areas of research . However, current methods to measure telomeres are cumbersome and not amenable to high-throughput analyses . Using a coupled pyrophosphorolysis/trans-phosphorylation reaction, we have developed a novel assay to quantitate telomere sequence content in a single tube or 96-well format . The method uses a telomere-specific oligonucleotide probe to sample nanogram quantities of DNA without PCR amplification . Polymerase and kinase enzymes drive the production of ATP, which is then monitored with a luciferase enzyme reporter system . Using this approach, we demonstrated that the luminescent output was linear across a 100-fold range of DNA input, and the assay was sensitive to 0.4-1 ng DNA . A control probe reaction and a DNA quantitation reaction were also designed using the same pyrophosphorolysis technology to correct for background activity and normalize the signal against variations in DNA input, respectively . Finally, we show that the normalized luminescent signal generated by this new method is highly correlated to the telomere restriction fragment length for six human cell lines. Biotechniques, 2002 Dec, 33(6), 1308 - 16 Proteomic approaches for the global analysis of proteins; Michaud GA et al.; Improvements in technology that allow miniaturization and high-throughput analyses of thousand of genes and gene products have changed the focus and scope of research and development in both academia and industry . It is now possible to study entire proteomes with the goals of elucidating protein expression, subcellular localization, biochemical activities, and their regulation . Alterations in different cell types and conditions and in normal and disease states can be revealed . This wealth of information not only has facilitated our basic understanding of many biological processes but also has enormous potential for drug discovery and development. Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 131 - 6 Epub 2002 Dec 26. Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARgamma agonist; McIntyre TM et al.; Lysophosphatidic acid (LPA) is a pluripotent lipid mediator acting through plasma membrane-associated LPA(x) receptors that transduce many, but not all, of its effects . We identify peroxisome proliferator-activated receptor gamma (PPARgamma) as an intracellular receptor for LPA . The transcription factor PPARgamma is activated by several lipid ligands, but agonists derived from physiologic signaling pathways are unknown . We show that LPA, but not its precursor phosphatidic acid, displaces the drug rosiglitazone from the ligand-binding pocket of PPARgamma . LPA and novel LPA analogs we made stimulated expression of a PPAR-responsive element reporter and the endogenous PPARgamma-controlled gene CD36, and induced monocyte lipid accumulation from oxidized low-density lipoprotein via the CD36 scavenger receptor . The synthetic LPA analogs were effective PPARgamma agonists, but were poor ones for LPA(1), LPA(2), or LPA(3) receptor transfected cells . Transfection studies in yeast, which lack nuclear hormone and LPA(x) receptors, show that LPA directly activates PPARgamma . A major growth factor of serum is LPA generated by thrombin-activated platelets, and media from activated platelets stimulated PPARgamma function in transfected RAW264.7 macrophages . This function was suppressed by ectopic LPA-acyltransferase expression . LPA is a physiologic PPARgamma ligand, placing PPARgamma in a signaling pathway, and PPARgamma is the first intracellular receptor identified for LPA . Moreover, LPA produced by stimulated plasma platelets activates PPARgamma in nucleated cells. Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 74 - 9 Epub 2002 Dec 26. Structural basis and prediction of substrate specificity in protein serine/threonine kinases; Brinkworth RI et al.; The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally . Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input . We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways . Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serinethreonine kinases and helps in silico construction of signaling pathways . The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches. Genes Dev, 2002 Dec 15, 16(24), 3236 - 52 ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase; Shimada K et al.; The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks . A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase . We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1 . Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2 . Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks. J Nat Prod, 2002 Dec, 65(12), 1930 - 2 DNA-damaging agents from Crypteronia paniculata; Deng JZ et al.; A survey of crude plant extracts using a new yeast strain designed to identify DNA-damaging agents resulted in the identification of an extract prepared from Crypteronia paniculata . Bioassay-guided fractionation resulted in the isolation of three active compounds . Two of these were ellagic acid derivatives, namely, 3,3'-di-O-methylellagic acid 4'-O-beta-d-xylopyranoside (1) and 3'-O-methyl-3,4-methylenedioxyellagic acid 4'-O-beta-d-glucopyranoside (2) . The third was identified as kaempferol-3-O-alpha-l-rhamnoside (3) . The three principles exhibited strong, selective cytotoxity toward the RAD52 repair-deficient yeast strain. Microbiol Res, 2002, 157(4), 337 - 44 Structural features of the glycogen branching enzyme encoding genes from aspergilli; Sasangka P et al.; A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose . The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity . The genomic gene and its cDNA encoding GBE (p78) were isolated from the A . nidulans genomic and cDNA libraries . Furthermore, a cDNA encoding A . oryzae GBE was entirely sequenced . A . nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A . oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55-56%). Sheng Li Ke Xue Jin Zhan, 1998 Jul, 29(3), 215 - 20 {Research progress on regulation of vascular endothelial barrier function}; Xiao ZL et al.; The regulatory mechanisms of vascular endothelial barrier function are complicated . Inflammatory mediators, such as alpha-thrombin, activate phospholipases to mediate generation of IP3 and other second messengers through receptor-coupled G protein . Protein kinase C and myosin light chain kinase are then activated, leading to phosphorylation of myosin light chains, rearrangement of F-actin skeleton, interendothelial cell gap formation and increased endothelial permeability. J Biol Chem, 2003 Feb 28, 278(9), 6779 - 86 Epub 2002 Dec 24. Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA; Klein J et al.; An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes . Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16 . We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors . In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA . Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histone-fold motifs. Biochemistry, 2002 Dec 31, 41(52), 15676 - 84 Probing backbone hydrogen bonds in the hydrophobic core of GCN4; Blankenship JW et al.; Backbone amide hydrogen bonds play a central role in protein secondary and tertiary structure . Previous studies have shown that substitution of a backbone ester (-COO-) in place of a backbone amide (-CONH-) can selectively destabilize backbone hydrogen bonds in a protein while maintaining a similar conformation to the native backbone structure . The majority of these studies have focused on backbone substitutions that were accessible to solvent . The GCN4 coiled coil domain is an example of a stable alpha-helical dimer that possesses a well-packed hydrophobic core . Amino acids in the a and d positions of the GCN4 helix, which pack the hydrophobic core, were replaced with the corresponding alpha-hydroxy acids in the context of a chemoselectively ligated heterodimer . While the overall structure and oligomerization state of the heterodimer were maintained, the overall destabilization of the ester analogues was greater (average DeltaDeltaG of 3+ kcal mol(-1)) and more variable than previous studies . Since burial of the more hydrophobic ester should stabilize the backbone and reduce the DeltaDeltaG, the increased destabilization must come from another source . However, the observed destabilization is correlated with the protection factors for individual amide hydrogens from previous hydrogen exchange experiments . Therefore, our results suggest that backbone engineering through ester substitution is a useful approach for probing the relative strength of backbone hydrogen bonds. Biol Pharm Bull, 2002 Dec, 25(12), 1528 - 32 Analysis of the gene encoding copper/zinc superoxide dismutase homolog in Dictyostelium discoideum; Akaza Y et al.; SodD, a Cu/Zn superoxide dismutase in Dictyostelium discoideum, shows 48% identity to the cytosolic Cu/Zn superoxide dismutase (SOD) of Saccharomyces cerevisiae (SOD1) . The sodD gene is expressed in D . discoideum cells at late-developmental stages . However, gene expression was not detected in the sporeless mutant, indicating that sodD is a spore cell-specific gene . The D . discoideum mutant, in which sodD was disrupted, grew and formed a multicellular structure normally, therefore the gene is not essential for growth and development . The mutant spores were sensitive to UV-light compared to the wild-type spores, indicating that SodD protects spores from cellular damage caused by UV-light. Glycobiology, 2002 Dec, 12(12), 803 - 11 Glycopeptide export from the endoplasmic reticulum into cytosol is mediated by a mechanism distinct from that for export of misfolded glycoprotein; Suzuki T et al.; When glycoproteins formed in the endoplasmic reticulum (ER) are misfolded, they are generally translocated into the cytosol for ubiquitination and are subsequently degraded by the proteasome . This system, the so-called ER-associated glycoprotein degradation, is important for eukaryotes to maintain the quality of glycoproteins generated in the ER . It has been established in yeast that several distinct proteins are involved in this translocation and degradation processes . Small glycopeptides formed in the ER are exported to the cytosol in a similar manner . This glycopeptide export system is conserved from yeast to mammalian cells, suggesting its basic biological significance for eukaryotic cells . These two export systems (for misfolded glycoproteins and glycopeptides) share some properties, such as a requirement for ATP and involvement of Sec61p, a central membrane protein presumably forming a dislocon channel for export of proteins . However, the machinery of glycopeptide export is poorly understood . In this study, various mutants known to have an effect on export/degradation of misfolded glycoproteins were examined for glycopeptide export activity with a newly established assay method . Surprisingly, most of the mutants were found not to exhibit a defect in glycopeptide export . The only gene that was found to be required on efficient export of both types of substrates was PMR1, the gene encoding the medial-Golgi Ca(2+)/Mn(2+)-ion pump . These results provide evidence that although the systems involved in export of misfolded glycoproteins and glycopeptides share some properties, they have exhibited distinct differences. J Biol Chem, 2003 Mar 14, 278(11), 9058 - 63 Epub 2002 Dec 23. Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition . Mutation of methionine 593 abolishes malonyl-CoA inhibition; Morillas M et al.; Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA . By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)) . To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A . In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT . Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E . The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased . In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished . Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity . Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gomez-Puertas, P., Rubi, B., Clotet, J., Arino, J., Valencia, A., Hegardt, F . G., Serra, D., and Asins, G . (2002) J . Biol . Chem . 277, 11473-11480) . Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported. J Biol Chem, 2003 Mar 7, 278(10), 7755 - 64 Epub 2002 Dec 23. Dynamic chromatin alterations triggered by natural and synthetic activation domains; Erkine AM et al.; The activation domains (ADs) of transcription activators recruit a multiplicity of enzymatic activities to gene promoters . The mechanisms by which such recruitment takes place are not well understood . Using chromatin immunoprecipitation, we demonstrate dynamic alterations in the abundance of histones H2A, H3, and H4 at promoters of genes regulated by the HSF and Gal4 activators of Saccharomyces cerevisiae . Transcriptional activation of these genes, particularly those regulated by HSF, is accompanied by a significant reduction in both acetylated and unacetylated histones at promoters and may involve the transient displacement of histone octamers . To gain insight into the function of ADs, we conducted a genetic screen to identify polypeptides that could substitute for the 340-residue C-terminal activator of HSF and rescue the temperature sensitivity caused by its deletion . We found that the ts(-) phenotype of HSF(1-493) could be complemented by peptides as short as 11 amino acids . Such peptides are enriched in acidic and hydrophobic residues, and exhibit both trans-activating and chromatin-modifying activities when fused to the Gal4 DNA-binding domain . We also demonstrate that a previously identified 14-amino acid histone H3-binding module of human CTF1/NF1, which is similar to synthetic ADs, can substitute for the HSF C-terminal activator in conferring temperature resistance and can mediate the modification of promoter chromatin structure . Possible mechanisms of AD function, including one involving direct interactions with histones, are discussed. J Cell Biol, 2002 Dec 23, 159(6), 1051 - 9 Epub 2002 Dec 23. The Cdc37 protein kinase-binding domain is sufficient for protein kinase activity and cell viability; Lee P et al.; Cdc37 is a molecular chaperone required for folding of protein kinases . It functions in association with Hsp90, although little is known of its mechanism of action or where it fits into a folding pathway involving other Hsp90 cochaperones . Using a genetic approach with Saccharomyces cerevisiae, we show that CDC37 overexpression suppressed a defect in v-Src folding in yeast deleted for STI1, which recruits Hsp90 to misfolded clients . Expression of CDC37 truncation mutants that were deleted for the Hsp90-binding site stabilized v-Src and led to some folding in both sti1Delta and hsc82Delta strains . The protein kinase-binding domain of Cdc37 was sufficient for yeast cell viability and permitted efficient signaling through the yeast MAP kinase-signaling pathway . We propose a model in which Cdc37 can function independently of Hsp90, although its ability to do so is restricted by its normally low expression levels . This may be a form of regulation by which cells restrict access to Cdc37 until it has passed through a triage involving other chaperones such as Hsp70 and Hsp90. J Cell Biol, 2002 Dec 23, 159(6), 993 - 1004 Epub 2002 Dec 23. Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin; Humphries CL et al.; Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies . Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin . Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro . The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments . The inhibitory activity of Crn1 resides in its coiled coil domain . Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain . Genetic studies provide in vivo evidence for these interactions and activities . Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops . These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex . Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction . This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin. J Cell Biol, 2002 Dec 23, 159(6), 915 - 21 Epub 2002 Dec 23. Concentrative sorting of secretory cargo proteins into COPII-coated vesicles; Malkus P et al.; Here, we show that efficient transport of membrane and secretory proteins from the ER of Saccharomyces cerevisiae requires concentrative and signal-mediated sorting . Three independent markers of bulk flow transport out of the ER indicate that in the absence of an ER export signal, molecules are inefficiently captured into coat protein complex II (COPII)-coated vesicles . A soluble secretory protein, glycosylated pro-alpha-factor (gpalphaf), was enriched approximately 20 fold in these vesicles relative to bulk flow markers . In the absence of Erv29p, a membrane protein that facilitates gpalphaf transport (Belden and Barlowe, 2001), gpalphaf is packaged into COPII vesicles as inefficiently as soluble bulk flow markers . We also found that a plasma membrane protein, the general amino acid permease (Gap1p), is enriched approximately threefold in COPII vesicles relative to membrane phospholipids . Mutation of a diacidic sequence present in the COOH-terminal cytosolic domain of Gap1p eliminated concentrative sorting of this protein. Bioinformatics, 2003 Jan, 19(1), 79 - 86 Mining gene expression databases for association rules; Creighton C et al.; MOTIVATION: Global gene expression profiling, both at the transcript level and at the protein level, can be a valuable tool in the understanding of genes, biological networks, and cellular states . As larger and larger gene expression data sets become available, data mining techniques can be applied to identify patterns of interest in the data . Association rules, used widely in the area of market basket analysis, can be applied to the analysis of expression data as well . Association rules can reveal biologically relevant associations between different genes or between environmental effects and gene expression . An association rule has the form LHS --> RHS, where LHS and RHS are disjoint sets of items, the RHS set being likely to occur whenever the LHS set occurs . Items in gene expression data can include genes that are highly expressed or repressed, as well as relevant facts describing the cellular environment of the genes (e.g . the diagnosis of a tumor sample from which a profile was obtained) . RESULTS: We demonstrate an algorithm for efficiently mining association rules from gene expression data, using the data set from Hughes et al . (2000, Cell, 102, 109-126) of 300 expression profiles for yeast . Using the algorithm, we find numerous rules in the data . A cursory analysis of some of these rules reveals numerous associations between certain genes, many of which make sense biologically, others suggesting new hypotheses that may warrant further investigation . In a data set derived from the yeast data set, but with the expression values for each transcript randomly shifted with respect to the experiments, no rules were found, indicating that most all of the rules mined from the actual data set are not likely to have occurred by chance . AVAILABILITY: An implementation of the algorithm using Microsoft SQL Server with Access 2000 is available at Our results from mining the yeast data set are available at http://dot.ped.med.umich.edu:2000/pub/assoc_rules/yeast_results.zip. Cancer Res, 2002 Dec 15, 62(24), 7230 - 3 8-Hydroxyguanosine repair is defective in some microsatellite stable colorectal cancer cells; Parker AR et al.; Mutator phenotypes are involved in the carcinogenesis of some cancers, e.g., defects in mismatch repair produce a mutator phenotype that drives carcinogenesis and causes microsatellite instability in hereditary nonpolyposis colon cancers and some sporadic colorectal cancers (CRC) . Less understood, however, is the potential role of mutator phenotypes in microsatellite stable (MSS) CRC carcinogenesis . A novel transversion mutator phenotype was reported recently in an MSS CRC cell line . We hypothesized that 8-hydroxyguanosine could be involved and found elevations in 5 of 15 (33%) MSS CRC cell lines analyzed . Repair of an adenine*8-hydroxyguanosine mispair was functionally defective in the same five cell lines . The human MutY homologue transcript and MutY homologue protein levels were also decreased . These findings may reflect a MSS mutator phenotype contributing to the development of CRC. Chem Biol, 2002 Dec, 9(12), 1323 - 8 Neoglycopeptides as inhibitors of oligosaccharyl transferase: insight into negotiating product inhibition; Peluso S et al.; Linear hexapeptides featuring the asparagine mimetics alanine-beta-hydrazide, alanine-beta-hydroxylamine, and 1,3-diaminobutanoic acid have been synthesized as oligosaccharyl transferase (OT) substrate mimetics and chemoselectively N-glycosylated to obtain the corresponding neoglycopeptides as OT product mimetics . The effect of glycosylation on the binding of these asparagine surrogates is in stark contrast with the effect of modification of native asparagine . In native N-linked glycosylation, product inhibition is minimal and glycopeptides show very low affinity for OT . In contrast, glycosylation of the substrate mimetics maintains or even improves affinity of the corresponding product mimetic for OT . Conformational considerations suggest that the flexibility of the N-glycosyl linkage in these neoglycopeptides allows them to be accommodated in the OT binding site while the native trans glycosyl amide linkage is rejected . These results provide insight into how OT minimizes product inhibition, thereby ensuring effective substrate turnover. Curr Biol, 2002 Dec 23, 12(24), R855 - 7 Polyadenylation: a tail of two complexes; Proudfoot N et al.; Recent studies have uncovered new connections between the enzymes of mRNA 3' end processing and RNA polymerase II . These connections improve the efficiency of polyadenylation and signal to the polymerase to terminate transcription; their discovery reveals another level of gene regulation. Curr Biol, 2002 Dec 23, 12(24), 2118 - 23 The Cdc20 homolog, FZY-1, and its interacting protein, IFY-1, are required for proper chromosome segregation in Caenorhabditis elegans; Kitagawa R et al.; Accurate chromosome segregation is achieved by a series of highly regulated processes that culminate in the metaphase-to-anaphase transition of the cell cycle . In the budding yeast Saccharomyces cerevisiae, the degradation of the securin protein Pds1 reverses the binding and inhibition of the separase protein Esp1 . Esp1 cleaves Scc1 . That cleavage promotes the dissociation of the cohesin complex from the chromosomes and leads the separation of sister chromatids . Proteolysis of Pds1 is regulated by the anaphase-promoting complex (APC), a large multi-subunit E3 ubiquitin ligase whose activity is regulated by Cdc20/Fizzy . We have previously shown that the Caenorhabditis elegans genes mdf-1/MAD1 and mdf-2/MAD2 encode key members of the spindle checkpoint . Loss of function of either gene leads to an accumulation of somatic and heritable defects and ultimately results in death . Here we show that a missense mutation in fzy-1/CDC20/Fizzy suppresses mdf-1 lethality . We identified a FZY-1-interacting protein, IFY-1, a novel destruction-box protein . IFY-1 accumulates in one-cell-arrested emb-30/APC4 embryos and interacts with SEP-1, a C . elegans separase, suggesting that IFY-1 functions as a C . elegans securin. Curr Biol, 2002 Dec 23, 12(24), 2098 - 110 Control of Lte1 localization by cell polarity determinants and Cdc14; Seshan A et al.; BACKGROUND: The putative guanine nucleotide exchange factor Lte1 plays an essential role in promoting exit from mitosis at low temperatures . Lte1 is thought to activate a Ras-like signaling cascade, the mitotic exit network (MEN) . MEN promotes the release of the protein phosphatase Cdc14 from the nucleolus during anaphase, and this release is a prerequisite for exit from mitosis . Lte1 is present throughout the cell during G1 but is sequestered in the bud during S phase and mitosis by an unknown mechanism . RESULTS: We show that anchorage of Lte1 in the bud requires septins, the cell polarity determinants Cdc42 and Cla4, and Kel1 . Lte1 physically associates with Kel1 and requires Kel1 for its localization in the bud, suggesting a role for Kel1 in anchoring Lte1 at the bud cortex . Our data further implicate the PAK-like protein kinase Cla4 in controlling Lte1 phosphorylation and localization . CLA4 is required for Lte1 phosphorylation and bud localization . Furthermore, when overexpressed, CLA4 induces Lte1 phosphorylation and localization to regions of polarized growth . Finally, we show that Cdc14, directly or indirectly, controls Lte1 dephosphorylation and delocalization from the bud during exit from mitosis . CONCLUSION: Restriction of Lte1 to the bud cortex depends on the cortical proteins Cdc42 and Kel1 and the septin ring . Cla4 and Cdc14 promote and demote Lte1 localization at and from the bud cortex, respectively, suggesting not only that the phosphorylation status of Lte1 controls its localization but also indicating that Cla4 and Cdc14 are key regulators of the spatial asymmetry of Lte1. Cancer Biol Ther, 2002 Sep-Oct, 1(5), 502 - 8 Mutations in the BRCT domain confer temperature sensitivity to BRCA1 in transcription activation; Carvalho MA et al.; BRCA1 is a tumor suppressor gene and germ line mutations account for the majority of familial cases of breast and ovarian cancer . There is mounting evidence that BRCA1 functions in DNA repair and transcriptional regulation . A major hurdle to dissect the role of BRCA1 is the lack of molecular reagents to carry out biochemical and genetic experiments . Therefore, we used random mutagenesis of the C-terminus of BRCA1 (aa 1560-1863) to generate temperature-sensitive (TS) mutants in transcription activation . We obtained 11 TS mutants in transcription that localized primarily to the hydrophobic core of the BRCT-N domain of BRCA1 . One of the mutants, H1686Q, also displayed temperature-dependent transcription activation in human cells . These conditional mutants represent valuable tools to assess the role of BRCA1 in transcription activation. Cancer Biol Ther, 2002 Sep-Oct, 1(5), 497 - 501 A naturally occurring allele of BRCA1 coding for a temperature-sensitive mutant protein; Worley T et al.; Recent evidence suggests that the breast and ovarian cancer susceptibility gene product BRCA1 is involved in at least two fundamental cellular processes: transcriptional regulation and DNA repair . However, the mechanism of action of BRCA1 in either of these processes is still unknown . Here, we report the characterization of a disease-predisposing allele of BRCA1, identified in a family with several cases of ovarian cancer, coding for a protein that displays temperature-sensitive activity in transcriptional activation . The mutant protein differs from the wild type protein at a single amino acid, R1699W that occurs in a region at the N-terminal BRCT domain that is highly conserved among BRCA1 homologs . When the C-terminus of the mutant protein (aa 1560-1863) was fused to a heterologous GAL4 DNA-binding domain and expressed in yeast or mammalian cells, it was able to activate transcription of a reporter gene to levels observed for wild type BRCA1 at the permissive temperature (30 degrees C) but exhibited significantly less transcription activity at the restrictive temperature (37 degrees C or 39 degrees C) . Our results indicate that the transcriptional activity of the R1699W mutant can be modulated as a function of temperature and provide a novel experimental approach which can be utilized to dissect the molecular mechanism(s) of BRCA1 in processes related to transcription. J Biol Chem, 2003 Feb 21, 278(8), 5963 - 9 Epub 2002 Dec 19. Studies with novel Pdr5p substrates demonstrate a strong size dependence for xenobiotic efflux; Golin J et al.; The yeast (Saccharomyces cerevisiae) multidrug transporter Pdr5p effluxes a broad range of substrates that are variable in structure and mode of action . Previous work suggested that molecular size and ionization could be important parameters . In this study, we compared the relative sensitivity of isogenic PDR5 and pdr5 strains toward putative substrates that are similar in chemical structure . Three series were used: imidazole-containing compounds, trialkyltin chlorides, and tetraalkyltin compounds . We demonstrate that the Pdr5p transporter is capable of mediating transport of substrates that neither ionize nor have electron pair donors and that are much simpler in structure than those transported by the human MDR1-encoded P-glycoprotein . Furthermore, the size of the substrate is critical and independent of any requirement for hydrophobicity . Substrates have surface volumes greater than 90 A(3) with an optimum response at approximately 200-225 A(3) as determined by molecular modeling . Assays measuring the efflux from cells of {(3)H}chloramphenicol and {(3)H}tritylimidazole were used . A concentration-dependent inhibition of chloramphenicol transport was observed with imidazole derivatives but not with either the organotin compounds or the antitumor agent doxorubicin . In contrast, several of the organotin compounds were potent inhibitors of tritylimidazole efflux, but the Pdr5p substrate tetrapropyltin was ineffective in both assays . This argues for the existence of at least three substrate-binding sites on Pdr5p that differ in behavior from those of the mammalian P-glycoprotein . Evidence also indicates that some substrates are capable of interacting at more than one site . The surprising observation that Pdr5p mediates resistance to tetraalkyltins suggests that one of the sites might use only hydrophobic interactions to bind substrates. J Biol Chem, 2003 Feb 28, 278(9), 7051 - 8 Epub 2002 Dec 19. Protein-protein interactions within an E2-RING finger complex . Implications for ubiquitin-dependent DNA damage repair; Ulrich HD; The RING finger protein RAD5 interacts and cooperates with the UBC13-MMS2 ubiquitin-conjugating enzyme in postreplication DNA damage repair in yeast . Previous observations implied that the function of UBC13 and MMS2 is dependent on the presence of RAD5, suggesting that the RING finger protein might act as a ubiquitin-protein ligase specific for the UBC13-MMS2 complex . In support of this notion it is shown here that the contact surfaces between the RAD5 RING domain and UBC13 correspond to those found in other pairs of ubiquitin-conjugating enzymes and ubiquitin-protein ligases . Mutations that compromise the protein-protein interactions either between the RING domain and UBC13 or within the UBC13-MMS2 dimer were found to have variable effects on repair activity in vivo that strongly depended on the expression levels of the corresponding mutants . Quantitative analysis of the affinity and kinetics of the UBC13-MMS2 interaction suggests a highly dynamic association model in which compromised mutual interactions result in phenotypic effects only under conditions where protein levels become limiting . Finally, this study demonstrates that beyond its cooperation with the UBC13-MMS2 dimer, RAD5 must have an additional role in DNA damage repair independent of its RING finger domain. J Biol Chem, 2003 Mar 7, 278(10), 8580 - 5 Epub 2002 Dec 20. Cleavage, but not read-through, stimulation activity is responsible for three biologic functions of transcription elongation factor S-II; Ubukata T et al.; Transcription elongation factor S-II stimulates cleavage of nascent transcripts generated by RNA polymerase II stalled at transcription arrest sites . In vitro experiments have shown that this action promotes RNA polymerase II to read through these transcription arrest sites . This S-II-mediated cleavage is thought to be necessary, but not sufficient, to promote read-through in the in vitro systems . Therefore, Saccharomyces cerevisiae strains expressing S-II mutant proteins with different in vitro activities were used to study both the cleavage and the read-through stimulation activities of S-II to determine which S-II functions are responsible for its biologic functions . Strains expressing mutant S-II proteins active in both cleavage and read-through stimulation were as resistant as wild type strains to 6-azauracil and mycophenolic acid . 6-Azauracil also induced IMD2 gene expression in both these mutant strains and the wild type . Furthermore, strains having a genotype consisting of one of these S-II mutations and the spt4 null mutation grew as well as the spt4 null mutant at 37 degrees C, a restrictive temperature for a strain bearing double null mutations of spt4 and S-II . In contrast, strains bearing S-II mutations defective in both cleavage and read-through stimulation had phenotypes similar to those of an S-II null mutant . However, one strain expressing a mutant S-II protein active only in cleavage stimulation had a phenotype similar to that of the wild type strain . These results suggest that cleavage, but not read-through, stimulation activity is responsible for all three biologic functions of S-II (i.e . suppression of 6-azauracil sensitivity, induction of the IMD2 gene, and suppression of temperature sensitivity of spt4 null mutant). Infect Immun, 2003 Jan, 71(1), 571 - 4 A single-copy gene encodes Kex1, a serine endoprotease of Pneumocystis jiroveci; Kutty G et al.; We have cloned and characterized the kex1 gene of Pneumocystis jiroveci . Unlike the case for Pneumocystis carinii, in which the homologous PRT-1 genes are multicopy, kex1 is a single-copy gene encoding a protein homologous to fungal serine endoproteases, which localize to the Golgi apparatus . Thus, substantial biological differences can be seen among Pneumocystis species. Biophys J, 2002 Dec, 83(6), 2906 - 17 Molecular dynamics simulations of a hydrated protein vectorially oriented on polar and nonpolar soft surfaces; Nordgren CE et al.; We present a collection of molecular dynamics computer simulation studies on a model protein-membrane system, namely a cytochrome c monolayer attached to an organic self-assembled monolayer (SAM) . Modifications of the system are explored, including the polarity of the SAM endgroups, the amount of water present for hydration, and the coordination number of the heme iron atom . Various structural parameters are measured, e.g., the protein radius of gyration and eccentricity, the deviation of the protein backbone from the x-ray crystal structure, the orientation of the protein relative to the SAM surface, and the profile structures of the SAM, protein, and water . The polar SAM appears to interact more strongly with the protein than does the nonpolar SAM . Increased hydration of the system tends to reduce the effects of other parameters . The choice of iron coordination model has a significant effect on the protein structure and the heme orientation . The overall protein structure is largely conserved, except at each end of the sequence and in one loop region . The SAM structure is only perturbed in the region of its direct contact with the protein . Our calculations are in reasonably good agreement with experimental measurements (polarized optical absorption/emission spectroscopy, x-ray interferometry, and neutron interferometry). Curr Opin Plant Biol, 2003 Feb, 6(1), 20 - 8 Chromatin regulation of plant development; Wagner D; Chromatin remodeling factors are being identified as genetic modifiers of developmental mutations in plants . These mutations result in lethality in metazoans, whereas in plants, they are viable and affect a wide range of developmental and physiological processes . Recent studies have begun to define the many functions of chromatin remodeling factors in plants and have revealed apparent differences between these factors in the two kingdoms. J Pharm Pharmacol, 2002 Nov, 54(11), 1521 - 8 Assessment of estrogenic activity in some common essential oil constituents; Howes MJ et al.; Estrogenic responses have not only been associated with endocrine function, but also with cognitive function . Several studies have indicated that estrogen replacement therapy has favourable effects on cognition, and may have potential in the prevention and treatment of Alzheimer's disease . Thus, ligands for the estrogen receptor, that have a better efficacy and adverse-effect profile than drugs currently available, require investigation . This study was undertaken to investigate the potential estrogenic activity of a number of essential oil constituents . Initially, estrogenic activity was determined by a sensitive and specific bioassay using recombinant yeast cells expressing the human estrogen receptor . At high concentrations, estrogenic activity was detected for citral (geranial and neral), geraniol, nerol and trans-anethole, while eugenol showed anti-estrogenic activity . Molecular graphics studies were undertaken to identify the possible mechanisms for the interaction of geranial, neral, geraniol, nerol and eugenol with the ligand-binding domain of the estrogen alpha-receptor, using the computer program HyperChem . Citral, geraniol, nerol and eugenol were also able to displace {(3)H}17beta-estradiol from isolated alpha- and beta-human estrogen receptors, but none of these compounds showed estrogenic or anti-estrogenic activity in the estrogen-responsive human cell line Ishikawa Var I at levels below their cytotoxic concentrations, and none showed activity in a yeast screen for androgenic and anti-androgenic activity . The potential in-vivo estrogenic effects of citral and geraniol were examined in ovariectomized mice, but neither compound showed any ability to stimulate the characteristic estrogenic responses of uterine hypertrophy or acute increase in uterine vascular permeability . These results show that very high concentrations of some commonly used essential oil constituents appear to have the potential to interact with estrogen receptors, although the biological significance of this is uncertain. Science, 2002 Dec 20, 298(5602), 2345 - 9 Finding genes that underlie complex traits; Glazier AM et al.; Phenotypic variation among organisms is central to evolutionary adaptations underlying natural and artificial selection, and also determines individual susceptibility to common diseases . These types of complex traits pose special challenges for genetic analysis because of gene-gene and gene-environment interactions, genetic heterogeneity, low penetrance, and limited statistical power . Emerging genome resources and technologies are enabling systematic identification of genes underlying these complex traits . We propose standards for proof of gene discovery in complex traits and evaluate the nature of the genes identified to date . These proof-of-concept studies demonstrate the insights that can be expected from the accelerating pace of gene discovery in this field. Nippon Yakurigaku Zasshi, 2002 Nov, 120(1), 47P - 50P {Drug discovery based on microarray}; Kuhara S et al.; Gene regulatory networks developed from full genome expression libraries from gene perturbation variant cell lines can be used to quickly and efficiently identify the molecular mechanism of action of drugs or lead compound molecules . We developed an extensive yeast gene expression library consisting of full-genome cDNA array data for over 500 yeast strains each with a single gene disruption . Using this data, combined with dose and time course expression experiments with the oral antifungal agent, we used Boolean network discovery techniques to determine the genes whose expression was most profoundly affected by this drug . Our system identified the gene as the most significantly suppressed target molecule due to exposure to the antifungal agent . This process for network based drug discovery can significantly decrease the time and resources necessary to make rational drug targeting decisions. Nucleic Acids Res . 2002 Dec 15;30(24):e141. Optimization of trans-splicing ribozyme efficiency and specificity by in vivo genetic selection; Ayre BG et al.; Trans-splicing ribozymes are RNA-based catalysts capable of splicing RNA sequences from one transcript specifically into a separate target transcript . In doing so, a chimeric mRNA can be produced, and new gene activities triggered in living cells dependent on the presence of the target mRNA . Based on this ability of trans-splicing ribozymes to deliver new gene activities, a simple and versatile plating assay was developed in Saccharomyces cerevisiae for assessing and optimizing constructs in vivo . Trans-splicing ribozymes were used to splice sequences encoding a GAL4-derived transcription activator into a target transcript from a prevalent viral pathogen . The transcription activator translated from this new mRNA in turn triggered the expression of genes under the regulatory control of GAL4 upstream-activating sequences . Two of the activated genes complemented metabolic deficiencies in the host strain, and allowed growth on selective media . A simple genetic assay based on phenotypic conversion from auxotrophy to prototrophy was established to select efficient and specific trans-splicing ribozymes from a ribozyme library . This simple assay may prove valuable for selecting optimal target sites for therapeutic agents such as ribozymes, antisense RNA and antisense oligodeoxyribonucleotides, and for optimizing the design of the therapeutic agents themselves, in higher eukaryotes. Nucleic Acids Res, 2002 Dec 15, 30(24), 5476 - 84 Mutations in U5 snRNA loop 1 influence the splicing of different genes in vivo; O'Keefe RT; The U5 snRNA loop 1 is characterized by the conserved sequence G1C2C3U4U5U6Y7A8Y9 and is essential for the alignment of exons during the second step of pre-mRNA splicing in Saccharo myces cerevisiae . Despite this sequence conservation the size, rather than sequence, of loop 1 is critical for exon alignment in vitro . To determine the in vivo requirements for U5 loop 1 a library of loop 1 sequences was transformed into a yeast strain where the endogenous U5 gene was deleted . Comparison of viable mutations in loop 1 revealed that position 6 was invariant and positions 5 and 7 displayed some sequence conservation . These data indicate positions 5, 6 and 7 in loop 1 are important for U5 function in vivo . A screen for mutations that suppress the temperature-sensitive phenotype of three loop 1 mutants produced eight intragenic suppressors all containing alterations in loop 1 . Further analysis of these temperature-sensitive mutants revealed that each displayed distinct cell cycle arrest phenotypes and pre-mRNA splicing inhibition patterns . The cell cycle arrest is likely attributed to inefficient splicing of alpha-tubulin pre-mRNA in one mutant and actin pre-mRNA in another . These results suggest that various mutations in loop 1 may affect the splicing of different pre-mRNAs in vivo. Nucleic Acids Res, 2002 Dec 15, 30(24), 5465 - 75 The centrosomal kinase Aurora-A/STK15 interacts with a putative tumor suppressor NM23-H1; Du J et al.; Alterations in the activity of the centrosomal kinase, Aurora-A/STK15, have been implicated in centrosome amplification, genome instability and cellular transformation . How STK15 participates in all of these processes remains largely mysterious . The activity of STK15 is regulated by phosphorylation and ubiquitin-mediated degradation, and physically interacts with protein phosphatase 1 (PP1) and CDC20 . However, the precise roles of these modifications and interactions have yet to be fully appreciated . Here we show that STK15 associates with a putative tumor and metastasis suppressor, NM23-H1 . STK15 and NM23 were initially found to interact in yeast in a two-hybrid assay . Association of these proteins in human cells was confirmed by co-immunoprecipitation from cell lysates and biochemical fractionation indicating that STK15 and NM23-H1 are present in a stable, physical complex . Notably, SKT15 and NM23 both localize to centrosomes throughout the cell cycle irrespective of the integrity of the microtubule network in normal human fibroblasts. Nucleic Acids Res, 2002 Dec 15, 30(24), 5416 - 24 Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability; West KL et al.; We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIbeta . We show that topo IIbeta is a DNA-dependent ATPase that appears to fit Michaelis-Menten kinetics . The ATPase activity is stimulated 44-fold by DNA . The k(cat) for ATP hydrolysis by human DNA topo IIbeta in the presence of DNA is 2.25 s(-1) . We have characterised a topo IIbeta derivative which carries a mutation in the ATPase domain (S165R) . S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s(-1), while not significantly altering the apparent K(m) . The specificity constant for the interaction between ATP and topo IIbeta (kcat/K(mapp)) showed a 90% reduction for betaS165R . The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation . However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis . The mutant enzyme is unable to complement ts yeast topo II in vivo . We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIbeta . Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation. Bioinformatics, 2002 Dec, 18(12), 1576 - 84 Comparing expression profiles of genes with similar promoter regions; Park PJ et al.; MOTIVATION: Gene regulatory elements are often predicted by seeking common sequences in the promoter regions of genes that are clustered together based on their expression profiles . We consider the problem in the opposite direction: we seek to find the genes that have similar promoter regions and determine the extent to which these genes have similar expression profiles . RESULTS: We use the data sets from experiments on Saccharomyces cerevisiae . Our similarity measure for the promoter regions is based on the set of common mapped or putative transcription factor binding sites and other regulatory elements in the upstream region of the genes, as contained in the Saccharomyces cerevisiae Promoter Database . We pair up the genes with high similarity scores and compare their expression levels in time-course experiment data . We find that genes with similar promoter regions on the average have significantly higher correlation, but it can vary widely depending on the genes . This confirms that the presence of similar regulatory elements often does not correspond to similarity in expression profiles and indicates that finding transcription factor binding sites or other regulatory elements starting with the expression patterns may be limited in many cases . Regardless of the correlation, the degree to which the profiles agree under different experimental conditions can be examined to derive hypotheses concerning the role of common regulatory elements . Overall, we find that considering the relationship between the promoter regions and the expression profiles starting with the regulatory elements is a difficult but useful process that can provide valuable insights. J Struct Biol, 2002 Oct-Dec, 140(1-3), 79 - 91 Myosin-like proteins 1 and 2 are not required for silencing or telomere anchoring, but act in the Tel1 pathway of telomere length control; Hediger F et al.; The positioning of chromosomal domains in interphase nuclei is thought to facilitate transcriptional repression in yeast . It has been reported that two large coiled-coil proteins of the nuclear envelope, myosin-like proteins 1 and 2, play direct roles in anchoring yeast telomeres to the nuclear periphery, thereby creating a subcompartment enriched for Sir proteins . We have created strains containing complete deletions of mlp1 and mlp2 genes, as well as the double null strain, and find no evidence for the disruption of telomere anchoring at the nuclear periphery in these cells . We also detect no disruption of telomere-associated gene silencing . We confirm, on the other hand, that mlp mutants are particularly sensitive to DNA-damaging agents, such as bleomycin . Moreover, we show that rather than having short telomeres as in yKu-deficient strains, the mlp1 mlp2 strains have extended telomeres, resembling phenotypes of mutations in rif1 . Whereas the mlp1 mlp2 mutations act on a pathway of telomere length regulation different from that of yKu70, the effects of the tel1 deletion are epistatic to the mlp mutations, suggesting that the Mlp proteins restrict telomere length in wild-type cells by influencing the Rif-Tel1 pathway of telomerase regulation. Org Lett, 2002 Dec 26, 4(26), 4655 - 7 Conformational stability of helical peptides containing a thioamide linkage; Miwa JH et al.; {structure: see text} Thioxo peptide analogues of the alpha-helical peptide GCN4-p1 were synthesized and evaluated for helicity and oligomeric state . Sedimentation equilibrium and CD measurements indicate that the thioxo peptides fold into parallel alpha-helical coiled coil structures essentially identical to the native structure . This work marks the first incorporation of a thioamide linkage into the backbone of an alpha-helix and demonstrates that a thioamide linkage is compatible with positions within the helix as well as near the C-terminus. Chromosome Res, 2002, 10(6), 455 - 66 Methylation of a euchromatin-heterochromatin transition region in Arabidopsis thaliana chromosome 5 left arm; Mathieu O et al.; Cytosine methylation was studied at the level of the euchromatin/heterochromatin transition genomic region of the Arabidopsis chromosome 5 left arm . It has been shown using a monoclonal antibody against 5-methylcytosines that the density of DNA methylation increases from the euchromatin towards the heterochromatin . YACs mapped along this region were characterized for their repeated sequences content . Some of them, corresponding to euchromatin, euchromatin/heterochromatin border and heterochromatin regions, were used as probes for a Southern blot analysis of methylation . This revealed that the degree of mCmCGG and GATmC methylation increases significantly from the euchromatin towards the heterochromatin . Moreover, an analysis of cytosine methylation levels (% of 5-methylcytosine) of different DNA fragments, inside the same genomic region, was performed using PCR and/or Southern blot approaches . There is a gradual increase of methylation along the genomic region analyzed: CpG methylation in the euchromatic fraction, CpG and CpNpG methylation at the euchromatin/heterochromatin transition and an additional asymmetrical methylation in the repeated-heterochromatic fraction . The most methylated repeated family at CpG, CpNpG and asymmetrical sites is the 5S ribosomal DNA, highly methylated even though it is transcribed. Biol Cell, 2002 Sep, 94(4-5), 217 - 31 Characterization of the poly(A) binding proteins expressed during oogenesis and early development of Xenopus laevis; Cosson B et al.; During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control . The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis . We showed that PABP1 and ePABP proteins are both present during oogenesis and early development . We selected ePABP as an eRF3 binding protein in a two-hybrid screening of a X . laevis cDNA library and demonstrated that this protein is associated with translational complexes . It can complement essential functions of the yeast homologue Pab1p . We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins. Biol Cell, 2002 Sep, 94(4-5), 205 - 16 Poly(A)-binding protein and eRF3 are associated in vivo in human and Xenopus cells; Cosson B et al.; An interaction between human poly(A)-binding protein (PABP) et human eRF3 has been demonstrated using a double-hybrid approach and in vitro assays . Here, we show that the binding of both proteins is conserved through evolution . We also demonstrate that the last 39 C-terminal amino acids of PABP contain the interface that interacts with eRF3 . This region includes helix 5, identified by RMN, which is conserved in all known PABPs . Lastly, we demonstrate that eRF3 et PABP molecules interact in vivo. J Biomed Biotechnol, 2002, 2(2), 55 - 60 Nucleotide Excision Repair, Genome Stability, and Human Disease: New Insight from Model Systems; Garfinkel DJ et al.; Nucleotide excision repair (NER) is one of several DNA repair pathways that are universal throughout phylogeny . NER has a broad substrate specificity and is capable of removing several classes of lesions to the DNA, including those that accumulate upon exposure to UV radiation . The loss of this activity in NER-defective mutants gives rise to characteristic sensitivities to UV that, in humans, is manifested as a greatly elevated sensitivity to exposure to the sun . Xeroderma pigmentosum (XP), Cockaynes syndrome (CS), and trichothiodystrophy (TTD) are three, rare, recessively inherited human diseases that are linked to these defects . Interestingly, some of the symptoms in afflicted individuals appear to be due to defects in transcription, the result of the dual functionality of several components of the NER apparatus as parts of transcription factor IIH (TFIIH) . Studies with several model systems have revealed that the genetic and biochemical features of NER are extraordinarily conserved in eukaryotes . One system that has been studied very closely is the budding yeast Saccharomyces cerevisiae . While many yeast NER mutants display the expected increases in UV sensitivity and defective transcription, other interesting phenotypes have also been observed . Elevated mutation and recombination rates, as well as increased frequencies of genome rearrangement by retrotransposon movement and recombination between short genomic sequences have been documented . The potential relevance of these novel phenotypes to disease in humans is discussed. J Mol Biol, 2003 Jan 10, 325(2), 399 - 408 Biogenesis of rat mitochondrial citrate carrier (CIC): the N-terminal presequence facilitates the solubility of the preprotein but does not act as a targeting signal; Zara V et al.; Most mitochondrial preproteins carry a cleavable N-terminal presequence that mediates targeting to mitochondria and translocation across the mitochondrial membranes . In this study, we characterized the presequence of the citrate carrier (CIC, tricarboxylate carrier) of rat liver mitochondria . The CIC presequence was found to be dispensable both for targeting to mitochondria and insertion into the inner membrane . Unlike the presequence of the related phosphate carrier, fusion of the CIC presequence to the cytosolic enzyme dihydrofolate reductase did not confer mitochondrial targeting, indicating that the CIC presequence does not act as a targeting signal . However, the presequence was required to keep the CIC in a soluble state . Mature CIC lacking the presequence was prone to aggregation . We conclude that mitochondrial presequences do not necessarily act as mediators of targeting . In the case of the CIC, the presequence appears to determine the folding state of the preprotein. J Med Food, 2002 Summer, 5(2), 79 - 83 Antioxidant properties of carboxymethyl glucan: comparative analysis; Babincova M et al.; Antioxidative capabilities of carboxymethylated (1 --> 3)-beta-D-glucan from Saccharomyces cerevisiae cell wall, alpha-tocopherol, and mannitol against lipid peroxidation in phosphatidylcholine liposomes induced by OH . radicals produced with Fenton's reagent (H(2)O(2)/Fe(2+)) were studied using absorption ultraviolet-visible spectrophotometry . It was found that (1 --> 3)-beta-D-glucan is an antioxidant with the scavenging ability lying between that of alpha-tocopherol, which is known to be incorporated in the lipid bilayer, and the water-soluble antioxidant, mannitol. Biochem J, 2003 Mar 15, 370(Pt 3), 1047 - 54 Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans; Cadinanos J et al.; Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue . In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH(2)Cl (tosylphenylalanylchloromethane; 'TPCK') respectively . A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C . elegans chromosome I, probably accounts for the EDTA-sensitive activity . An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH(2)Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism . We have identified a previously overlooked gene in C . elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1 . We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a -factor in vivo and to cleave a farnesylated peptide in vitro . These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16875 - 80 Epub 2002 Dec 16. Genome-wide coexpression dynamics: theory and application; Li KC; High-throughput expression profiling enables the global study of gene activities . Genes with positively correlated expression profiles are likely to encode functionally related proteins . However, all biological processes are interlocked, and each protein may play multiple cellular roles . Thus the coexpression of any two functionally related genes may depend on the constantly varying, yet often-unknown cellular state . To initiate a systematic study on this issue, a theory of coexpression dynamics is presented . This theory is used to rationalize a strategy of conducting a genome-wide search for the most critical cellular players that may affect the coexpression pattern of any two genes . In one example, using a yeast data set, our method reveals how the enzymes associated with the urea cycle are expressed to ensure proper mass flow of the involved metabolites . The correlation between ARG2 and CAR2 is found to change from positive to negative as the expression level of CPA2 increases . This delicate interplay in correlation signifies a remarkable control on the influx and efflux of ornithine and reflects well the intrinsic cellular demand for arginine . In addition to the urea cycle, our examples include SCH9 and CYR1 (both implicated in a recent longevity study), cytochrome c1 (mitochondrial electron transport), calmodulin (main calcium-binding protein), PFK1 and PFK2 (glycolysis), and two genes, ECM1 and YNL101W, the functions of which are newly revealed . The complexity in computation is eased by a new result from mathematical statistics. J Biol Chem, 2003 Feb 21, 278(8), 5854 - 63 Epub 2002 Dec 16. The auto-inhibitory function of importin alpha is essential in vivo; Harreman MT et al.; Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin alpha and beta . The importin alpha subunit recognizes classical NLS sequences, and the importin beta subunit directs the complex to the nuclear pore . Recent work shows that the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding in the absence of importin beta in vitro . To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha protein . These mutants dissect the two functions of the N-terminal IBB domain, importin beta binding and auto-inhibition . One of these importin alpha mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta or the importin alpha export receptor, Cse1p . We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin alpha remains bound to NLS-cargo within the nucleus . We propose a model where the auto-inhibitory activity of importin alpha is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin alpha to the cytoplasm. Comput Biol Med, 2003 Jan, 33(1), 1 - 15 A neural network-based similarity index for clustering DNA microarray data; Sawa T et al.; A common approach to the analysis of gene expression data is to define clusters of genes that have similar expression . A critical step in cluster analysis is the determination of similarity between the expression levels of two genes . We introduce a neural network-based similarity index as a non-linear similarity index and compare the results with other proximity measures for Saccharomyces cerevisiae gene expression data . We show that the clusters obtained using Euclidean distance, correlation coefficients, and mutual information were not significantly different . The clusters formed with the neural network-based index were more in agreement with those defined by functional categories and common regulatory motifs. Arch Biochem Biophys, 2002 Dec 1, 408(1), 17 - 32 Interaction of phosphoinositolglycan(-peptides) with plasma membrane lipid rafts of rat adipocytes; Muller G et al.; Insulin receptor-independent activation of the insulin signal transduction cascade in insulin-responsive target cells by phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) is accompanied by redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI proteins) and dually acylated nonreceptor tyrosine kinases from detergent/carbonate-resistant glycolipid-enriched plasma membrane raft domains of high-cholesterol content (hcDIGs) to rafts of lower cholesterol content (lcDIGs) . Here we studied the nature and localization of the primary target of PIG(-P) in isolated rat adipocytes . Radiolabeled PIG-P (Tyr-Cys-Asn-NH-(CH(2))(2)-O-PO(OH)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manalpha1-4GluN1-6Ino-1,2-(cyclic)-phosphate) prepared by chemical synthesis or a radiolabeled lipolytically cleaved GPI protein from Saccharomyces cerevisiae, which harbors the PIG-P moiety, bind to isolated hcDIGs but not to lcDIGs . Binding is saturable and abolished by pretreatment of intact adipocytes with trypsin followed by NaCl or with N-ethylmaleimide, indicating specific interaction of PIG-P with a cell surface protein . A 115-kDa polypeptide released from the cell surface by the trypsin/NaCl-treatment is labeled by {(14)C}N-ethylmaleimide . The labeling is diminished upon incubation of adipocytes with PIG-P which can be explained by direct binding of PIG-P to the 115-kDa protein and concomitant loss of its accessibility to N-ethylmaleimide . Binding of PIG-P to hcDIGs is considerably increased after pretreatment of adipocytes with (glycosyl)phosphatidylinositol-specific phospholipases compatible with lipolytic removal of endogenous ligands, such as GPI proteins/lipids . These data demonstrate that in rat adipocytes synthetic PIG(-P) as well as lipolytically cleaved GPI proteins interact specifically with hcDIGs . The interaction depends on the presence of a trypsin/NaCl/NEM-sensitive 115-kDa protein located at hcDIGs which thus represents a candidate for a binding protein for exogenous insulin-mimetic PIG(-P) and possibly endogenous GPI proteins/lipids. Oncogene, 2002 Dec 16, 21(58), 9022 - 32 Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors; Nishino T et al.; DNA nucleases catalyze the cleavage of phosphodiester bonds . These enzymes play crucial roles in various DNA repair processes, which involve DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and double strand break repair . In recent years, new nucleases involved in various DNA repair processes have been reported, including the Mus81 : Mms4 (Eme1) complex, which functions during the meiotic phase and the Artemis : DNA-PK complex, which processes a V(D)J recombination intermediate . Defects of these nucleases cause genetic instability or severe immunodeficiency . Thus, structural biology on various nuclease actions is essential for the elucidation of the molecular mechanism of complex DNA repair machinery . Three-dimensional structural information of nucleases is also rapidly accumulating, thus providing important insights into the molecular architectures, as well as the DNA recognition and cleavage mechanisms . This review focuses on the three-dimensional structure-function relationships of nucleases crucial for DNA repair processes. Oncogene, 2002 Dec 16, 21(58), 8967 - 80 Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability; Tauchi H et al.; DNA double-strand breaks represent the most potentially serious damage to a genome and hence, at least two pathways of DNA repair have evolved; namely, homologous recombination repair and non-homologous end joining . Defects in both rejoining processes result in genomic instability including chromosome rearrangements, LOH and gene mutations, which may lead to development of malignancies . Nijmegen breakage syndrome is a recessive genetic disorder, characterized by elevated sensitivity to ionizing radiation that induces double-strand breaks, and high frequency of malignancies . NBS1, the product of the gene underlying the disease, forms a multimeric complex with hMRE11/hRAD50 nuclease and recruits them to the vicinity of sites of DNA damage by direct binding to phosphorylated histone H2AX . The combination of the highly-conserved NBS1 forkhead associated domain and BRCA1 C-terminus domain has a crucial role for recognition of damaged sites . Thereafter, the NBS1-complex proceeds to rejoin double-strand breaks predominantly by homologous recombination repair in vertebrates . This process collaborates with cell-cycle checkpoints at S and G2 phase to facilitate DNA repair . NBS1 is also associated with telomere maintenance and DNA replication . Based on recent knowledge regarding NBS1, we propose here a two-step binding mechanism for damage recognition by repair proteins, and describe the molecular links to factors for genome stability. Nature, 2003 Jan 9, 421(6919), 177 - 82 Epub 2002 Dec 11. Rhythmic histone acetylation underlies transcription in the mammalian circadian clock; Etchegaray JP et al.; In the mouse circadian clock, a transcriptional feedback loop is at the centre of the clockwork mechanism . Clock and Bmal1 are essential transcription factors that drive the expression of three period genes (Per1-3) and two cryptochrome genes (Cry1 and Cry2) . The Cry proteins feedback to inhibit Clock/Bmal1-mediated transcription by a mechanism that does not alter Clock/Bmal1 binding to DNA . Here we show that transcriptional regulation of the core clock mechanism in mouse liver is accompanied by rhythms in H3 histone acetylation, and that H3 acetylation is a potential target of the inhibitory action of Cry . The promoter regions of the Per1, Per2 and Cry1 genes exhibit circadian rhythms in H3 acetylation and RNA polymerase II binding that are synchronous with the corresponding steady-state messenger RNA rhythms . The histone acetyltransferase p300 precipitates together with Clock in vivo in a time-dependent manner . Moreover, the Cry proteins inhibit a p300-induced increase in Clock/Bmal1-mediated transcription . The delayed timing of the Cry1 mRNA rhythm, relative to the Per rhythms, is due to the coordinated activities of Rev-Erbalpha and Clock/Bmal1, and defines a new mechanism for circadian phase control. Mol Cell Biol, 2003 Jan, 23(1), 382 - 8 The stalling of transcription at abasic sites is highly mutagenic; Yu SL et al.; Abasic (AP) sites represent one of the most frequently formed lesions in DNA . Here, we examine the consequences of the stalling of RNA polymerase II at AP sites in DNA in Saccharomyces cerevisiae . A severe inhibition of transcription occurs in strains that are defective in the removal of AP sites and that also lack the RAD26 gene, a homolog of the human Cockayne syndrome group B (CSB) gene, and, importantly, a dramatic rise in mutagenesis is incurred in such strains . From the various observations presented here, we infer that the stalling of transcription at AP sites is highly mutagenic. Mol Cell Biol, 2003 Jan, 23(1), 306 - 21 Two different Drosophila ADA2 homologues are present in distinct GCN5 histone acetyltransferase-containing complexes; Muratoglu S et al.; We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4 . Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b) . The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains . The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development . Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3 . Both dADA2s further potentiate transcriptional activation in insect and mammalian cells . Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs) . Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs . Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes . These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes. Mol Cell Biol, 2003 Jan, 23(1), 289 - 305 The Drosophila SNR1 (SNF5/INI1) subunit directs essential developmental functions of the Brahma chromatin remodeling complex; Marenda DR et al.; The Drosophila melanogaster Brahma (Brm) complex, a counterpart of the Saccharomyces cerevisiae SWI/SNF ATP-dependent chromatin remodeling complex, is important for proper development by maintaining specific gene expression patterns . The SNR1 subunit is strongly conserved with yeast SNF5 and mammalian INI1 and is required for full activity of the Brm complex . We identified a temperature-sensitive allele of snr1 caused by a single amino acid substitution in the conserved repeat 2 region, implicated in a variety of protein-protein interactions . Genetic analyses of snr1(E1) reveal that it functions as an antimorph and that snr1 has critical roles in tissue patterning and growth control . Temperature shifts show that snr1 is continuously required, with essential functions in embryogenesis, pupal stages, and adults . Allele-specific genetic interactions between snr1(E1) and mutations in genes encoding other members of the Brm complex suggest that snr1(E1) mutant phenotypes result from reduced Brm complex function . Consistent with this view, SNR1(E1) is stably associated with other components of the Brm complex at the restrictive temperature . SNR1 can establish direct contacts through the conserved repeat 2 region with the SET domain of the homeotic regulator Trithorax (TRX), and SNR1(E1) is partially defective for functional TRX association . As truncating mutations of INI1 are strongly correlated with aggressive cancers, our results support the view that SNR1, and specifically the repeat 2 region, has a critical role in mediating cell growth control functions of the metazoan SWI/SNF complexes. Mol Cell Biol, 2003 Jan, 23(1), 229 - 37 Targeting the MEF2-like transcription factor Smp1 by the stress-activated Hog1 mitogen-activated protein kinase; de Nadal E et al.; Exposure of Saccharomyces cerevisiae to increases in extracellular osmolarity activates the stress-activated Hog1 mitogen-activated protein kinase (MAPK), which is essential for cell survival upon osmotic stress . Yeast cells respond to osmotic stress by inducing the expression of a very large number of genes, and the Hog1 MAPK plays a critical role in gene transcription upon stress . To understand how Hog1 controls gene expression, we designed a genetic screen to isolate new transcription factors under the control of the MAPK and identified the MEF2-like transcription factor, Smp1, as a target for Hog1 . Overexpression of SMP1 induced Hog1-dependent expression of osmoresponsive genes such as STL1, whereas smp1Delta cells were defective in their expression . Consistently, smp1Delta cells displayed reduced viability upon osmotic shock . In vivo coprecipitation and phosphorylation studies showed that Smp1 and Hog1 interact and that Smp1 is phosphorylated upon osmotic stress in a Hog1-dependent manner . Hog1 phosphorylated Smp1 in vitro at the C-terminal region . Phosphorylation of Smp1 by the MAPK is essential for its function, since a mutant allele unable to be phosphorylated by the MAPK displays impaired stress responses . Thus, our data indicate that Smp1 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK . Moreover, Smp1 concentrates in the nucleus during the stationary phase, and the lack of SMP1 results in cells that lose viability in the stationary phase . Localization of Smp1 depends on HOG1, and consistently, hog1Delta cells also lose viability during this growth phase . These data suggest that Smp1 could be mediating a role for the Hog1 MAPK during the stationary phase. Mol Cell Biol, 2003 Jan, 23(1), 216 - 28 Novel p27(kip1) C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions; McAllister SS et al.; Hepatocyte growth factor (HGF) signaling via its receptor, the proto-oncogene Met, alters cell proliferation and motility and has been associated with tumor metastasis . HGF treatment of HepG2 human hepatocellular carcinoma cells induces cell migration concomitant with increased levels of the p27(kip1) cyclin-cdk inhibitor . HGF signaling resulted in nuclear export of endogenous p27 to the cytoplasm, via Ser-10 phosphorylation, where it colocalized with F-actin . Introduction of transducible p27 protein (TATp27) was sufficient for actin cytoskeletal rearrangement and migration of HepG2 cells . TATp27 mutational analysis identified a novel p27 C-terminal domain required for cell migration, distinct from the N-terminal cyclin-cyclin-dependent kinase (cdk) binding domain . Loss or disruption of the p27 C-terminal domain abolished both actin rearrangement and cell migration . The cell-scattering activity of p27 occurred independently of its cell cycle arrest functions and required cytoplasmic localization of p27 via Ser-10 phosphorylation . Furthermore, Rac GTPase was necessary for p27-dependent migration but alone was insufficient for HepG2 cell migration . These results predicted a migration defect in p27-deficient cells . Indeed, p27-deficient primary fibroblasts failed to migrate, and reconstitution with TATp27 rescued the motility defect . These observations define a novel role for p27 in cell motility that is independent of its function in cell cycle inhibition. Mol Cell Biol, 2003 Jan, 23(1), 163 - 77 Induction of apoptosis by sphingoid long-chain bases in Aspergillus nidulans; Cheng J et al.; Sphingolipid metabolism is implicated to play an important role in apoptosis . Here we show that dihydrosphingosine (DHS) and phytosphingosine (PHS), two major sphingoid bases of fungi, have potent fungicidal activity with remarkably high structural and stereochemical specificity against Aspergillus nidulans . In fact, only naturally occurring DHS and PHS are active . Further analysis revealed that DHS and PHS induce rapid DNA condensation independent of mitosis, large-scale DNA fragmentation, and exposure of phosphatidylserine, all common morphological features characteristic of apoptosis, suggesting that DHS and PHS induce apoptosis in A . nidulans . The finding that DNA fragmentation requires protein synthesis, which implies that an active process is involved, further supports this proposition . The induction of apoptosis by DHS and PHS is associated with the rapid accumulation of reactive oxygen species (ROS) . However, ROS are not required for apoptosis induced by DHS and PHS, as scavenging of ROS by a free radical spin trap has no effect . We further demonstrate that apoptosis induced by DHS and PHS is independent of metacaspase function but requires mitochondrial function . Together, the results suggest that DHS and PHS induce a type of apoptosis in A . nidulans most similar to the caspase-independent apoptosis observed in mammalian systems . As A . nidulans is genetically tractable, this organism should be an ideal model system for dissecting sphingolipid signaling in apoptosis and, importantly, for further elucidating the molecular basis of caspase-independent apoptosis. Mol Cell Biol, 2003 Jan, 23(1), 140 - 9 Activating signal cointegrator 2 belongs to a novel steady-state complex that contains a subset of trithorax group proteins; Goo YH et al.; Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner . These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors . In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex {ASCOM}) in HeLa nuclei . ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/beta-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2 . In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities . Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo . Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors . Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation. Mol Cell Biol, 2003 Jan, 23(1), 38 - 54 The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis; McBurney MW et al.; The yeast Sir2p protein has an essential role in maintaining telomeric and mating type genes in their transcriptionally inactive state . Mammalian cells have a very large proportion of their genome inactive and also contain seven genes that have regions of homology with the yeast sir2 gene . One of these mammalian genes, sir2alpha, is the presumptive mammalian homologue of the yeast sir2 gene . We set out to determine if sir2alpha plays a role in mammalian gene silencing by creating a strain of mice carrying a null allele of sir2alpha . Animals carrying two null alleles of sir2alpha were smaller than normal at birth, and most died during the early postnatal period . In an outbred background, the sir2alpha null animals often survived to adulthood, but both sexes were sterile . We found no evidence for failure of gene silencing in sir2alpha null animals, suggesting that either SIR2alpha has a different role in mammals than it does in Saccharomyces cerevisiae or that its role in gene silencing in confined to a small subset of mammalian genes . The phenotype of the sir2alpha null animals suggests that the SIR2alpha protein is essential for normal embryogenesis and for normal reproduction in both sexes. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16934 - 9 Epub 2002 Dec 13. A genome-wide screen for methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in the presence of DNA damage; Chang M et al.; We performed a systematic screen of the set of approximately 5,000 viable Saccharomyces cerevisiae haploid gene deletion mutants and have identified 103 genes whose deletion causes sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS) . In total, 40 previously uncharacterized alkylation damage response genes were identified . Comparison with the set of genes known to be transcriptionally induced in response to MMS revealed surprisingly little overlap with those required for MMS resistance, indicating that transcriptional regulation plays little, if any, role in the response to MMS damage . Clustering of the MMS response genes on the basis of their cross-sensitivities to hydroxyurea, UV radiation, and ionizing radiation revealed a DNA damage core of genes required for responses to a broad range of DNA-damaging agents . Of particular significance, we identified a subset of genes that show a specific MMS response, displaying defects in S phase progression only in the presence of MMS . These genes may promote replication fork stability or processivity during encounters between replication forks and DNA damage. J Cell Sci, 2003 Jan 15, 116(Pt 2), 415 - 27 COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle; Siddiqi SA et al.; The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins . The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown . To study the dependence of PCTV-ER budding on COPII proteins we examined protein and PCTV budding by using ER prepared from rat small intestinal mucosal cells prelabeled with (3)H-oleate or (14)C-oleate and (3)H-leucine . Budded (3)H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles . Our results showed the following: (1) Proteinase K treatment did not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 was added . (2) PCTV budding was dependent on cytosol and ATP . (3) The COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 were associated with PCTVs . (4) Isolated PCTVs were able to fuse with intestinal Golgi . (5) Antibodies to Sar1 completely inhibited protein vesicle budding but increased the generation of PCTV; these changes were reversed by the addition of recombinant Sar1 . (6) PCTVs formed in the absence of Sar1 did not contain the COPII proteins Sar1, Sec24 or Sec31 and did not fuse with the Golgi complex . Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi. J Biol Chem, 2003 Feb 21, 278(8), 6495 - 502 Epub 2002 Dec 12. Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis; Fransson A et al.; The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome . Here we report the characterization of a new family of Rho GTPases with atypical features . These proteins, which were called Miro-1 and Miro-2 (for mitochondrial Rho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs . Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early during evolution . Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria . Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induced an aggregation of the mitochondrial network and resulted in an increased apoptotic rate of the cells expressing activated Miro-1 . These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis. J Biol Chem, 2003 Feb 21, 278(8), 6355 - 62 Epub 2002 Dec 12. Cyclin D3 is a cofactor of retinoic acid receptors, modulating their activity in the presence of cellular retinoic acid-binding protein II; Despouy G et al.; Ligand-induced transcription activation of retinoic acid (RA) target genes by nuclear receptors (retinoic acid (RAR) and retinoid X (RXR) receptors) depends on the recruitment of coactivators . We have previously demonstrated that the small 15-kDa cellular RA-binding protein II (CRABPII) is a coactivator present in the RA-dependent nuclear complex . As identifying cell-specific partners of CRABPII might help to understand the novel control of RA signaling, we performed a yeast two-hybrid screen of a hematopoietic HL-60 cDNA library using human CRABPII as bait and have subsequently identified human cyclin D3 as a partner of CRABPII . Cyclin D3 interacted with CRABPII in a ligand-independent manner and equally bound RAR alpha, but not RXR alpha, and only in the presence of RA . We further show that cyclin D3 positively modulated RA-mediated transcription through CRABPII . Therefore, cyclin D3 may be part of a ternary complex with CRABPII and RAR . Finally, we show that cyclin D3 expression paralleled HL-60 differentiation and arrest of cell growth . These findings led us to speculate that control of cell proliferation during induction of differentiation may directly involve, at the transcriptional level, nuclear receptors, coactivators, and proteins of the cell cycle in a cell- and nuclear receptor-specific manner. J Biol Chem, 2003 Feb 14, 278(7), 4943 - 8 Epub 2002 Dec 13. Factors governing nonoverlapping substrate specificity by mitochondrial inner membrane peptidase; Luo W et al.; At least three peptidases are involved in cleaving presequences from imported mitochondrial proteins . One of the peptidase, the inner membrane peptidase, has two catalytic subunits, Imp1p and Imp2p, which are structurally related but functionally distinct in the yeast Saccharomyces cerevisiae . Whereas both subunits are members of the type I signal peptidase family, they exhibit nonoverlapping substrate specificities . A clue to the substrate specificity mechanism has come from our discovery of the importance not only of the -1 and -3 residues in the signal peptides cleaved by Imp1p and Imp2p but also the +1 cargo residues attached to the signal peptides . We specifically find that Imp1p prefers substrates having a negatively charged residue (Asp or Glu) at the +1 position, whereas Imp2p prefers substrates having the Met residue at the +1 position . We further suggest that the conformation of the cargo is important for substrate recognition by Imp2p . A role for the cargo in presequence recognition distinguishes Imp1p and Imp2p from other type I signal peptidases. Mol Plant Microbe Interact, 2002 Dec, 15(12), 1268 - 76 The mitogen-activated protein kinase gene MAF1 is essential for the early differentiation phase of appressorium formation in Colletotrichum lagenarium; Kojima K et al.; Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium . In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation . In this study, the goal was to investigate roles of other MAPKs in C . lagenarium . The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized . The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity . The M . grisea mps1 mutant forms appressoria, but conidia of the C . lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces . On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria . The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar . The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants . These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria. Mol Plant Microbe Interact, 2002 Dec, 15(12), 1253 - 67 Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea; Soanes DM et al.; The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation . The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression . The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress . The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions . During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes . Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation . Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection. Mol Plant Microbe Interact, 2002 Dec, 15(12), 1213 - 8 The P34 syringolide elicitor receptor interacts with a soybean photorespiration enzyme, NADH-dependent hydroxypyruvate reductase; Okinaka Y et al.; The syringolide receptor P34 mediates avrD-Rpg4 gene-for-gene complementarity in soybean . However, the mechanism underlying P34 signal transmission after syringolide binding is unknown . In an effort to identify a second messenger for P34, soybean leaf proteins were run though a P34-affinity column . A 42-kDa protein which specifically bound to the column was identified as a putative plant NADH-dependent hydroxypyruvate reductase (HPR) by N-terminal peptide sequencing . HPR is an important enzyme involved in the plant photorespiration system . Screening of a soybean cDNA library yielded two distinct HPR clones that encoded proteins with 97% identity (P42-1 and P42-2) . Surprisingly, only P42-2 displayed good binding with P34 in a yeast two-hybrid assay, indicating that P42-2, but not P42-1, is a potential second messenger for P34 . Glycerate and its analogs, which are utilized in the photorespiration system, were tested for their inhibitory effect on syringolide-induced hypersensitive response (HR) to evaluate the biological significance of P42-2 . Interestingly, the downstream products of HPR (glycerate and 3-phosphoglycerate) inhibited HR but the upstream compounds (hydroxypyruvate or serine) did not have a significant effect on HR . These results suggest that P42-2 is a primary target for a P34/syringolide complex and that P42-2 binding with the complex probably induces HR by inhibiting one or more HPR functions in soybean. Plant Physiol, 2002 Dec, 130(4), 2152 - 63 Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells; Klusener B et al.; A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure . Our study addresses commonalities in the signaling pathways that these stimuli trigger . A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca(2+) influx current in Arabidopsis guard cells termed "I(Ca)" (Z.M . Pei, Y . Murata, G . Benning, S . Thomine, B . Klusener, G.J . Allen, E . Grill, J.I . Schroeder, Nature {2002} 406: 731-734) . We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H . These elicitors also induced elevations in the concentration of free cytosolic calcium ({Ca(2+)}(cyt)) and stomatal closure in guard cells . ABA and ROS elicited {Ca(2+)}(cyt) oscillations in guard cells only when extracellular Ca(2+) was present . In a 5 mM KCl extracellular buffer, 45% of guard cells exhibited spontaneous {Ca(2+)}(cyt) oscillations that differed in their kinetic properties from ABA-induced Ca(2+) increases . These spontaneous {Ca(2+)}(cyt) oscillations also required the availability of extracellular Ca(2+) and depended on the extracellular potassium concentration . Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of {Ca(2+)}(cyt) elevations in 62 of 101 cells, revealing a new mode of ABA signaling . These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane I(Ca) channels and support a requirement for extracellular Ca(2+) for elicitor and ABA signaling, as well as for cellular {Ca(2+)}(cyt) oscillation maintenance. Plant Physiol, 2002 Dec, 130(4), 2076 - 84 The sac mutants of Chlamydomonas reinhardtii reveal transcriptional and posttranscriptional control of cysteine biosynthesis; Ravina CG et al.; Algae and vascular plants are cysteine (Cys) prototrophs . They are able to import, reduce, and assimilate sulfate into Cys, methionine, and other organic sulfur-containing compounds . Characterization of genes encoding the enzymes required for Cys biosynthesis from the unicellular green alga Chlamydomonas reinhardtii reveals that transcriptional and posttranscriptional mechanisms regulate the pathway . The derived amino acid sequences of the C . reinhardtii genes encoding 5'-adenylylsulfate (APS) reductase and serine (Ser) acetyltransferase are orthologous to sequences from vascular plants . The Cys biosynthetic pathway of C . reinhardtii is regulated by sulfate availability . The steady-state level of transcripts and activity of ATP sulfurylase, APS reductase, Ser acetyltransferase, and O-acetyl-Ser (thiol) lyase increase when cells are deprived of sulfate . The sac1 mutation, which impairs C . reinhardtii ability to acclimate to sulfur-deficient conditions, prevents the increase in accumulation of the transcripts encoding these enzymes and also prevents the increase in activity of all the enzymes except APS reductase . The sac2 mutation, which does not affect accumulation of APS reductase transcripts, blocks the increase in APS reductase activity . These results suggest that APS reductase activity is regulated posttranscriptionally in a SAC2-dependent process. Plant Physiol, 2002 Dec, 130(4), 2027 - 38 Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung . Implications for the evolution of plant fatty acid diversity; Dyer JM et al.; The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil . Here, we describe the cloning and functional analysis of two closely related Delta(12) oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid . Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity . Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds . Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1delta(9cis)) into linoleic acid (18:2delta(9cis,12cis)) and that FADX converted linoleic acid into alpha-eleostearic acid . Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and delta(12)-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates . Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration . Phylogenetic analysis revealed that tung FADX is grouped with delta(12) fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity . Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity. Plant Physiol, 2002 Dec, 130(4), 1837 - 51 Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar; Ro DK et al.; NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases . We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa x Populus deltoides) . Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity) . CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevi