J Biol Chem, 2003 Jun 27, 278(26), 24225 - 32 Epub 2003 Apr 17. Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism; Gallardo M et al.; THP1 is a conserved eukaryotic gene whose null mutations confer, in yeast, transcription and genetic instability phenotypes and RNA export defects similar to those of the THO/TREX complex null mutations . In a search for multicopy suppressors of the transcription defect of thp1Delta cells, we identified the poly(A)+ RNA-binding heterogeneous nuclear ribonucleoprotein Nab2p . Multicopy NAB2 also suppressed the RNA export defect of thp1Delta cells . This result suggests a functional relationship between Thp1p and Nab2p . Consistently, the leaky mutation nab2-1 conferred a transcription defect and hyper-recombination phenotype similar to those of thp1Delta, although to a minor degree . Reciprocally, a purified His6-tagged Thp1p fusion bound RNA in vitro . In a different approach, we show by Western analyses that a highly purified Thp1p-Sac3p complex does not contain components of THO/TREX and that sac3Delta confers a transcription defect and hyper-recombination phenotype identical to those of thp1Delta . mRNA degradation was not affected in thp1Delta mutants, implying that their expression defects are not due to mRNA decay . This indicates that Thp1p-Sac3p is a structural and functional unit . Altogether, our results suggest that Thp1p-Sac3p and Nab2p are functionally related heterogeneous nuclear ribonucleoproteins that define a further link between mRNA metabolism and transcription. FEMS Yeast Res, 2003 Mar, 3(1), 27 - 34 The Hansenula polymorpha PDD7 gene is essential for macropexophagy and microautophagy; Komduur JA et al.; Hansenula polymorpha PDD genes are involved in the selective degradation of peroxisomes via macropexophagy . We have isolated various novel pdd mutants by a gene-tagging method . Here we describe the isolation and characterisation of PDD7, which encodes a protein with high sequence similarity (40% identity) to Saccharomyces cerevisiae Apg1p/Aut3p, previously described to be involved in random autophagy and the cytoplasm-to-vacuole targeting pathway . Our data indicate that HpPdd7p is essential for two processes that degrade peroxisomes, namely the highly selective process of macropexophagy and microautophagy, which occurs in H . polymorpha upon nitrogen starvation. Mol Cell Proteomics, 2003 Mar, 2(3), 173 - 81 Epub 2003 Apr 07. Genome-wide Analyses of Carboxyl-terminal Sequences; Chung JJ et al.; Sequence motifs at the protein carboxyl termini in linear polypeptides are uniquely positioned and functionally capable of serving as recognition signatures for a variety of cellular and biochemical processes . At the proteome level, it is unknown whether and what carboxyl-terminal sequences might be particularly conserved, which may be directly related to specific biological functions shared among certain groups of proteins . To investigate this question, we analyzed the terminal sequences of reported yeast open reading frames, which presumably constitute the predicted, entire proteome of Saccharomyces cerevisiae . The results show that there are both known and novel terminal sequences . They are conserved at a frequency similar to that of functionally important, experimentally confirmed signals such as the HDEL sequence that mediates the endoplasmic reticulum retention and/or retrieval . The findings support the notion that there may be additional carboxyl-terminal signals, and the conserved motifs could be experimentally tested for currently unknown biological functions . Similar analyses were also applied to the limited proteome databases of other organisms with overall consistent findings . Therefore, indexing a proteome according to its carboxyl-terminal sequences may provide a means for functional classification and determination of proteins. EMBO J, 2003 Apr 15, 22(8), 1846 - 56 Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes; Simic R et al.; Transcription in eukaryotes is influenced by the chromatin state of the template, and chromatin remodeling factors have well-documented roles in regulating transcription initiation by RNA polymerase (pol) II . Chromatin also influences transcription elongation; however, little is known about the role of chromatin remodeling factors in this process . Here, we present evidence that the Saccharomyces cerevisiae chromatin remodeling factor Chd1 functions during transcription elongation . First, we identified Chd1 in a two-hybrid screen for proteins that interact with Rtf1, a member of the Paf1 complex that associates with RNA pol II and regulates transcription elongation . Secondly, we show through co-immunoprecipitation studies that Chd1 also interacts with components of two essential elongation factors, Spt4-Spt5 and Spt16-Pob3 . Thirdly, we demonstrate that deletion of CHD1 suppresses a cold-sensitive spt5 mutation that is also suppressed by defects in the Paf1 complex and RNA pol II . Finally, we demonstrate that Chd1, Rtf1 and Spt5 associate with actively transcribed regions of chromatin . Collectively, these findings suggest an important role for Chd1 and chromatin remodeling in the control of transcription elongation. Curr Biol, 2003 Apr 15, 13(8), R326 - 37 Mechanisms of protein import into mitochondria; Truscott KN et al.; Apart from a handful of proteins encoded by the mitochondrial genome, most proteins residing in this organelle are nuclear-encoded and synthesised in the cytosol . Thus, delivery of proteins to their final destination depends on a network of specialised import components that form at least four main translocation complexes . The import machinery ensures that proteins earmarked for the mitochondrion are recognised and delivered to the organelle, transported across membranes, sorted to the correct compartment and assisted in overcoming energetic barriers. Drugs, 2003, 63(10), 1021 - 51 Recombinant hepatitis B vaccine (Engerix-B): a review of its immunogenicity and protective efficacy against hepatitis B; Keating GM et al.; Engerix-B (Hep-B{Eng}) is a noninfectious recombinant DNA vaccine containing hepatitis B surface antigen (HBsAg) . It is produced from genetically engineered yeast (Saccharomyces cerevisiae) . Intramuscular Hep-B(Eng) {0-, 1-, 6-month schedule} has excellent immunogenicity in healthy neonates and infants, children, adolescents and adults, with seroprotection rates of 85-100% seen approximate, equals 1 month after the final dose of vaccine; seroprotection was defined as an antibody against HBsAg (anti-HBs) titre of > or =10 IU/L . The use of alternative Hep-B(Eng) immunisation schedules (e.g . a 0-, 1-, 2-, 12-month schedule in neonates and infants, 0-, 12-, 24-month or two-dose schedules in children and adolescents, and accelerated schedules in adults) have also been associated with high rates of seroprotection . Seroprotection rates were generally similar with Hep-B(Eng) and the recombinant vaccine Recombivax HB (Hep-B{Rax}) or plasma-derived vaccines (PDVs) approximate, equals 1 month after the final dose (although anti-HBs geometric mean titres were significantly higher with Hep-B{Eng} than with Hep-B{Rax}) . One month after the final dose, adults had significantly higher seroprotection rates with the recombinant triple-antigen vaccine Bio-Hep-B (Hep-B{Bio}) than with Hep-B(Eng), although seroprotection rates in healthy infants were similar with Hep-B(Eng) and Hep-B(Bio) . Hep-B(Eng) had excellent immunogenicity in several groups considered at high risk of acquiring hepatitis B (e.g . neonates born to hepatitis B carrier mothers and healthcare workers) . The immunogenicity of Hep-B(Eng) was reduced in patients with conditions associated with impaired immune function (e.g . patients undergoing haemodialysis or being treated for malignancy), although it had good immunogenicity in patients with diabetes mellitus.Hep-B(Eng) had excellent protective efficacy against HBsAg carriage in healthy infants and children, and in neonates born to hepatitis B carrier mothers (protective efficacy of 95-99%) . Hep-B(Eng) also demonstrated good protective efficacy in a number of other high-risk groups . Hep-B(Eng) is generally well tolerated with a tolerability profile similar to that of Hep-B(Rax), Hep-B(Bio) and PDVs . In conclusion, Hep-B(Eng) is a well established, highly immunogenic hepatitis B vaccine with good tolerability and excellent protective efficacy; it offers flexibility through a variety of immunisation schedules . In addition, it appears that Hep-B(Eng) confers immunity for at least 10 years . Hep-B(Eng) has an important role in mass vaccination campaigns against hepatitis B, as well as in groups considered at high risk of acquiring hepatitis B. Mol Cell Biol, 2003 May, 23(9), 3339 - 51 Regulation of the bone-specific osteocalcin gene by p300 requires Runx2/Cbfa1 and the vitamin D3 receptor but not p300 intrinsic histone acetyltransferase activity; Sierra J et al.; p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors . p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation . In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D(3) via the vitamin D(3) receptor complex . Therefore, we addressed p300 control of basal and vitamin D(3)-enhanced activity of the OC promoter . We find that transient overexpression of p300 results in a significant dose-dependent increase of both basal and vitamin D(3)-stimulated OC gene activity . This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element . In addition, by coimmunoprecipitation, we show that the endogenous Runx2/Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations . We also demonstrate by chromatin immunoprecipitation assays that p300, Runx2/Cbfa1, and 1alpha,25-dihydroxyvitamin D(3) receptor interact with the OC promoter in intact osteoblastic cells expressing this gene . The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, as a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300 . On the basis of these results, we propose that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D(3). Mol Cell Biol, 2003 May, 23(9), 3329 - 38 Loss of the Rpb4/Rpb7 subcomplex in a mutant form of the Rpb6 subunit shared by RNA polymerases I, II, and III; Tan Q et al.; We have identified a conditional mutation in the shared Rpb6 subunit, assembled in RNA polymerases I, II, and III, that illuminated a new role that is independent of its assembly function . RNA polymerase II and III activities were significantly reduced in mutant cells before and after the shift to nonpermissive temperature . In contrast, RNA polymerase I was marginally affected . Although the Rpb6 mutant strain contained two mutations (P75S and Q100R), the majority of growth and transcription defects originated from substitution of an amino acid nearly identical in all eukaryotic counterparts as well as bacterial omega subunits (Q100R) . Purification of mutant RNA polymerase II revealed that two subunits, Rpb4 and Rpb7, are selectively lost in mutant cells . Rpb4 and Rpb7 are present at substoichiometric levels, form a dissociable subcomplex, are required for RNA polymerase II activity at high temperatures, and have been implicated in the regulation of enzyme activity . Interaction experiments support a direct association between the Rpb6 and Rpb4 subunits, indicating that Rpb6 is one point of contact between the Rpb4/Rpb7 subcomplex and RNA polymerase II . The association of Rpb4/Rpb7 with Rpb6 suggests that analogous subunits of each RNA polymerase impart class-specific functions through a conserved core subunit. Mol Cell Biol, 2003 May, 23(9), 3141 - 51 The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex; Melville MW et al.; The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell . Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex . VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells . Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells . Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70 . VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex . Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70 . In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC . We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC. Mol Cell Biol, 2003 May, 23(9), 3126 - 40 Cell cycle activation of the Swi6p transcription factor is linked to nucleocytoplasmic shuttling; Queralt E et al.; The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in the regulation of cell division . In the present work, we have characterized the function of the karyopherin Msn5p in the control of the cell cycle of Saccharomyces cerevisiae . Phenotypic analysis of the msn5 mutant revealed an increase in cell size and a functional interaction between Msn5p and the cell cycle transcription factor SBF (composed of the Swi4p and Swi6p proteins), indicating that Msn5p is involved in Start control . In fact, we have shown that the level of Cln2p protein is drastically reduced in an msn5 mutant . The effect on CLN2 expression is mediated at a transcriptional level, Msn5p being necessary for proper SBF-dependent transcription . On the contrary, loss of MSN5 has no effect on the closely related transcription factor MBF (composed of the Mbp1p and Swi6p proteins) . Regulation of SBF by Msn5p is exerted by control of the localization of the regulatory subunit Swi6p . Swi6p shuttles between the nucleus and the cytoplasm during the cell cycle, and we have found that Msn5p is required for Swi6p export from the nucleus during the G(2)-M phase . What is more important, we have demonstrated that export of Swi6p to the cytoplasm is required for SBF activity, providing evidence for a functional switch of Swi6p linked to its nucleocytoplasmic shuttling during the cell cycle. J Biol Chem, 2003 Jun 20, 278(25), 22574 - 7 Epub 2003 Apr 14. REX1, a novel gene required for DNA repair; Cenkci B et al.; Nucleotide excision repair is a major pathway for repairing UV light-induced DNA damage in most organisms . Using insertional mutagenesis, we isolated a UV-sensitive mutant of Chlamydomonas reinhardtii that is blocked in the excision of cyclobutane pyrimidine dimers . The mutant is also sensitive to the alkylating agent, methyl methanesulphonate . We have cloned REX1, a novel gene that rescues the mutant . The gene is unusual in a eukaryotic organism in that it is predicted to encode two different proteins, a small protein (8.9 kDa) and a larger protein (31.8 kDa) . Neither protein is homologous to known DNA repair proteins . Partial complementation is achieved with subclones of the gene encoding only the 8.9-kDa protein . The 8.9-kDa protein has homologues in many organisms including Saccharomyces cerevisiae, Arabidopsis, and humans . The 31.8-kDa protein appears to be less conserved . These findings may be of general importance for DNA repair in other organisms. J Biol Chem, 2003 Jun 27, 278(26), 23410 - 7 Epub 2003 Apr 14. The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein; Kantake N et al.; Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination . Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication . We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)) . This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex . RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA . However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11) . Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA . These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation. BMC Dev Biol . 2003 Apr 09;3(1):3. IfkA, a presumptive eIF2 alpha kinase of Dictyostelium, is required for proper timing of aggregation and regulation of mound size; Fang R et al.; BACKGROUND: The transition from growth to development in Dictyostelium is initiated by amino acid starvation of growing amobae . In other eukaryotes, a key sensor of amino acid starvation and mediator of the resulting physiological responses is the GCN2 protein, an eIF2alpha kinase . GCN2 downregulates the initiation of translation of bulk mRNA and enhances translation of specific mRNAs by phosphorylating the translation initiation factor eIF2alpha . Two eIF2alpha kinases were identified in Dictyostelium and studied herein . RESULTS: Neither of the eIF2alpha kinases appeared to be involved in sensing amino acid starvation to initiate development . However, one of the kinases, IfkA, was shown to phosphorylate eIF2alpha from 1 to 7 hours after the onset of development, resulting in a shift from polysomes to free ribosomes for bulk mRNA . In the absence of the eIF2alpha phosphorylation, ifkA null cells aggregated earlier than normal and formed mounds and ultimately fruiting bodies that were larger than normal . The early aggregation phenotype in ifkA null cells reflected an apparent, earlier than normal establishment of the cAMP pulsing system . The large mound phenotype resulted from a reduced extracellular level of Countin, a component of the counting factor that regulates mound size . In wild type cells, phosphorylation of eIF2alpha by IfkA resulted in a specific stabilization and enhanced translational efficiency of countin mRNA even though reduced translation resulted for bulk mRNA . CONCLUSIONS: IfkA is an eIF2alpha kinase of Dictyostelium that normally phosphorylates eIF2alpha from 1 to 7 hours after the onset of development, or during the preaggregation phase . This results in an overall reduction in the initiation of protein synthesis during this time frame and a concomitant reduction in the number of ribosomes associated with most mRNAs . For some mRNAs, however, initiation of protein synthesis is enhanced or stabilized under the conditions of increased eIF2alpha phosphorylation . This includes countin mRNA. Cell Cycle, 2003 Mar-Apr, 2(2), 105 - 11 Vascular smooth muscle polyploidization--from mitotic checkpoints to hypertension; Hixon ML et al.; Aging and hypertension are accompanied by an increase in mass and rigidity of arterial walls . At capacitance arteries, the enlargement and stiffness of the medial smooth muscle layer promote systolic hypertension and contribute to left ventricular hypertrophy and cardiovascular morbidity . Morphological studies have demonstrated that vascular smooth muscle cell (VSMC) hypertrophy, with minimal hyperplasia, causes the enlargement of vascular smooth muscle at capacitance arteries, and that VSMC hypertrophy is strongly associated with VSMC polyploidization . Recent studies demonstrate that hypertrophic signals, such as those elicited by Angiotensin II, abrogate the mechanisms of control of M phase in VSMC and induce cell cycle re-entry and polyploidization . These polyploid VSMC have a lower replicative rate, but a higher mass, protein content and matrix production than their diploid counterparts . Both, the protein kinase Aktl and the cyclin kinase-associated protein CKsl, have been implicated in the mechanism of VSMC polyploidization during hypertension . Here, we review the function of these proteins at the mitotic spindle cell cycle checkpoint and their role in the process of VSMC polyploidization. J Biol Chem, 2003 Jun 20, 278(25), 22453 - 9 Epub 2003 Apr 14. A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants; Venema K et al.; In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill . cv . Moneymaker), which is a member of the intracellular NHX exchanger protein family . The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment . LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments . The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants . A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro . These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers. Genome Res, 2003 May, 13(5), 896 - 904 Epub 2003 Apr 14. Predicting gene function from patterns of annotation; King OD et al.; The Gene Ontology (GO) Consortium has produced a controlled vocabulary for annotation of gene function that is used in many organism-specific gene annotation databases . This allows the prediction of gene function based on patterns of annotation . For example, if annotations for two attributes tend to occur together in a database, then a gene holding one attribute is likely to hold the other as well . We modeled the relationships among GO attributes with decision trees and Bayesian networks, using the annotations in the Saccharomyces Genome Database (SGD) and in FlyBase as training data . We tested the models using cross-validation, and we manually assessed 100 gene-attribute associations that were predicted by the models but that were not present in the SGD or FlyBase databases . Of the 100 manually assessed associations, 41 were judged to be true, and another 42 were judged to be plausible. Eur J Cardiothorac Surg, 2003 Apr, 23(4), 497 - 502 Improvements in lung compliance after pulmonary transplantation: correlation with interleukin 8 expression; Rao JN et al.; OBJECTIVE: Previous studies have suggested reductions in lung reperfusion injury when initial reperfusion is undertaken with the addition of pharmacological modulators . We investigated three pharmacological agents in a porcine model of left single lung transplantation to determine the effect on lung compliance and its relationship with the expression of the cytokine, interleukin-8 (IL-8) . METHODS: Donor lungs were preserved with modified Euro-Collins for a mean ischaemic time of 18.6 h . Pulmonary venous oxygenation, lung compliance and IL-8 expression were assessed over a 12-h period . Group A (n=5) was a control group with no interventions added, Group B was reperfused with the addition of intravenous inositol hexakisphosphate (InSP6) (0.02 mg/kg per min), Group C received the nitric oxide donor, 3-morpholinosydnonimine (SIN-1) (0.02 mg/kg per min) and Group D received intravenous Pentoxifylline (2 mg/kg per h) . All interventions were administered at a pulmonary artery pressure of 20 mmHg . RESULTS: Group D yielded the best oxygenation (P=0.0041) while Groups B and C were similar . All were superior to Group A (P<0.001) . Lung compliance was significantly improved in Groups B, C and D compared to group A . In Group D, the greatest improvements in lung compliance were observed (P<0.0001) . Similar observations were seen with regard to pulmonary vascular resistance . IL-8 expression was delayed until after 30 min of reperfusion in Group D, but was evident after 10 min in all the other groups . This correlates with the compliance and oxygenation data . CONCLUSIONS: The addition of InSP6 or SIN-1 at reperfusion significantly attenuates reperfusion injury compared with controls and improves lung compliance . The unique comparison with Pentoxifylline afforded by this study indicates that at the doses studied Pentoxifylline appears to be superior, correlating with a greater inhibition of IL-8 expression. Mol Microbiol, 2003 May, 48(3), 823 - 32 Opi1p, Ume6p and Sin3p control expression from the promoter of the INO2 regulatory gene via a novel regulatory cascade; Kaadige MR et al.; The INO2 gene of Saccharomyces cerevisiae is required for expression of most of the phospholipid biosynthetic genes . INO2 expression is regulated by a complex cascade that includes autoregulation, Opi1p-mediated repression and Ume6p-mediated activation . To screen for mutants with altered INO2 expression directly, we constructed an INO2-HIS3 reporter that provides a plate assay for INO2 promoter activity . This reporter was used to isolate mutants (dim1) that fail to repress expression of the INO2 gene in an otherwise wild-type strain . The dim1 mutants contain mutations in the OPI1 gene . To define further the mechanism for Ume6p regulation of INO2 expression, we isolated suppressors (rum1, 2, 3) of the ume6Delta mutation that overexpress the INO2-HIS3 gene . Two of the rum mutant groups contain mutations in the OPI1 and SIN3 genes showing that opi1 and sin3 mutations are epistatic to the ume6Delta mutation . These results are surprising given that Ume6p, Sin3p and Rpd3p are known to form a complex that represses the expression of a diverse set of yeast genes . This prompted us to examine the effect of sin3Delta and rpd3Delta mutants on INO2-cat expression . Surprisingly, the sin3Delta allele overexpressed INO2-cat, whereas the rpd3Delta mutant had no effect . We also show that the UME6 gene does not affect the expression of an OPI1-cat reporter . This suggests that Ume6p does not regulate INO2 expression indirectly by regulating OPI1 expression. Genes Cells, 2003 May, 8(5), 493 - 500 AKRL1 and AKRL2 activate the JNK pathway; Harada T et al.; BACKGROUND: c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, is activated by specific cytokines and various environmental stresses . MKK4 and MKK7 are shown to be direct activators of JNK . Although several upstream components of the JNK pathway, including members of the MAPKKK family have been described, the components lying between the receptors or sensors and JNK have not been fully characterized . RESULTS: We have identified AKRL1 and AKRL2 (Akr1p-like 1 and 2) as novel activators of the JNK pathway . AKRL1 and AKRL2 proteins have a considerable sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae . Expression of AKRL1 or AKRL2 activates JNK and its activators MKK4 and MKK7 . This AKRL1/2-induced JNK activation is significantly suppressed by the expression of a kinase-negative mutant of TAK1, a member of the MAPKKK family . AKRL1 and AKRL2 localize to the Golgi . Both the N-terminal half and the C-terminal transmembrane domain of AKRL1/2 are required for the JNK activation . The C-terminal transmembrane domain of AKRL1/2 is required for localization to the Golgi . CONCLUSION: AKRL1 and AKRL2 are localized to Golgi and the novel activators of the JNK pathway. Genes Cells, 2003 May, 8(5), 413 - 21 Mammalian Mcm2/4/6/7 complex forms a toroidal structure; Yabuta N et al.; BACKGROUND: The Mcm proteins are a family of six homologous proteins (Mcm2-7) that play an important role in DNA replication . They form Mcm4/6/7 and Mcm2/4/6/7 complexes, but their structures are not known . RESULTS: We found that the human Mcm2/4/6/7 tetramer forms a toroidal structure, with a central cavity about 3-4 nm in diameter . Observations were made using electron microscopy, employing the image analysis of single particles . The most predominant averaged image displayed a toroid harbouring four bulges forming corners, one of which was larger than the others . This structure was very similar to the mouse Mcm2/4/6/7 tetramer that was independently prepared and analysed by electron microscopy . These toroidal structures are distinct from that of the Mcm4/6/7 hexamer, which was also examined by electron microscopy . GST(glutathione S-transferase)-pull down and two hybrid experiments suggest that a putative Mcm6-Mcm6 hinge contributes to the formation of the Mcm7/4/6/6/4/7 heterohexamer . CONCLUSIONS: The Mcm2/4/6/7 tetramer forms a toroidal structure that is distinct from that of the Mcm4/6/7 hexamer in size and shape. Biochemistry (Mosc), 2003 Feb, 68(2), 247 - 51 Induced absorption band of holotransketolase and its interpretation; Kovina MV et al.; It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band . Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band . The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps . As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown . The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids . They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme . However none of these hypotheses had been substantiated experimentally . According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1;-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445. Biochemistry, 2003 Apr 22, 42(15), 4311 - 20 New insights into the mechanisms of protein palmitoylation; Linder ME et al.; Since its discovery more than 30 years ago, protein palmitoylation has been shown to have a role in protein-membrane interactions, protein trafficking, and enzyme activity . Until recently, however, the molecular machinery that carries out reversible palmitoylation of proteins has been elusive . In fact, both enzymatic and nonenzymatic S-acylation reaction mechanisms have been proposed . Recent reports of protein palmitoyltransferases in Saccharomyces cerevisiae and Drosophila provide the first glimpse of enzymes that carry out protein palmitoylation . Equally important is the mechanism of depalmitoylation . Two major classes of protein palmitoylthioesterases have been described . One family is lysosomal and is involved in protein degradation . The second is cytosolic and removes palmitoyl moieties preferentially from proteins associated with membranes . This review discusses recent advances in the understanding of mechanisms of addition of palmitate to proteins and removal of palmitate from proteins. Plant Physiol, 2003 Apr, 131(4), 1737 - 47 Identification, expression, and import of components 17 and 23 of the inner mitochondrial membrane translocase from Arabidopsis; Murcha MW et al.; Characterization of components 17 and 23 of the inner mitochondrial membrane translocase (TIM17:23) from Arabidopsis indicated that there were three genes present for TIM17 and TIM23 and two for TIM44 . AtTIM17 differed from the yeast (Saccharomyces cerevisiae) and mammalian homologs in that two genes encoded proteins that were longer and one gene encoded a shorter protein . All Arabidopsis TIM23 predicted proteins appeared to lack the first 34 amino acids compared with yeast TIM23 . All AtTIM17 and AtTIM23 genes were expressed but displayed different tissue and developmental profiles . Complementation of deletion mutants in yeast indicated that for AtTIM17, the extension at the C terminus not present in yeast had to be removed to achieve complementation, whereas for TIM23, a preprotein and amino acid transporter domain had to be present for complementation . Import assays with AtTIM17 and AtTIM23 indicated that they both contained internal signals for integration into the inner mitochondrial membrane in a membrane potential-dependent manner . The C terminus of imported AtTIM17-2 was susceptible to degradation by externally added protease with intact mitochondria . Removal of the 85 C-terminal amino acids resulted in import and full protection of the truncated protein . This suggests that the novel extension at the C terminus of AtTIM17-2 links the outer and inner membrane in a manner analogous to yeast TIM23. Plant Physiol, 2003 Apr, 131(4), 1648 - 60 New insight into Phaeodactylum tricornutum fatty acid metabolism . Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases; Domergue F et al.; In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)) . In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants . These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively . Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6) . The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA . In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed . Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P . tricornutum was highly specific for 16:1(Delta9) . Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P . tricornutum (16:3(Delta6,9,12)) is of plastidial origin. Plant Physiol, 2003 Apr, 131(4), 1566 - 75 Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues; Gao Z et al.; The acyclic polyol sorbitol is a primary photosynthetic product and the principal photosynthetic transport substance in many economically important members of the family Rosaceace (e.g . almond {Prunus dulcis (P . Mill.) D.A . Webber}, apple {Malus pumila P . Mill.}, cherry {Prunus spp.}, peach {Prunus persica L . Batsch}, and pear {Pyrus communis}) . To understand key steps in long-distance transport and particularly partitioning and accumulation of sorbitol in sink tissues, we have cloned two sorbitol transporter genes (PcSOT1 and PcSOT2) from sour cherry (Prunus cerasus) fruit tissues that accumulate large quantities of sorbitol . Sorbitol uptake activities and other characteristics were measured by heterologous expression of PcSOT1 and PcSOT2 in yeast (Saccharomyces cerevisiae) . Both genes encode proton-dependent, sorbitol-specific transporters with similar affinities (K(m) sorbitol of 0.81 mM for PcSOT1 and 0.64 mM for PcSOT2) . Analyses of gene expression of these transporters, however, suggest different roles during leaf and fruit development . PcSOT1 is expressed throughout fruit development, but especially when growth and sorbitol accumulation rates are highest . In leaves, PcSOT1 expression is highest in young, expanding tissues, but substantially less in mature leaves . In contrast, PcSOT2 is mainly expressed only early in fruit development and not in leaves . Compositional analyses suggest that transport mediated by PcSOT1 and PcSOT2 plays a major role in sorbitol and dry matter accumulation in sour cherry fruits . Presence of these transporters and the high fruit sorbitol concentrations suggest that there is an apoplastic step during phloem unloading and accumulation in these sink tissues . Expression of PcSOT1 in young leaves before completion of the transition from sink to source is further evidence for a role in determining sink activity. J Biol Chem, 2003 Jun 27, 278(26), 24125 - 31 Epub 2003 Apr 13. Importance of the conserved aromatic residues in the scorpion alpha-like toxin BmK M1: the hydrophobic surface region revisited; Sun YM et al.; About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare . In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1 . Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47 . The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis . Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1 . The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded beta-sheet seem to be essential for the structure and function of the toxin . Trp38 and Tyr42 (located in the beta2-sheet and in the loop between the beta2- and beta3-sheets, respectively) are most likely involved in the pharmacological function of the toxin. Receptors Channels, 2002, 8(5-6), 297 - 308 Functional assays for identifying ligands at orphan G protein-coupled receptors; Szekeres PG; The superfamily of G protein-coupled receptors (GPCRs; 7TMs) is one of the largest families of genes identified in humans, and has a proven history of being an excellent source of drug targets . The near completion of the human genome sequencing project has allowed the identification of a plethora of sequences encoding "orphan" GPCRs--putative receptors whose natural ligand(s) remain to be discovered . In many cases, the level of sequence homology with known receptors is insufficient to be able to predict the natural ligand for these orphan receptors, although it is usually possible to determine the likely nature of the cognate ligand e.g . peptide, lipid, nucleotide etc . Deorphanizing these novel GPCRs and evaluating their biological function has become a major target of many of the major pharmaceutical companies as well as several academic groups . Since 1995 more than 50 ligands for orphan GPCRs have been discovered by using the orphan receptor as a biosensor and screening candidate compounds looking for a biological response (the so-called "reverse pharmacology" approach) . Identification of the natural ligands for these receptors marks the beginning of the process of understanding the biology of these newly discovered signalling systems and the development of novel therapies targeted at them . This article will focus on the functional assays which have been used to discover ligands for orphan GPCRs. BMC Biochem . 2003 Mar 18;4(1):3. Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system; Schulze WX et al.; BACKGROUND: The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4 . The carriers differ in their kinetic properties . Many transport proteins are known to exist as oligomers . The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact . RESULTS: Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants . All three fusion genes are co-expressed in companion cells . Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system . Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers . The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters . Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters . CONCLUSIONS: The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits . Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers . The biochemical approaches are required to confirm the in planta interaction. BMC Bioinformatics . 2003 Mar 27;4(1):11. PreBIND and Textomy--mining the biomedical literature for protein-protein interactions using a support vector machine; Donaldson I et al.; BACKGROUND: The majority of experimentally verified molecular interaction and biological pathway data are present in the unstructured text of biomedical journal articles where they are inaccessible to computational methods . The Biomolecular interaction network database (BIND) seeks to capture these data in a machine-readable format . We hypothesized that the formidable task-size of backfilling the database could be reduced by using Support Vector Machine technology to first locate interaction information in the literature . We present an information extraction system that was designed to locate protein-protein interaction data in the literature and present these data to curators and the public for review and entry into BIND . RESULTS: Cross-validation estimated the support vector machine's test-set precision, accuracy and recall for classifying abstracts describing interaction information was 92%, 90% and 92% respectively . We estimated that the system would be able to recall up to 60% of all non-high throughput interactions present in another yeast-protein interaction database . Finally, this system was applied to a real-world curation problem and its use was found to reduce the task duration by 70% thus saving 176 days . CONCLUSIONS: Machine learning methods are useful as tools to direct interaction and pathway database back-filling; however, this potential can only be realized if these techniques are coupled with human review and entry into a factual database such as BIND . The PreBIND system described here is available to the public at Current capabilities allow searching for human, mouse and yeast protein-interaction information. Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Mar;67(3 Pt 1):031902 . Epub 2003 Mar 11. Iterative signature algorithm for the analysis of large-scale gene expression data; Bergmann S et al.; We present an approach for the analysis of genome-wide expression data . Our method is designed to overcome the limitations of traditional techniques, when applied to large-scale data . Rather than alloting each gene to a single cluster, we assign both genes and conditions to context-dependent and potentially overlapping transcription modules . We provide a rigorous definition of a transcription module as the object to be retrieved from the expression data . An efficient algorithm, which searches for the modules encoded in the data by iteratively refining sets of genes and conditions until they match this definition, is established . Each iteration involves a linear map, induced by the normalized expression matrix, followed by the application of a threshold function . We argue that our method is in fact a generalization of singular value decomposition, which corresponds to the special case where no threshold is applied . We show analytically that for noisy expression data our approach leads to better classification due to the implementation of the threshold . This result is confirmed by numerical analyses based on in silico expression data . We discuss briefly results obtained by applying our algorithm to expression data from the yeast Saccharomyces cerevisiae. J Immunol, 2003 Apr 15, 170(8), 4139 - 47 Communication between NF-kappa B and Sp1 controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein 1 gene; Boekhoudt GH et al.; The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence . To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions . An E1a-sensitive coactivator was found to be required for expression . In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1 . Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65 . Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation . These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF. FEBS Lett, 2003 Apr 10, 540(1-3), 86 - 90 The apoptosis-associated protein BNIPL interacts with two cell proliferation-related proteins, MIF and GFER; Shen L et al.; Bcl-2/adenovirus E1B 19 kDa interacting protein 2-like, BNIP-2-like (BNIPL) is a recently cloned and characterized apoptosis-associated protein that shares 72% homology with BNIP-2 . It is highly expressed in human placenta and lung . A yeast two-hybrid system was used to obtain two BNIPL-interacting proteins, MIF (macrophage migration inhibitory factor) and GFER (growth factor erv1 (Saccharomyces cerevisiae)-like) . The interactions were confirmed by glutathione S-transferase pull-down assay in vitro and co-immunoprecipitation assay in vivo . Colony formation assay and cell proliferation test suggest that overexpression of BNIPL could inhibit the growth of BEL-7402 cells . These findings suggest that BNIPL may physically bind to cell proliferation-related proteins, MIF and GFER . Mol Biol Cell, 2003 Apr, 14(4), 1610 - 23 Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p; Conibear E et al.; Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly . However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown . We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi . This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst . Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p . All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN . vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role . Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p . The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery. Mol Biol Cell, 2003 Apr, 14(4), 1583 - 96 Composition and dynamics of human mitochondrial nucleoids; Garrido N et al.; The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae . Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA . However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm . In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids . The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine . Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM . The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network . In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance . Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery. Mol Biol Cell, 2003 Apr, 14(4), 1501 - 16 Synthetic lethal analysis implicates Ste20p, a p21-activated potein kinase, in polarisome activation; Goehring AS et al.; The p21-activated kinases Ste20p and Cla4p carry out undefined functions that are essential for viability during budding in Saccharomyces cerevisiae . To gain insight into the roles of Ste20p, we have used a synthetic lethal mutant screen to identify additional genes that are required in the absence of Cla4p . Altogether, we identified 65 genes, including genes with roles in cell polarity, mitosis, and cell wall maintenance . Herein, we focus on a set that defines a function carried out by Bni1p and several of its interacting proteins . We found that Bni1p and a group of proteins that complex with Bni1p (Bud6p, Spa2p, and Pea2p) are essential in a cla4delta mutant background . Bni1p, Bud6p, Spa2, and Pea2p are members of a group of polarity determining proteins referred to as the polarisome . Loss of polarisome proteins from a cla4delta strain causes cells to form elongated buds that have mislocalized septin rings . In contrast, other proteins that interact with or functionally associate with Bni1p and have roles in nuclear migration and cytokinesis, including Num1p and Hof1p, are not essential in the absence of Cla4p . Finally, we have found that Bni1p is phosphorylated in vivo, and a substantial portion of this phosphorylation is dependent on STE20 . Together, these results suggest that one function of Ste20p may be to activate the polarisome complex by phosphorylation of Bni1p. Eur J Clin Pharmacol, 2003 Jun, 59(2), 103 - 9 Epub 2003 Apr 09. Bioactivation of cyclophosphamide: the role of polymorphic CYP2C enzymes; Griskevicius L et al.; Several-fold differences have been observed among patients in the biotransformation of cyclophosphamide . The aim of this study was to investigate the contribution of CYP2C9 and CYP2C19 and their polymorphisms to the variability of cyclophosphamide activation . The formation of 4-hydroxycyclophosphamide was studied in microsomes from a total of 32 different genotyped human livers, as well as in yeast microsomes expressing different genetic variants of CYP2C9 and CYP2C19 . The kinetic data obtained in the yeast system revealed that the intrinsic clearance (V(max)/K(m)) of cyclophosphamide by CYP2C9.2 and CYP2C9.3 samples was approximately threefold lower than that by CYP2C9.1 . However, in liver microsomes, there were no statistically significant differences in the intrinsic clearance of 4-hydroxycyclophosphamide formation between the group of seven CYP2C9*1/*1 livers and the remaining nine with one or two variant CYP2C9 alleles ( P>0.7) . We found a statistically significant correlation ( r(s)=0.65, P=0.003) between 4-hydroxylation of cyclophosphamide and 5'-hydroxylation of R-omeprazole, a measure of CYP2C19 activity in human liver microsomes ( n=19) . No correlation was found between 4-hydroxylation of cyclophosphamide and the formation rate of hydroxycelecoxib, mainly catalysed by CYP2C9 ( r(s)=0.17, P=0.55, n=32) . In conclusion, based on the correlation with the formation of R-5'-hydroxyomeprazole, CYP2C19 may partly contribute to the bioactivation of cyclophosphamide in human liver microsomes, while the role of CYP2C9 appears minor. J Biol Chem, 2003 Jun 20, 278(25), 22498 - 505 Epub 2003 Apr 08. A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis; Thiel C et al.; Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii . The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities . An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient . Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol . Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog . Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2 . Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells . hALG2 was shown to act as an alpha1,3-mannosyltransferase . The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase. Eukaryot Cell, 2003 Apr, 2(2), 265 - 73 Isolation of mutations in the catalytic domain of the snf1 kinase that render its activity independent of the snf4 subunit; Leech A et al.; Activation of the Snf1 kinase requires at least two events, phosphorylation of the activation loop on threonine 210 and an Snf4-dependent process that is not completely defined . Snf4 directly interacts with a region of the regulatory domain of Snf1 that may otherwise act as an autoinhibitory domain . In order to gain insight into the regulation of Snf1 kinase by Snf4, deletions in the regulatory domain of the catalytic subunit were engineered and tested for their effect on Snf1 function in the absence of Snf4 . Deletion of residues 381 to 488 from the Snf1 protein resulted in a kinase that was activated by glucose limitation even in the absence of the Snf4 protein . A larger deletion (amino acids 381 to 608) encompassing virtually the entire regulatory domain resulted in complete inactivation of the Snf1 kinase even in the presence of Snf4 . A genetic screen for amino acid substitutions that conferred an Snf4-independent phenotype identified four point mutations in the Snf1 catalytic domain . One very conservative mutation, leucine 183 to isoleucine, conferred nearly wild-type levels of Snf1 kinase function in the absence of the Snf4 protein . Purified Snf1 kinase was inactive when isolated from snf4Delta cells, whereas the Snf1-L183I kinase exhibited significant activity in the absence of Snf4 . Our data support the idea that Snf1 kinase activity is constrained in cis by an autoinhibitory domain and that the Snf4-mediated activation of Snf1 can be bypassed by subtle conformational changes in the catalytic domain of the Snf1 kinase. J Mol Biol, 2003 Apr 18, 328(1), 157 - 66 A novel heme and peroxide-dependent tryptophan-tyrosine cross-link in a mutant of cytochrome c peroxidase; Bhaskar B et al.; The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51 . We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52 . This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link . Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144 . Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron . Plant Mol Biol, 2003 Mar, 51(5), 731 - 43 Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus; Iturbe-Ormaetxe I et al.; Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens . Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus . These include a beta-amyrin synthase from M . truncatula (MtAMY1) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2) . A partial cDNA predicted to encode a beta-amyrin synthase (LjAMY1) was also isolated from L . japonicus . The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M . truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development. Nat Cell Biol, 2003 May, 5(5), 486 - 8 Reconstitution of gamma-secretase activity; Edbauer D et al.; gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD) . Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity . Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro . Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast. Theor Appl Genet, 2003 Jun, 107(1), 155 - 61 Epub 2003 Apr 01. Characterization of a DRE-binding transcription factor from a halophyte Atriplex hortensis; Shen YG et al.; Environmental stresses, such as salinity, drought and cold, can induce the expression of a large amount of genes . Among these are many transcription factors that regulate the expression of downstream genes by specifically binding to cis-elements or forming transcriptional complexes with other proteins . In the present study, a DREB-like transcription factor gene, named AhDREB1, was isolated from a halophyte Atriplex hortensis . AhDREB1 encoded a protein containing a conserved EREBP/AP2 domain featuring the DREB family . In yeast one-hybrid analysis AhDREB1 protein was specifically bound to DRE elements and activated the expression of the reporter genes of HIS3 and LacZ . The AhDREB1 gene was expressed in roots, stems and leaves of A . hortensis . Salinity induced its expression in roots, but not in other organs . Overexpression of AhDREB1 in transgenic tobacco led to the accumulation of its putative downstream genes . The performance of the transgenic lines was also tested under stressed conditions and two lines were found to be stress-tolerant . These results suggest that the AhDREB1 protein functions as a DRE-binding transcription factor and play roles in the stress-tolerant response of A . hortensis. Science, 2003 Apr 4, 300(5616), 139 - 41 Regulated cycling of mitochondrial Hsp70 at the protein import channel; Liu Q et al.; Hsp70 of the mitochondrial matrix (mtHsp70) provides a critical driving force for the import of proteins into mitochondria . Tim44, a peripheral inner-membrane protein, tethers it to the import channel . Here, regulated interactions were found to maximize occupancy of the active, adenosine 5'-triphosphate (ATP)-bound mtHsp70 at the channel through its intrinsic high affinity for Tim44, as well as through release of adenosine diphosphate (ADP)-bound mtHsp70 from Tim44 by the cofactor Mge1 . A model peptide substrate rapidly released mtHsp70 from Tim44, even in the absence of ATP hydrolysis . In vivo, the analogous interaction of translocating polypeptide would release mtHsp70 from the channel . Consistent with the ratchet model of translocation, subsequent hydrolysis of ATP would trap the polypeptide, driving import by preventing its movement back toward the cytosol. Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4372 - 6 Epub 2003 Apr 03. Assessing experimentally derived interactions in a small world; Goldberg DS et al.; Experimentally determined networks are susceptible to errors, yet important inferences can still be drawn from them . Many real networks have also been shown to have the small-world network properties of cohesive neighborhoods and short average distances between vertices . Although much analysis has been done on small-world networks, small-world properties have not previously been used to improve our understanding of individual edges in experimentally derived graphs . Here we focus on a small-world network derived from high-throughput (and error-prone) protein-protein interaction experiments . We exploit the neighborhood cohesiveness property of small-world networks to assess confidence for individual protein-protein interactions . By ascertaining how well each protein-protein interaction (edge) fits the pattern of a small-world network, we stratify even those edges with identical experimental evidence . This result promises to improve the quality of inference from protein-protein interaction networks in particular and small-world networks in general. J Biol Chem, 2003 Jun 6, 278(23), 20457 - 60 Epub 2003 Apr 02. Rapamycin-induced translational derepression of GCN4 mRNA involves a novel mechanism for activation of the eIF2 alpha kinase GCN2; Kubota H et al.; When starved for amino acids, Saccharomyces cerevisiae accumulates uncharged tRNAs to activate its sole eukaryotic initiation factor (eIF) 2alpha kinase GCN2 . Subsequent phosphorylation of eIF2alpha impedes general translation, but translationally derepresses the transcription factor GCN4, which induces expression of various biosynthetic genes to elicit general amino acid control response . By contrast, when supplied with enough nutrients, the yeast activates the target of rapamycin signaling pathway to stimulate translation initiation by facilitating the assembly of eIF4F . A cross-talk was suggested between the two pathways by rapamycin-induced translation of GCN4 mRNA . Here we show that rapamycin causes an increase in phosphorylated eIF2alpha to translationally derepress GCN4 . This increment is not observed in the cells expressing mammalian non-GCN2 eIF2alpha kinases in place of GCN2 . It is thus suggested that rapamycin does not inhibit dephosphorylation of eIF2alpha but rather activates the kinase GCN2 . This activation seems to require an interaction between the kinase and uncharged tRNAs, because rapamycin, similar to amino acid starvation, fails to induce eIF2alpha phosphorylation in the cells with GCN2 defective in tRNA binding . However, in contrast with amino acid starvation, rapamycin activates GCN2 without increasing the amount of uncharged tRNAs, but presumably by modifying the tRNA binding affinity of GCN2. Pharmacol Res, 2003 May, 47(5), 401 - 7 Signaling and biological actions of sphingosine 1-phosphate; Hla T; Sphingosine 1-phosphate (S1P) is a product of sphingomyelin (SM) metabolism . It is present in most eukaryotic organisms; however, in higher vertebrates (bony fishes to mammals), it is utilized as a regulator of cell function by binding to extracellular receptors . Its role as a second messenger function was originally proposed; however, the unequivocal role of S1P as a second messenger in higher eukaryotes is lacking at present . As an extracellular mediator, S1P binds to a family of G-protein-coupled receptors named S1P(1-5), originally referred to as (EDG-1, 3, 5, 6 and 8) . These receptors couple to multiple G-proteins and regulate intracellular signaling pathways . Most tissues express one or more S1P receptor subtypes; therefore, S1P is known as a multifunctional physiologic mediator . Its precise physiologic roles in multiple organ systems are just beginning to be elucidated; however, at present, regulation of cardiogenesis, vascular system formation, oocyte survival and immune cell trafficking has been recently reported . Currently, a S1P receptor agonist, is undergoing clinical trials as an agent to fight organ transplant rejection . Since S1P is a fundamental multifunctional mediator, better understanding of the biology of S1P holds great promise to develop novel tools to control various diseases. Biol Chem, 2003 Jan, 384(1), 105 - 16 Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster; Grebe M et al.; Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP) . The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding . Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648) . Some amino acids are suboptimal for ligand binding (L615, T619), but mediate ligand-dependent dimerization . We conclude that the enhanced regulatory potential by ligand-dependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding . Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations . Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligand-binding pocket is modified by heterodimerization. Biol Chem, 2003 Jan, 384(1), 39 - 49 Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit; Stohwasser R et al.; PA28 is a modulator of the 20S proteasome . The PA28 binding sites on the 20S proteasome are still not well defined . Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome alpha4 subunit is one of the PA28 binding sites . This finding is supported by the observation that a hepatitis B virus X protein-derived polypeptide habouring the alpha4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28. Cancer Biol Ther, 2003 Jan-Feb, 2(1), 92 - 9 A potential therapeutic strategy to combat leukemia virus infection; Pan J et al.; To test the concept that a replication-competent retrovirus carrying a suicide gene could have potential utility in the control of the natural virus infection in mammalian species, we constructed derivatives of a feline leukemia virus (FeLV) that is commonly associated with leukemia-lymphomas in this species . The FeLV, Rickard strain, subgroup A (FRA) genome contained at the 3' end of the envgene, an insert of an internal ribosomal entry site (IRES) linked to cDNA sequence of either herpes simplex virus thymidine kinase (HSV-TK) or a truncated HSV-TK (HSV-ATK) or yeast cytosine deaminase (CD) . These constructs were transfected into feline fibroblast cells (H927) . The viruses produced were determined to be replication-competent . The stable propagation of the full-length transgene was, however, dependent on the size of the insert, IRES-CD being the smallest in size (1031 bp) exhibiting maximal stability for at least up to six months . The protein products of the transgenes could be detected, despite the appearance of deleted proviruses at late passages . The transduced cells were susceptible to cytotoxic killing when the appropriate prodrug, ganciclovir (GCV), acyclovir (ACV) or 5-fluorocytosine (5-FC) was added to the culture medium . H927 cells, infected with another subgroup of FeLV, namely, FeLV-B or FeLV-C, could be superinfected by the FRA-suicide gene viruses and thus, subjected to killing . Interestingly, at an early stage of infection by the parental FRA, H927 cells could also be reinfected by the same subgroup FRA constructs to induce the suicide effect . Among the three constructs, the vector with the CD gene was determined to be superior to others in terms of stability, therapeutic index and bystander effect in the cell culture test system . While the in vivo correlates of the therapeutic effect in the feline model remain to be determined, our results do encourage investigation of the same concept in the control of HTLV and, perhaps even, HIV infection in humans. Cancer Biol Ther, 2003 Jan-Feb, 2(1), 22 - 9 Chromatin remodeling and cancer; Davis PK et al.; The fate of the cell relies on a delicate balance between gene expression and repression . The transcriptional control of the genome is maintained not only by transcription factors but also chromatin remodeling proteins . The purpose of the chromatin remodeling proteins is to alter the nucleosome architecture such that genes are exposed to or hidden from the transcriptional machinery . The nucleosome can be restructured by two mechanisms: 1 . the movement of nucleosomes along DMA which is carried out by ATP-dependent chromatin remodeling complexes; and 2 . the modification of core histones by histone acetyltransferases, deactylases, methyltrans-ferases, and kinases . Since these chromatin remodeling proteins play an essential role in transcriptional regulation, it is not surprising that they have been linked to cancer . In this review, we provide a general overview on chromatin remodeling and describe known genetic alterations of chromatin remodeling proteins in human cancers . We also discuss potential other, as yet unexplored strategies that cancers might take to manipulate the chromatin remodeling machinery. Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4527 - 32 Epub 2003 Apr 02. Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition; Scheibel T et al.; Recent research in the field of nanometer-scale electronics has focused on the operating principles of small-scale devices and schemes to realize useful circuits . In contrast to established "top-down" fabrication techniques, molecular self-assembly is emerging as a "bottom-up" approach for fabricating nanostructured materials . Biological macromolecules, especially proteins, provide many valuable properties, but poor physical stability and poor electrical characteristics have prevented their direct use in electrical circuits . Here we describe the use of self-assembling amyloid protein fibers to construct nanowire elements . Self-assembly of a prion determinant from Saccharomyces cerevisiae, the N-terminal and middle region (NM) of Sup35p, produced 10-nm-wide protein fibers that were stable under a wide variety of harsh physical conditions . Their lengths could be roughly controlled by assembly conditions in the range of 60 nm to several hundred micrometers . A genetically modified NM variant that presents reactive, surface-accessible cysteine residues was used to covalently link NM fibers to colloidal gold particles . These fibers were placed across gold electrodes, and additional metal was deposited by highly specific chemical enhancement of the colloidal gold by reductive deposition of metallic silver and gold from salts . The resulting silver and gold wires were approximately 100 nm wide . These biotemplated metal wires demonstrated the conductive properties of a solid metal wire, such as low resistance and ohmic behavior . With such materials it should be possible to harness the extraordinary diversity and specificity of protein functions to nanoscale electrical circuitry. Physiol Genomics, 2003 Jun 24, 14(1), 35 - 46 Clustering gene expression data using adaptive double self-organizing map; Ressom H et al.; This paper presents a novel clustering technique known as adaptive double self-organizing map (ADSOM) . ADSOM has a flexible topology and performs clustering and cluster visualization simultaneously, thereby requiring no a priori knowledge about the number of clusters . ADSOM is developed based on a recently introduced technique known as double self-organizing map (DSOM) . DSOM combines features of the popular self-organizing map (SOM) with two-dimensional position vectors, which serve as a visualization tool to decide how many clusters are needed . Although DSOM addresses the problem of identifying unknown number of clusters, its free parameters are difficult to control to guarantee correct results and convergence . ADSOM updates its free parameters during training, and it allows convergence of its position vectors to a fairly consistent number of clusters provided that its initial number of nodes is greater than the expected number of clusters . The number of clusters can be identified by visually counting the clusters formed by the position vectors after training . A novel index is introduced based on hierarchical clustering of the final locations of position vectors . The index allows automated detection of the number of clusters, thereby reducing human error that could be incurred from counting clusters visually . The reliance of ADSOM in identifying the number of clusters is proven by applying it to publicly available gene expression data from multiple biological systems such as yeast, human, and mouse . ADSOM's performance in detecting number of clusters is compared with a model-based clustering method. J Biol Chem, 2003 Jun 13, 278(24), 22090 - 101 Epub 2003 Apr 01. Cu(I)-dependent biogenesis of the galactose oxidase redox cofactor; Whittaker MM et al.; Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272 . Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen . We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II) . Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7) . In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h) . EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction . The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction . The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading . The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2 . Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation . These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism. J Biol Chem, 2003 Jun 13, 278(24), 21361 - 9 Epub 2003 Apr 02. Characterization of the auto-inhibitory sequence within the N-terminal domain of importin alpha; Harreman MT et al.; Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta . An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha . In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p . We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo . In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function . We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p . We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism. Curr Opin Genet Dev, 2003 Apr, 13(2), 207 - 14 Looking at mRNA decay pathways through the window of molecular evolution; Culbertson MR et al.; In eukaryotes, mRNAs are monitored for errors in gene expression by RNA surveillance where untranslatable mRNAs are selectively degraded by the nonsense-mediated mRNA decay (NMD) pathway . Depending on the organism, three to seven genes are required for NMD . Besides RNA surveillance, the genes required for NMD serve a second purpose by controlling the overall abundance of a substantial fraction of the transcriptome. Curr Opin Genet Dev, 2003 Apr, 13(2), 191 - 8 Stretching it: putting the CEN(P-A) in centromere; Mellone BG et al.; The centromere is the locus responsible for the segregation of chromosomes during mitosis and meiosis . The number of newly characterised centromere-associated proteins continues to increase . The kinetochore complex assembles at this site and in many organisms is visible as the primary constriction . In several systems the location of the site of kinetochore assembly is known to vary and the site is not specified by a strict cis-acting primary sequence . It is proposed that tension between bioriented sister centromeres may act to imprint the site. Curr Opin Genet Dev, 2003 Apr, 13(2), 143 - 53 Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression; Yang XJ et al.; The flexible N-terminal tails of core histones are subject to dynamic, reversible lysine acetylation . At least 10 histone deacetylases have been identified in Saccharomyces cerevisiae and 19 in humans . Emerging themes regarding the function and regulation of these enzymes include the following: targeted and non-targeted chromatin deacetylation; their collaboration with each other and with other chromatin regulators to promote transcriptional repression and silencing; deacetylation of transcription factors and other non-histone proteins; and regulation by subcellular compartmentalization and subunit association . Histone deacetylases are important targets for drugs with potential therapeutic value in the treatment of cancer, neurodegenerative disorders, cardiac hypertrophy and other human diseases. Int J Biochem Cell Biol, 2003 May, 35(5), 553 - 61 Role of the Apg12 conjugation system in mammalian autophagy; Mizushima N et al.; The Apg12 system is one of the ubiquitin-like protein conjugation systems conserved in eukaryotes . It was first discovered in yeast during systematic analyses of the apg mutants defective in autophagy, which is the intracellular bulk degradation system . Covalent attachment of Apg12-Apg5 is essential for autophagy . Enzymes catalyzing this conjugation reaction were also identified based on the apg mutant analyses . These are Apg7 and Apg10, corresponding to E1 and E2 enzymes, respectively . Studies using mammalian cells further revealed the function of the Apg12 system . The Apg12-Apg5 conjugate localizes to elongating autophagic isolation membranes . Apg12 conjugation of Apg5 is required for elongation of the isolation membrane to form a complete spherical autophagosome . Discovery of the Apg12 system has facilitated our understanding of the molecular mechanism of autophagosome formation. Phytother Res, 2003 Mar, 17(3), 259 - 64 Antinociceptive and antipyretic activities of Pongamia pinnata leaves; Srinivasan K et al.; In the present study, the antinociceptive activity of a 70% ethanol extract of Pongamia pinnata leaves (PLE) was investigated in different models of pain in mice and rats . Further, PLE was also evaluated for its antipyretic activity in Brewer's yeast-induced pyrexia in rats . Per os (p.o.) administration of the PLE (100-1000 mg/kg) produced significant antinociceptive activity in the hotplate and tail flick (central) as well as in acetic acid writhing and Randall-Selitto (peripheral) nociceptive tests suggesting the involvement of both central and peripheral mechanisms in alleviating the pain response . In addition, PLE also exhibited a significant antipyretic response in Brewer's yeast-induced pyrexia in rats . These results demonstrated that PLE possesses marked antinociceptive as well as antipyretic activities and thus scientifically validated its use in the treatment of pain and pyretic disorders . EMBO Rep, 2003 Apr, 4(4), 419 - 24 Self-assembly of minimal COPII cages; Antonny B et al.; The small G-protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles . Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self-assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40-60 nm . Self-assembly is favoured by lowering the pH, the ionic strength and/or the temperature . Electron microscopy reveals the formation of spherical particles 60-120 nm in diameter with a tight, rough mesh on their surfaces . We suggest that these structures, which represent a minimal COPII cage, mimic the molecular organization of the membrane-associated COPII coat. EMBO Rep, 2003 Apr, 4(4), 405 - 11 Glycopeptide specificity of the secretory protein folding sensor UDP-glucose glycoprotein:glucosyltransferase; Taylor SC et al.; Secretory and membrane N-linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle . UDP-glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP-glucose to N-linked Man9-GlcNAc2 glycans . To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9-GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry . A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT . The preference for amino-acid residues close to N-linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT. EMBO Rep, 2003 Apr, 4(4), 387 - 93 Monitoring global messenger RNA changes in externally controlled microarray experiments; van de Peppel J et al.; Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression . To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced . When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts . This does not reflect absolute changes if global shifts in messenger RNA populations occur . Using external RNA controls, we have set up microarray experiments to monitor global changes . The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included . Even small global changes had a significant effect on the number of genes reported as being differentially expressed . This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression. J Biol Chem, 2003 Jun 27, 278(26), 23714 - 9 Epub 2003 Apr 01. Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits; Hirata T et al.; Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells . In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase . Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit . V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase) . The rotation was inhibited by concanamycin and nitrate but not by azide . These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis. Genes Dev, 2003 Apr 1, 17(7), 896 - 911 Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1; Wysocka J et al.; The abundant and chromatin-associated protein HCF-1 is a critical player in mammalian cell proliferation as well as herpes simplex virus (HSV) transcription . We show here that separate regions of HCF-1 critical for its role in cell proliferation associate with the Sin3 histone deacetylase (HDAC) and a previously uncharacterized human trithorax-related Set1/Ash2 histone methyltransferase (HMT) . The Set1/Ash2 HMT methylates histone H3 at Lys 4 (K4), but not if the neighboring K9 residue is already methylated . HCF-1 tethers the Sin3 and Set1/Ash2 transcriptional regulatory complexes together even though they are generally associated with opposite transcriptional outcomes: repression and activation of transcription, respectively . Nevertheless, this tethering is context-dependent because the transcriptional activator VP16 selectively binds HCF-1 associated with the Set1/Ash2 HMT complex in the absence of the Sin3 HDAC complex . These results suggest that HCF-1 can broadly regulate transcription, both positively and negatively, through selective modulation of chromatin structure. FEMS Microbiol Lett, 2003 Mar 28, 220(2), 241 - 6 Activation of chsC transcription by AbaA during asexual development of Aspergillus nidulans; Park BC et al.; The temporal and spatial regulation of chitin synthesis plays an important role in morphogenesis during fungal growth and development . Northern blot analysis showed that the transcription level of chsA, chsC, and chsD was significantly decreased in an Aspergillus nidulans abaA mutant . Electrophoretic mobility shift assays revealed that AbaA bound tightly to all three AREs (AbaA response elements) in the chsC promoter region . Experiments with the Saccharomyces cerevisiae heterologous expression system confirmed AbaA-dependent transcriptional activation of chsC . Taken together, these data suggest that AbaA plays an important role in chitin biosynthesis during conidiophore development by controlling the transcription level of certain chitin synthase genes. Biochem Biophys Res Commun, 2003 Apr 11, 303(3), 978 - 84 High expression of Cks1 in human non-small cell lung carcinomas; Inui N et al.; Enhanced degradation of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is known to be a powerful prognostic marker in many types of human cancers . Human CDK subunit 1 (Cks1) and S-phase kinase associated protein 2 (Skp2) are components of the SCF(Skp2) complex, which acts as a ubiquitin ligase for p27(Kip1) . There are no reports about the involvement of Cks1 in the pathogenesis of human cancer . Here we show high expression of Cks1 in non-small cell lung cancers (NSCLCs) using Western blotting and quantitative real-time RT-PCR . The Skp2 mRNA expression level was high in squamous cell carcinomas and was inversely related with the p27(Kip1) protein level in individual clinical samples . In contrast, Cks1 mRNA expression had no such relationship with p27(Kip1), although Cks1 mRNA was significantly elevated in adenocarcinomas . These results suggest that high expression of Skp2 and Cks1 may be involved in the pathogenesis of NSCLCs via different mechanisms. Plant Cell Physiol, 2003 Mar, 44(3), 223 - 32 The sucrose transporter gene family in rice; Aoki N et al.; In this paper we report the identification, cloning and expression analysis of four putative sucrose transporter (SUT) genes from rice, designated OsSUT2, 3, 4 and 5 . Three of the four genes were identified through extensive searches of the recently published draft sequence of the rice genome . Along with the previously reported OsSUT1 we propose that these five genes comprise the rice SUT gene family . Complementary DNA clones were isolated for the four newly identified genes . The deduced proteins of all five SUT genes were predicted to contain 12 membrane-spanning helices and a domain highly conserved throughout all known plant SUTs, suggesting the four additional OsSUT genes encode functional SUTs . Reverse transcription-PCR analysis was performed in order to investigate the expression pattern of each member of the SUT family in rice . A differing but overlapping expression pattern was observed for each member of the SUT family at different stages through plant development . These results, together with the structural variations apparent from the deduced protein sequences, suggest that the five SUTs possess diverse roles in both sink and source tissues . We also discuss the classification and evolution of the rice SUT gene family, using a comparison of the gene structures and deduced amino acid sequences with other known plant SUT genes. J Biol Chem, 2003 Jun 20, 278(25), 22284 - 9 Epub 2003 Mar 31. Regulation of Ste7 ubiquitination by Ste11 phosphorylation and the Skp1-Cullin-F-box complex; Wang Y et al.; Ste7 is a mitogen-activated protein kinase kinase that mediates pheromone signaling in Saccharomyces cerevisiae . We showed previously that Ste7 is ubiquitinated upon prolonged stimulation by pheromone and that accumulation of ubiquitinated Ste7 results in enhanced transcription and cell division arrest responses (Wang, Y., and Dohlman, H . G . (2002) J . Biol . Chem . 277, 15766-15772) . We now report that ubiquitination of Ste7 requires Ste11 kinase and Skp1/Cullin/F-box (SCF) ubiquitin-conjugating activities . Ste7 is not ubiquitinated in Ste11-deficient cells or when the Ste11 phosphorylation sites have been mutated . Ste7 ubiquitination and degradation (but not phosphorylation) is specifically blocked in mutants defective for the E2 ubiquitin-conjugating enzyme Cdc34 or the cullin homologue Cdc53 . Both are components of the SCF complex that ubiquitinates proteins during the G1-S transition of the cell cycle . Our findings suggest that SCF promotes the ubiquitination and degradation of Ste7, thereby favoring the resumption of cell division cycling after pheromone-induced growth arrest. Arch Biochem Biophys, 2003 Apr 15, 412(2), 267 - 71 Hydrogen isotope tracing in the reaction of orotidine-5'-monophosphate decarboxylase; Smiley JA et al.; The mechanism of the enzyme orotidine-5(')-monophosphate decarboxylase (OMP decarboxylase, ODCase) is not fully characterized; some of the proposed mechanisms suggest the possibility of hydrogen rearrangement (shift from C5 to C6 or loss of H5 to solvent) during catalysis . In this study, we sought mechanistic information for the ODCase reaction by examining the extent of hydrogen exchange in the product uridine-5(')-monophosphate, in combination with ODCase, at the H5 and H6 positions . In a subsequent experiment, partially deuterated OMP was prepared, and the extent of 2H5 rearrangement or loss to solvent was examined by integration of 1H nuclear magnetic resonance signals in the substrate and the resulting enzymatically decarboxylated product . The absence of detectable hydrogen exchange in these experiments limits somewhat the possible mechanisms for ODCase catalysis. Mol Cell, 2003 Mar, 11(3), 827 - 35 PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break; Leroy C et al.; Saccharomyces cells suffering a DNA double-strand break (DSB) ultimately escape checkpoint-mediated G2/M arrest either by recovery once the lesion is repaired or by adaptation if the lesion proves irreparable . Cells lacking the PP2C-like phosphatases Ptc2 and Ptc3 are unable to adapt to a HO-induced DSB and are also defective in recovering from a repairable DSB . In contrast, overexpression of PTC2 rescues adaptation-defective yku80Delta and cdc5-ad mutants . These effects are not explained by alterations either in the processing of DSB ends or in DSB repair . In vivo and in vitro evidence suggests that phosphorylated forms of Ptc2 and Ptc3 specifically bind to the Rad53 FHA1 domain and inactivate Rad53-dependent pathways during adaptation and recovery by dephosphorylating Rad53. Mol Cell, 2003 Mar, 11(3), 721 - 9 The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation; Krogan NJ et al.; Methylation of histone proteins is one of their many modifications that affect chromatin structure and regulate gene expression . Methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively, is required for silencing of expression of genes located near chromosome telomeres in yeast . We report that the Paf1 protein complex, which is associated with the elongating RNA polymerase II, is required for methylation of lysines 4 and 79 of histone H3 and for silencing of expression of a telomere-associated gene . We show that the Paf1 complex is required for recruitment of the COMPASS methyltransferase to RNA polymerase II and that the subunits of these complexes interact physically and genetically . Collectively, our results suggest that the Paf1 complex is required for histone H3 methylation, therefore linking transcriptional elongation to chromatin methylation. Mol Cell, 2003 Mar, 11(3), 709 - 19 Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity; Ng HH et al.; Set1, the yeast histone H3-lysine 4 (H3-K4) methylase, is recruited by the Pol II elongation machinery to a highly localized domain at the 5' portion of active mRNA coding regions . Set1 association depends upon the TFIIH-associated kinase that phosphorylates the Pol II C-terminal domain (CTD) and mediates the transition between initiation and elongation, and Set1 interacts with the form of Pol II whose CTD is phosphorylated at serine 5 but not serine 2 . The Rtf1 and Paf1 components of the Pol II-associated Paf1 complex are also important for Set1 recruitment . Although the level of dimethylated H3-K4 is fairly uniform throughout the genome, the pattern of trimethylated H3-K4 strongly correlates with Set1 occupancy . Hypermethylated H3-K4 within the mRNA coding region persists for considerable time after transcriptional inactivation and Set1 dissociation from the chromatin, indicating that H3-K4 hypermethylation provides a molecular memory of recent transcriptional activity. Mol Cell, 2003 Mar, 11(3), 635 - 46 Pex8p: an intraperoxisomal organizer of the peroxisomal import machinery; Agne B et al.; Peroxisomes transport folded and oligomeric proteins across their membrane . Two cytosolic import receptors, Pex5p and Pex7p, along with approximately 12 membrane-bound peroxins participate in this process . While interactions among individual peroxins have been described, their organization into functional units has remained elusive . We have purified and defined two core complexes of the peroxisomal import machinery: the docking complex comprising Pex14p and Pex17p, with the loosely associated Pex13p, and the RING finger complex containing Pex2p, Pex10p, and Pex12p . Association of both complexes into a larger import complex requires Pex8p, an intraperoxisomal protein . We conclude that Pex8p organizes the formation of the larger import complex from the trans side of the peroxisomal membrane and thus might enable functional communication between both sides of the membrane. Biochemistry, 2003 Apr 8, 42(13), 3751 - 8 Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13; Anderson EM et al.; The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity . The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres . We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA) . The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop . Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues . A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity . Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed . The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding . While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface . This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity. Biochemistry, 2003 Apr 8, 42(13), 3716 - 24 Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase; Turek-Etienne TC et al.; Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein . The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets . While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood . To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods . Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM . Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase . However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate . Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site . Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein . Taken together, these data do not support the "molecular ruler hypotheses" . Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product. Biopolymers, 2003 Apr, 68(4), 528 - 38 Role of C-terminal domain phosphorylation in RNA polymerase II transcription through the nucleosome; Liu YV et al.; End-initiated transcription of a 256 base-pair (bp) template containing a single uniquely positioned nucleosome by yeast and calf thymus nuclear RNA polymerases II (pol II) was analyzed in vitro . The nucleosome-specific pausing pattern is similar to the pattern observed in the case of transcription of the same nucleosome by yeast RNA polymerase III . However, the pausing pattern is clearly different from the patterns observed previously during transcription by promoter-initiated and assembled pol II . This suggests that end-initiated and promoter-initiated RNA polymerases differ in the way they progress through the nucleosome . The rates of transcription through the nucleosome by pol II are significantly lower than the rates observed in the case of SP6 polymerase and RNA polymerase III . Using calf thymus pol II, we have investigated the possibility that phosphorylation of the C-terminal domain (CTD) facilitates transcription through the nucleosome . The rates of transcription through the nucleosome by phosphorylated (IIO) and nonphosphorylated (IIA) forms of calf thymus pol II are very similar . This suggests that CTD phosphorylation is not sufficient to facilitate transcription through the nucleosome by end-initiated pol II . Mol Cell Biol, 2003 Apr, 23(8), 3008 - 12 Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases; Johnson RE et al.; Although DNA polymerase eta (Pol eta) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains . Here, we examine the role in Saccharomyces cerevisiae Pol eta of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation . We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably . The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family . Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively. Mol Cell Biol, 2003 Apr, 23(8), 2999 - 3007 Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p, a cohesion establishment factor, associates with three different replication factor C complexes; Kenna MA et al.; CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion . The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C {RFC} homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies . To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery . Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication . Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes . By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p . The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation . Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes . These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways . Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment . The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication . Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment. Mol Cell Biol, 2003 Apr, 23(8), 2942 - 52 Novel SWI/SNF chromatin-remodeling complexes contain a mixed-lineage leukemia chromosomal translocation partner; Nie Z et al.; The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin . Several complexes of this family have been reported for humans . In this study, two additional complexes are described that belong to the same SWI/SNF family . These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity . But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes . This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3 . Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia . The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL . Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins. Mol Cell Biol, 2003 Apr, 23(8), 2844 - 58 Isotype-restricted corepressor recruitment: a constitutively closed helix 12 conformation in retinoic acid receptors beta and gamma interferes with corepressor recruitment and prevents transcriptional repression; Farboud B et al.; Retinoic acid receptors (RARs) are ligand-regulated transcription factors that play multiple roles in vertebrate development and differentiation . RARs as a class are capable of both repressing and activating target gene expression . Transcriptional repression is mediated through the recruitment of corepressor proteins such as SMRT . Notably, vertebrates encode three major forms of RARs, alpha, beta, and gamma, and these distinct RAR isotypes differ in the ability to recruit a corepressor . RAR alpha strongly interacts with SMRT and can repress target gene transcription, whereas RAR beta and -gamma interact with SMRT only weakly and fail to repress . We report here the use of a genetic suppressor approach, based on a yeast two-hybrid interaction assay using Saccharomyces cerevisiae, for the isolation of RAR beta mutants that have gained the RAR alpha-like corepressor phenotype, i.e., a strong interaction with SMRT and the ability to repress gene expression in vertebrate cells . Analysis of these gain-of-function mutants indicates that the different corepressor interaction properties of RAR alpha, -beta and -gamma are determined by a gating mechanism through which amino acid differences in the helix 3 region of these receptors influence the position of the receptor C-terminal helix 12 domain . As a consequence, the RAR beta and RAR gamma receptors appear to adopt a constitutively closed helix 12 conformation in the absence of hormone that may approximate the conformation of RAR alpha when bound to hormone agonist . This closed helix 12 conformation in RAR beta and RAR gamma blocks corepressor binding, prevents repression, and permits significant levels of target gene activation even in the absence of hormone . We refer to this phenomenon as a "gate-latch" model of corepressor regulation. Mol Cell Biol, 2003 Apr, 23(8), 2800 - 20 A multiplicity of coactivators is required by Gcn4p at individual promoters in vivo; Swanson MJ et al.; Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes . Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p . We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces