Biochemistry, 2002 Feb 5, 41(5), 1512 - 9 A single glycine mutation in the equilibrative nucleoside transporter gene, hENT1, alters nucleoside transport activity and sensitivity to nitrobenzylthioinosine; SenGupta DJ et al.; The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues . hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases . Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides . We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides . Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system . Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity . Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity . Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM) . Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR . In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane . This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR. Biochemistry, 2002 Feb 5, 41(5), 1451 - 6 Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP; Jacobsen MD et al.; To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain . Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding . The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation . The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP {K(1/2)(ATP) 12 nM} . Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol . Mutations of Cys(549) did not affect ATP binding . In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369). Cell Mol Life Sci, 2001 Dec, 58(14), 2108 - 16 Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation; Matuoka K et al.; Aging appears to be an irreversible process . Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated beta-galactosidase activity . Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system . The effects are unique to NAA: none ofthe NAA-related compounds examined (an NAD precursor/niacin, NAD analogs, and poly(ADP-ribose) polymerase inhibitors) exerted similar effects . Thus, NAD-related metabolism and poly(ADP-ribosyl)ation are unlikely related to the NAA action . On the other hand, histone acetyltransferase (HAT) activity was elevated in NAA-exposed cells, while in aged cells, HAT activity and histone H4 acetylation were lowered . Taken together, the results suggest that NAA may cause rejuvenation by restoring, at least in part, altered gene expression in aged cells through its activation of HAT. Nat Cell Biol, 2002 Feb, 4(2), 134 - 9 Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48; Jarosch E et al.; Endoplasmic reticulum (ER)-associated protein degradation by the ubiquitin-proteasome system requires the dislocation of substrates from the ER into the cytosol . It has been speculated that a functional ubiquitin proteasome pathway is not only essential for proteolysis, but also for the preceding export step . Here, we show that short ubiquitin chains synthesized on proteolytic substrates are not sufficient to complete dislocation; the size of the chain seems to be a critical determinant . Moreover, our results suggest that the AAA proteins of the 26S proteasome are not directly involved in substrate export . Instead, a related AAA complex Cdc48, is required for ER-associated protein degradation upstream of the proteasome. Nucleic Acids Res, 2001 Dec 15, 29(24), 5216 - 25 The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity; Hayman ML et al.; RBP16 is a guide RNA (gRNA)-binding protein that was shown through immunoprecipitation experiments to interact with approximately 30% of total gRNAs in Trypanosoma brucei mitochondria . To gain insight into the biochemical function of RBP16, we used affinity chromatography and immunoprecipitation to identify RBP16 protein binding partners . By these methods, RBP16 does not appear to stably interact with the core editing machinery . However, fractionation of mitochondrial extracts on MBP-RBP16 affinity columns consistently isolated proteins of 12, 16, 18 and 22 kDa that were absent from MBP control columns . We describe here our analysis of one RBP16-associated protein, p22 . The predicted p22 protein has significant sequence similarity to a family of multimeric, acidic proteins that includes human p32 and Saccharomyces cerevisiae mam33p . Glutaraldehyde crosslinking of recombinant p22 identified homo-multimeric forms of the protein, further substantiating its homology to p32 . We confirmed the p22-RBP16 interaction and demonstrated that the two proteins bind each other directly by ELISA utilizing recombinant p22 and RBP16 . p32 family members have been reported to modulate viral and cellular pre-mRNA splicing, in some cases by perturbing interaction of their binding partners with RNA . To determine whether p22 similarly affects the gRNA binding properties of RBP16, we titrated recombinant p22 into UV crosslinking assays . These experiments revealed that p22 significantly stimulates the gRNA binding capacity of RBP16 . Thus, p22 has the potential to be a regulatory factor in T.brucei mitochondrial gene expression by modulating the RNA binding properties of RBP16. J Biol Chem, 2002 Apr 12, 277(15), 12632 - 41 Epub 2002 Jan 25. Conserved enzymatic production and biological effect of O-acetyl-ADP-ribose by silent information regulator 2-like NAD+-dependent deacetylases; Borra MT et al.; Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging . Yeast Sir2 and several homologues have been shown to be NAD(+)-dependent histone/protein deacetylases . Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD(+) and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite . We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human . Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose . By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres . This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects . In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose. J Mol Biol, 2002 Jan 25, 315(4), 809 - 18 Protein-protein interactions of hCsl4p with other human exosome subunits; Raijmakers R et al.; The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA . We demonstrate that the two human proteins hCsl4p and hRrp42p, which have been identified on the basis of their sequence homology with Saccharomyces cerevisiae proteins, are associated with the human exosome . By mammalian two-hybrid and GST pull-down assays, we show that the hCsl4p protein interacts directly with two other exosome proteins, hRrp42p and hRrp46p . Mutants of hCsl4p that fail to interact with either hRrp42p or hRrp46p are also not able to associate with exosome complexes in vivo . These results indicate that the association of hCsl4p with the exosome is mediated by protein-protein interactions with hRrp42p and hRrp46p . Anal Biochem, 2002 Feb 1, 301(1), 35 - 48 Characterization of monomeric and dimeric forms of recombinant Sml1p-histag protein by electrospray mass spectrometry; Uchiki T et al.; Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR)3, a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair . In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p) . Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway . Neither the structure of Sml1p nor its complex to Rnr1p is well known . In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry . Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: (1) confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS-PAGE) and (2) verifying the presence or absence of PTM, chemical modifications, or metal-ion binding to the protein species, which may alter the function and binding of the protein partners. Mol Genet Genomics, 2001 Dec, 266(4), 657 - 63 Epub 2001 Oct 06. The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants; Thumm M et al.; A P-element line ( P0997) of Drosophila melanogaster in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut ( ct) modifiers . The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy . The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants . These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway. Nucleic Acids Res, 2002 Feb 1, 30(3), 732 - 9 Suppression of genetic defects within the RAD6 pathway by srs2 is specific for error-free post-replication repair but not for damage-induced mutagenesis; Broomfield S et al.; srs2 was isolated during a screen for mutants that could suppress the UV-sensitive phenotype of rad6 and rad18 cells . Genetic analyses led to a proposal that Srs2 acts to prevent the channeling of DNA replication-blocking lesions into homologous recombination . The phenotypes associated with srs2 indicate that the Srs2 protein acts to process lesions through RAD6-mediated post-replication repair (PRR) rather than recombination repair . The RAD6 pathway has been divided into three rather independent subpathways: two error-free (represented by RAD5 and POL30) and one error-prone (represented by REV3) . In order to determine on which subpathways Srs2 acts, we performed comprehensive epistasis analyses; the experimental results indicate that the srs2 mutation completely suppresses both error-free PRR branches . Combined with UV-induced mutagenesis assays, we conclude that the Polzeta-mediated error-prone pathway is functional in the absence of Srs2; hence, Srs2 is not required for mutagenesis . Furthermore, we demonstrate that the helicase activity of Srs2 is probably required for the phenotypic suppression of error-free PRR defects . Taken together, our observations link error-free PRR to homologous recombination through the helicase activity of Srs2. Nucleic Acids Res, 2002 Feb 1, 30(3), 667 - 74 Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction; Huang J et al.; The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells . Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay . To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair . We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts . Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence . Factor activity was inhibited by anti-Mre11 antibody . The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified . Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation. Mol Cell Biol, 2002 Feb, 22(4), 1253 - 65 Functional multimerization of human telomerase requires an RNA interaction domain in the N terminus of the catalytic subunit; Moriarty TJ et al.; Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase {hTERT}) containing reverse transcriptase (RT)-like motifs . The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms . We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization . Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity . We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif . Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70% . Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity . Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus. Mol Cell Biol, 2002 Feb, 22(4), 1246 - 52 Convergence of TOR-nitrogen and Snf1-glucose signaling pathways onto Gln3; Bertram PG et al.; Carbon and nitrogen are two basic nutrient sources for cellular organisms . They supply precursors for energy metabolism and metabolic biosynthesis . In the yeast Saccharomyces cerevisiae, distinct sensing and signaling pathways have been described that regulate gene expression in response to the quality of carbon and nitrogen sources, respectively . Gln3 is a GATA-type transcription factor of nitrogen catabolite-repressible (NCR) genes . Previous observations indicate that the quality of nitrogen sources controls the phosphorylation and cytoplasmic retention of Gln3 via the target of rapamycin (TOR) protein . In this study, we show that glucose also regulates Gln3 phosphorylation and subcellular localization, which is mediated by Snf1, the yeast homolog of AMP-dependent protein kinase and a cytoplasmic glucose sensor . Our data show that glucose and nitrogen signaling pathways converge onto Gln3, which may be critical for both nutrient sensing and starvation responses. Mol Cell Biol, 2002 Feb, 22(4), 1233 - 45 Developmental defects and male sterility in mice lacking the ubiquitin-like DNA repair gene mHR23B; Ng JM et al.; mHR23B encodes one of the two mammalian homologs of Saccharomyces cerevisiae RAD23, a ubiquitin-like fusion protein involved in nucleotide excision repair (NER) . Part of mHR23B is complexed with the XPC protein, and this heterodimer functions as the main damage detector and initiator of global genome NER . While XPC defects exist in humans and mice, mutations for mHR23A and mHR23B are not known . Here, we present a mouse model for mHR23B . Unlike XPC-deficient cells, mHR23B(-/-) mouse embryonic fibroblasts are not UV sensitive and retain the repair characteristics of wild-type cells . In agreement with the results of in vitro repair studies, this indicates that mHR23A can functionally replace mHR23B in NER . Unexpectedly, mHR23B(-/-) mice show impaired embryonic development and a high rate (90%) of intrauterine or neonatal death . Surviving animals display a variety of abnormalities, including retarded growth, facial dysmorphology, and male sterility . Such abnormalities are not observed in XPC and other NER-deficient mouse mutants and point to a separate function of mHR23B in development . This function may involve regulation of protein stability via the ubiquitin/proteasome pathway and is not or only in part compensated for by mHR23A. Mol Cell Biol, 2002 Feb, 22(4), 1116 - 25 A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine; McCulloch V et al.; A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication . Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA . However, based on studies in model systems, the existence of a second human mitochondrial transcription factor has been postulated . Here we report the isolation of a cDNA encoding h-mtTFB, the human homolog of Saccharomyces cerevisiae mitochondrial transcription factor B (sc-mtTFB) and the first metazoan member of this class of transcription factors to which a gene has been assigned . Recombinant h-mtTFB is capable of binding mtDNA in a non-sequence-specific fashion and activates transcription from the human mitochondrial light-strand promoter in the presence of h-mtTFA in vitro . Remarkably, h-mtTFB and its fungal homologs are related in primary sequence to a superfamily of N6 adenine RNA methyltransferases . This observation, coupled with the ability of recombinant h-mtTFB to bind S-adenosylmethionine in vitro, suggests that a structural, and perhaps functional, relationship exists between this class of transcription factors and this family of RNA modification enzymes and that h-mtTFB may perform dual functions during mitochondrial gene expression. Int Immunol, 2002 Feb, 14(2), 189 - 200 Cis elements for transporter associated with antigen-processing-2 transcription: two new promoters and an essential role of the IFN response factor binding element in IFN-gamma-mediated activation of the transcription initiator; Guo Y et al.; Expression of cell surface MHC class I:peptide complex requires coordinated expression of multiple genes such as MHC class I heavy chain, beta(2)-microglobulin (beta(2)m), transporters associated with antigen-processing (TAP)-1 and TAP-2, and proteosomal components low-molecular weight polypeptide (LMP)-2 and LMP-7 . All of these genes are expressed at defined and distinct levels in normal tissues, and are inducible by IFN-gamma . While the cis elements involved in transcription of the MHC class I heavy chain, beta(2)m, TAP-1 and LMP-2 have been analyzed extensively, those for TAP-2 and LMP-7 have not been well studied . Here we systematically analyzed the cis elements for TAP-2 transcription . We found at least two independent elements that are sufficient to activate transcription of a reporter gene . One (hereby called TAP-2 P1) is located 5' to the TAP-2 exon 1, while the other (hereby called TAP-2 P2) is a transcription initiator residing in intron 1 . Analysis of the 5' sequence of TAP-2 mRNA indicates that both promoters are active . Moreover, while the TAP-2 promoter region contains cis elements that can mediate TAP-2 induction by IFN-gamma, such as gamma-activation site and IFN response factor binding element (IRFE), only the IRFE is required for IFN-gamma induction of TAP-2 promoter in vitro . The IRFE appears to work as an enhancer for the initiator (P2) . Together with another promoter recently identified by others, TAP-2 therefore has three independent promoters that can be differentially regulated. J Nat Prod, 2002 Jan, 65(1), 65 - 8 A new triterpene saponin from Pittosporum viridiflorum from the Madagascar rainforest; Seo Y et al.; A novel triterpenoid saponin, pittoviridoside (1), which possesses an unusual 2,3,4-trisubstituted glycosidic linkage, has been isolated from Pittosporum viridiflorum using the engineered yeast strains 1138, 1140, 1353, and Sc-7 for bioactivity-guided fractionation . The structure of this compound was determined to be 3-O-{beta-D-glucopyranosyl(1-->2)}-{alpha-D-arabinopyranosyl(1-->3)},{alpha-l-arabinofuranosyl(1-->4)-beta-D-glucuronopyranosyl-21-angeloyl-22-senecioylolean-12-en-3beta,15alpha,16alpha,21beta,22alpha,28-hexol by spectral, chemical, and GC analyses . This compound showed weak cytotoxicity against the A2780 human ovarian cancer cell line. Nature, 2002 Jan 24, 415(6870), 447 - 50 Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein; Helenius J et al.; N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway . The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER . The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate . This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion . The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins . The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells . The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive . Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane. J Cell Biol, 2002 Jan 21, 156(2), 315 - 26 Epub 2002 Jan 21. Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p; Gladfelter AS et al.; At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence . Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring . The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP) . Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs . In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization . Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly . However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type . Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p . These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure. Curr Genet, 2001 Dec, 40(4), 234 - 42 Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function; Wu X et al.; Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II . Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5 . Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD . Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation . In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI . We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1 . Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect . Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD . The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription. Sci STKE . 2002 Jan 22;2002(116):PE4. A new gun in town: the U box is a ubiquitin ligase domain; Patterson C; Ubiquitin ligases determine protein stability in a highly regulated manner by coordinating the addition of polyubiquitin chains to proteins that are then targeted to the proteasome for degradation . Ubiquitin ligases have generally been separated into two groups--those containing HECT domains and those with RING finger domains . Recently, a third group of ubiquitin ligases has emerged: those containing a U-box domain . Patterson discusses what is known about the few U-box-containing proteins that have been characterized, although the general properties of U-box proteins that distinguish them from other ubiquitin ligases are still a matter of speculation. J Biol Chem, 2002 Apr 5, 277(14), 11691 - 5 Epub 2002 Jan 22. Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23; Rao H et al.; Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates . The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation . The major function of Ub is to regulate the in vivo half-life of its substrates . Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it . Molecular mechanisms of the postubiquitylation processes are poorly understood . Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA) . Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation . We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis . Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome . Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events. J Biol Chem, 2002 Mar 29, 277(13), 10852 - 60 Epub 2002 Jan 22. Adenosine monophosphoramidase activity of Hint and Hnt1 supports function of Kin28, Ccl1, and Tfb3; Bieganowski P et al.; The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases . Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily . Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5'-monophosphoramidate (AMPNH(2)) in an active-site-dependent manner at second order rates exceeding 1,000,000 m(-1) s(-1) . Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures . Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28 . The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers . Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity. J Biol Chem, 2002 Mar 29, 277(13), 10753 - 5 Epub 2002 Jan 22. COMPASS, a histone H3 (Lysine 4) methyltransferase required for telomeric silencing of gene expression; Krogan NJ et al.; The trithorax (Trx) family of proteins is required for maintaining a specific pattern of gene expression in some organisms . Recently we reported the isolation and characterization of COMPASS, a multiprotein complex that includes the Trx-related protein Set1 of the yeast Saccharomyces cerevisiae . Here we report that COMPASS catalyzes methylation of the fourth lysine of histone H3 in vitro . Set1 and several other components of COMPASS are also required for histone H3 methylation in vivo and for transcriptional silencing of a gene located near a chromosome telomere. Genetics, 2002 Jan, 160(1), 137 - 48 Meiotic deletion at the BUF1 locus of the fungus Magnaporthe grisea is controlled by interaction with the homologous chromosome; Farman ML; The Magnaporthe grisea BUF1 gene suffers high-frequency mutation in certain genetic crosses, resulting in buff-colored progeny . Analysis of 16 buf1 mutants arising from a cross with a mutation frequency of 25% revealed that, in every case, the BUF1 gene was deleted . The deletions occurred in only one of the parental chromosomes and were due to intrachromosomal recombination . Tetrad analysis revealed that deletions occurred in 44% of meioses and usually affected both chromatids of the mutable chromosome . This suggests that they happen before the premeiotic round of DNA synthesis . However, they were also almost entirely restricted to heteroallelic crosses . This, together with the discovery of numerous repetitive elements that were present only in the mutable BUF1 locus, suggests that the deletion process is sensitive to pairing interactions between homologous chromosomes, such that only unpaired loci are subject to deletion . Given that karyogamy is not supposed to occur until after premeiotic DNA replication in Pyrenomycetous fungi such as M . grisea, this latter observation would place the time of deletion during, or after, DNA synthesis . These conflicting results suggest that karyogamy might actually precede DNA replication in Pyrenomycetous fungi or that parts of the genome remain unreplicated until after karyogamy and subsequent chromosome pairing have taken place. Mol Cell, 2002 Jan, 9(1), 8 - 9 The spliceosome: no assembly required? Nilsen TW. In this issue of Molecular Cell, Stevens et al . purify a large particle from yeast extracts that contains all five of the U snRNPs required for pre-mRNA splicing . The existence of this "penta-snRNP" suggests the provocative possibility that spliceosome assembly does not depend upon a pre-mRNA substrate. Eur J Pharm Sci, 2002 Feb, 15(1), 39 - 47 Compromised integrity of excised porcine intestinal epithelium obtained from the abattoir affects the outcome of in vitro particle uptake studies; Pietzonka P et al.; Excised porcine intestinal tissue obtained from the local abattoir was studied for its suitability to examine the uptake and transport of poly(lactic-co-glycolic acid) (PLGA) nanoparticles in Peyer's (PP) and non-Peyer's patch (NPP) tissue in vitro . Incubation of such tissue with fluorescent PLGA and polystyrene particles revealed negligible uptake into the intercellular space with no noticeable difference between PP and NPP tissue . Similarly, yeast cells, which were used as a positive control for selective uptake into PP tissue, were found in the subepithelial area of both PP and NPP tissue . Therefore we examined the morphological integrity of the tissue for the duration of the experiments . For this purpose, excised intestinal tissue from the abattoir transported to the laboratory was examined for morphological changes by light microscopy and compared to intestinal tissue from freshly slaughtered piglets . Already after 25 min postmortem, we observed lysis and defoliation of the epithelial cell layer followed by a complete loss of villus architecture and, consequently, resulting in a complete loss of the integrity of the intestinal tissue . This may explain the limited and non-selective particle uptake when using excised intestinal tissue from the abattoir . It is suggested to avoid small intestine obtained from the abattoir and to use tissue from freshly sacrificed animals within a few minutes postmortem . Experiments should then be performed under adequate oxygenation of the excised intestinal tissue. Nat Cell Biol, 2002 Feb, 4(2), 140 - 7 IFN-Stimulated transcription through a TBP-free acetyltransferase complex escapes viral shutoff; Paulson M et al.; Type I interferon (IFN) stimulates transcription through a heteromeric transcription factor that contains tyrosine-phosphorylated STAT2 . We show that STAT2 recruits histone acetyltransferases (HAT) through its transactivation domain, resulting in localized transient acetylation of histones . GCN5, but not p300/CBP or PCAF, is required for STAT2 function . However, GCN5 function is impaired by the transcriptional antagonist, adenovirus E1A oncoprotein . The TFIID component TAF(II)130 potentiates STAT2 function, but TAF(II)28 or the HAT activity of TAF(II)250 do not, and transcriptional induction can proceed independently of the TATA-binding protein, TBP . Moreover, IFN-stimulated transcription was resistant to poliovirus-targeted degradation by TBP, and continued despite host-cell transcriptional shutoff during poliovirus infection . We conclude that a non-classical transcriptional mechanism combats an anticellular action of poliovirus, through a TBP-free TAF-containing complex and GCN5. Mol Biol Evol, 2002 Feb, 19(2), 189 - 200 Evolution of eukaryotic translation elongation and termination factors: variations of evolutionary rate and genetic code deviations; Moreira D et al.; Translation is carried out by the ribosome and several associated protein factors through three consecutive steps: initiation, elongation, and termination . Termination remains the least understood of them, partly because of the nonuniversality of the factors involved . To get some insights on the evolution of eukaryotic translation termination, we have compared the phylogeny of the release factors eRF1 and eRF3 to that of the elongation factors EF-1alpha and EF-2, with special focus on ciliates . Our results show that these four translation proteins have experienced different modes of evolution . This is especially evident for the EF-1alpha, EF-2, and eRF1 ciliate sequences . Ciliates appear as monophyletic in the EF-2 phylogenetic tree but not in the EF-1alpha and eRF1 phylogenetic trees . This seems to be mainly because of phylogeny reconstruction artifacts (the long-branch attraction) produced by the acceleration of evolutionary rate of ciliate EF-1alpha and eRF1 sequences . Interaction with the highly divergent actin found in ciliates, or on the contrary, loss of interaction, could explain the acceleration of the evolutionary rate of the EF-1alpha sequences . In the case of ciliate eRF1 sequences, their unusually high evolutionary rate may be related to the deviations in the genetic code usage found in diverse ciliates . These deviations involve a relaxation (or even abolition) of the recognition of one or two stop codons by eRF1 . To achieve this, structural changes in eRF1 are needed, and this may affect its evolutionary rate . Eukaryotic translation seems to have followed a mosaic evolution, with its different elements governed by different selective pressures . However, a correlation analysis shows that, beneath the disagreement shown by the different translation proteins, their concerted evolution can still be made apparent when they are compared with other proteins that are not involved in translation. Mol Biol Evol, 2002 Feb, 19(2), 179 - 88 Low nucleotide diversity at the pal1 locus in the widely distributed Pinus sylvestris; Dvornyk V et al.; Nucleotide polymorphism in Scots pine (Pinus sylvestris) was studied in the gene encoding phenylalanine ammonia-lyase (Pal, EC 4.3.1.5) . Scots pine, like many other pine species, has a large current population size . The observed levels of inbreeding depression suggest that Scots pine may have a high mutation rate to deleterious alleles . Many Scots pine markers such as isozymes, RFLPs, and microsatellites are highly variable . These observations suggest that the levels of nucleotide variation should be higher than those in other plant species . A 2,045-bp fragment of the pal1 locus was sequenced from five megagametophytes each from a different individual from each of four populations, from northern and southern Finland, central Russia, and northern Spain . There were 12 segregating sites in the locus . The synonymous site overall nucleotide diversity was only 0.0049 . In order to compare pal1 with other pine genes, sequence was obtained from two alleles of 11 other loci (total length 4,606 bp) . For these, the synonymous nucleotide diversity was 0.0056 . These estimates are lower than those from other plants . This is most likely because of a low mutation rate, as estimated from between-pine species synonymous site divergence . In other respects, Scots pine has the characteristics of a species with a large effective population . There was no linkage disequilibrium even between closely linked sites . This resulted in high haplotype diversity (14 different haplotypes among 20 sequences) . This could also give rise to high per locus diversity at the protein level . Divergence between populations in the main range was low, whereas an isolated Spanish population had slightly lower diversity and higher divergence than the remaining populations. J Cell Sci, 2002 Jan 1, 115(Pt 1), 131 - 40 Roles of the N- and C-termini of GLUT4 in endocytosis; Al-Hasani H et al.; In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane . To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells . Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4 . However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states . Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis . Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins mu1, mu2, and mu3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles. J Biol Chem, 2002 Apr 12, 277(15), 13016 - 28 Epub 2002 Jan 18. Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase; Menguy T et al.; We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process . In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment . We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase . Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum . All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion . Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C . (2001) J . Biol . Chem . 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane. Reprod Fertil Dev, 2001, 13(4), 221 - 9 Protein coregulators that mediate estrogen receptor function; Ratajczak T; The recent discovery of estrogen receptor beta as a biological partner with estrogen receptor alpha in mediating the estrogen response has come at precisely the same time as intensive research is revealing the role played by downstream coregulator proteins in linking nuclear hormone receptor activity to general transcription machinery involved in gene transcriptional activation . In what is a rapidly evolving area of research, findings to date have led to a proposed model of hormonal action, in which a receptor activated by estrogen or cell-membrane-derived phosphorylation-dependent signaling pathways promotes recruitment of selected members of the multifunctional steroid receptor coactivator family and the cointegrators, p300/CBP and P/CAF . The intrinsic histone acetylase activity mediated by these coactivator and cointegrator proteins, alters chromatin structure giving rise to increased transcriptional efficiency . On the other hand, antiestrogen-bound receptors favour the assembly of receptor-corepressor complexes containing the sequence-related corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoid and thyroid hormone receptors), localizing histone deacetylase activity to the promoter and leading to transcriptional repression . The model predicts that a change in the balance between corepressor and coactivator expression in favour of coactivators, might result in antiestrogen resistance . Together with available crystal structure data for estrogen- and antiestrogen-bound receptors, these studies have provided valuable insights into events that occur subsequent to receptor interaction with specific DNA sequences and have helped define the molecular basis of estrogen and antiestrogen activity. Biochem Cell Biol, 2001, 79(6), 681 - 92 Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery; McMaster CR; The movement of lipids from their sites of synthesis to ultimate intracellular destinations must be coordinated with lipid metabolic pathways to ensure overall lipid homeostasis is maintained . Thus, lipids would be predicted to play regulatory roles in the movement of vesicles within cells . Recent work has highlighted how specific lipid metabolic events can affect distinct vesicle trafficking steps and has resulted in our first glimpses of how alterations in lipid metabolism participate in the regulation of intracellular vesicles . Specifically, (i) alterations in sphingolipid metabolism affect the ability of SNAREs to fuse membranes, (ii) sterols are required for efficient endocytosis, (iii) glycerophospholipids and phosphorylated phosphatidylinositols regulate Golgi-mediated vesicle transport, (iv) lipid acylation is required for efficient vesicle transport mediated membrane fission, and (v) the addition of glycosylphosphatidylinositol lipid anchors to proteins orders them into distinct domains that result in their preferential sorting from other vesicle destined protein components in the endoplasmic reticulum . This review describes the experimental evidence that demonstrates a role for lipid metabolism in the regulation of specific vesicle transport events. J Biol Chem, 2002 Mar 29, 277(13), 11401 - 9 Epub 2002 Jan 17. Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway; Yoo JS et al.; The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown . Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S . I., Bannykh, G . I., Fish, K . N., Moyer, B . D., Riordan, J . R., and Balch, W . E . (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway . We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested . Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi . Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation . We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells. Genes Dev, 2002 Jan 15, 16(2), 183 - 97 The mitotic spindle is required for loading of the DASH complex onto the kinetochore; Li Y et al.; A role for the mitotic spindle in the maturation of the kinetochore has not been defined previously . Here we describe the isolation of a novel and conserved essential gene, ASK1, from Saccharomyces cerevisiae involved in this process . ask1 mutants display either G(2)/M arrest or segregation of DNA masses without the separation of sister chromatids, resulting in massive nondisjunction and broken spindles . Ask1 localizes along mitotic spindles and to kinetochores, and cross-links to centromeric DNA . Microtubules are required for Ask1 binding to kinetochores, and are partially required to maintain its association . We found Ask1 is part of a multisubunit complex, DASH, that contains approximately 10 components, including several proteins essential for mitosis including Dam1, Duo1, Spc34, Spc19, and Hsk1 . The Ipl1 kinase controls the phosphorylation of Dam1 in the DASH complex and may regulate its function . We propose that DASH is a microtubule-binding complex that is transferred to the kinetochore prior to mitosis, thereby defining a new step in kinetochore maturation. Zhonghua Yi Xue Za Zhi, 2000 Jun, 80(6), 456 - 60 {Function of one novel gene identified by SSH PCR differentially expressed in HBX transfected HepG2 cells}; Liu J et al.; OBJECTIVE: To clone full length differentially expressed genes which are related with HBxAg . METHODS: HepG2-cells were infected with prepared recombinant retroviruses encoding the X antigen . The differences in gene expression between HepG2 x and HepG2Cat cells were evaluated by suppression subtractive hybridization and PCR . In situ hybridization (ISH) and Northern blot analysis were carried out to screen the differentially expressed genes . The full length cDNA clone of the gene was obtained by 5' and 3' rapid amplification of cDNA ends(race) PCR . HepG2 cells transiently transfected with the new full length gene were subjected to fluorescence activated cell sorting (FACS) analysis for DNA content . HepG2 cells stably transfected with the new full length gene were tested for anchorage independent growth in soft agar and for tumorigenicity in nude mice . RESULTS: The expression of multiple genes were turned on (8) or off (2) in HepG2X compared to HepG2CAT cells . One differentially expressed gene C2, the human homology of Sui1, encoded a translation initiation factor whose expression was suppressed by X antigen in HepG(2) cells . The full length of this gene was 1.35 kb, which encoded a small protein of 113 amino acids . Introduction of C2 into HepG2 cells could inhibit cell growth in culture, in soft agar, and partially inhibit tumor formation in nude mice . Cells transfected with pcDNA3-HBx showed little or no detectable C2, which was consistent with the suppression of this protein in the presence of HBxAg . C2 was also expressed in nontumor liver, but not in tumor cells from patients with hepatocellular carcinoma . CONCLUSIONS: HBX can regulate the expression of genes whose products may be positive or negative regulators of cell growth . Our work for the first time demonstrates that the mechanism of DNA virus associated carcinogenesis involves altered patterns of gene expression regulated at the level of translation initiation. IUBMB Life, 2001 Sep-Nov, 52(3-5), 101 - 12 Protein import into mitochondria; Paschen SA et al.; Most mitochondrial proteins are encoded by the nuclear genome and thus have to be imported into mitochondria from the cytosol . Protein translocation across and into the mitochondrial membranes is a multistep process facilitated by the coordinated action of at least four specialized translocation systems in the outer and inner membranes of mitochondria . The outer membrane contains one general translocase, the TOM complex, whereas three distinct translocases are located in the inner membrane, which facilitates translocation of different classes of preproteins . The TIM23 complex mediates import of matrix-targeted preproteins with N-terminal presequences, whereas hydrophobic preproteins with internal targeting signals are inserted into the inner membrane via the TIM22 complex . The OXA translocase mediates the insertion of preproteins from the matrix space into the inner membrane . This review focuses on the structural organization and function of the import machinery of the model organisms of Saccharomyces cerevisiae and Neurospora crassa . In addition, the molecular basis of a new human mitochondrial disorder is discussed, the Mohr-Tranebjaerg syndrome . This is the first known disease, which is caused by an impaired mitochondrial protein import machinery leading to progressive neurodegeneration. J Biol Chem, 2002 Mar 22, 277(12), 9741 - 8 Epub 2002 Jan 16. Studies on the mode of Ku interaction with DNA; Arosio D et al.; The Ku heterodimer plays a central role in non-homologous end-joining . The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays . The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (D25) is added, indicating that only one Ku molecule binds to a 25-bp fragment . The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm . If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d) . The k(d) is not affected by ATP, Mg(2+), or ionic strength . However, the addition of bovine serum albumin decreases the k(d) by 2-fold . DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate . Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the k(d) values for the binding of Ku to both ends of these substrates are the same . Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates. J Biol Chem, 2002 Apr 19, 277(16), 13848 - 55 Epub 2002 Jan 16. Transient inhibition of translation initiation by osmotic stress; Uesono Y et al.; Cells respond and adapt to changes in the environment . In this study, we examined the effect of environmental stresses on protein synthesis in the yeast Saccharomyces cerevisiae . We found that osmotic stress causes irreversible inhibition of methionine uptake, transient inhibition of uracil uptake, transient stimulation of glucose uptake, transient repression of ribosomal protein (RP) genes such as CYH2 and RPS27, and the transient inhibition of translation initiation . Rapid inhibition of translation initiation by osmotic stress requires a novel pathway, different from the amino acid-sensing pathway, the glucose-sensing pathway, and the TOR pathway . The Hog1 MAP kinase pathway is not involved in the inhibition of either methionine uptake or translation initiation but is required for the adaptation of translation initiation after inhibition and the repression of RP genes by osmotic stress . These results suggest that the transient inhibition of translation initiation occurs as a result of a combination of both acute inhibition of translation and the long-term activation of translation by the Hog1 pathway. Hum Mutat, 2002 Feb, 19(2), 173 - 7 A robust method for detecting CHK2/RAD53 mutations in genomic DNA; Sodha N et al.; While screening for germline CHK2 mutations in cancer cases by heteroduplex CSGE, we observed that additional PCR fragments were generated from the 3' end region of the gene that includes exons 11-14 . Direct sequencing of these fragments suggested that homologous loci (possibly pseudogenes) were concomitantly being amplified . Searches of public sequence databases showed that a number of areas of the genome show a high degree of homology to exons 10-14 of the CHK2 gene . The presence of these homologous regions means that standard screening methods for detecting mutations in CHK2, based on PCR of genomic DNA, are prone to error . To circumvent this problem, we have developed a strategy, based on long-range PCR, to screen the functional copy of CHK2 . Using this approach it is possible to carry out a comprehensive mutational analysis of CHK2 from genomic DNA . Funct Integr Genomics, 2001 Mar, 1(4), 269 - 78 Dynamic models of gene expression and classification; Dewey TG et al.; Powerful new methods, like expression profiles using cDNA arrays, have been used to monitor changes in gene expression levels as a result of a variety of metabolic, xenobiotic or pathogenic challenges . This potentially vast quantity of data enables, in principle, the dissection of the complex genetic networks that control the patterns and rhythms of gene expression in the cell . Here we present a general approach to developing dynamic models for analyzing time series of whole genome expression . In this approach, a self-consistent calculation is performed that involves both linear and non-linear response terms for interrelating gene expression levels . This calculation uses singular value decomposition (SVD) not as a statistical tool but as a means of inverting noisy and near-singular matrices . The linear transition matrix that is determined from this calculation can be used to calculate the underlying network reflected in the data . This suggests a direct method of classifying genes according to their place in the resulting network . In addition to providing a means to model such a large multivariate system this approach can be used to reduce the dimensionality of the problem in a rational and consistent way, and suppress the strong noise amplification effects often encountered with expression profile data . Non-linear and higher-order Markov behavior of the network are also determined in this self-consistent method . In data sets from yeast, we calculate the Markov matrix and the gene classes based on the linear-Markov network . These results compare favorably with previously used methods like cluster analysis . Our dynamic method appears to give a broad and general framework for data analysis and modeling of gene expression arrays. Funct Integr Genomics, 2000 Nov, 1(3), 174 - 85 Constraint structure analysis of gene expression; Rifkin SA et al.; A microarray experiment gives a snapshot of the state of an organism in terms of the relative abundances of its mRNA transcripts, locating the organism at a point in a high dimensional state space where each axis represents the relative expression level of a single gene . Multiple experiments generate a cloud of points in this gene expression space . We present a geometric approach to analyzing the covariational properties of such a cloud and use a dataset from Saccharomyces cerevisiae as an illustration . In particular, we use singular value decomposition to identify significant linear sub-structures in the data and analyze the contributions of both individual genes and functional classes of genes to these major directions of variation . Analyzing the publicly available yeast expression data, we show that under all experimental conditions the variation in expression is limited to a small number of linear dimensions . Projections of individual gene axes onto the significant dimensions can order the contribution of individual genes to variation in expression within an experiment . We show that no particular groups of genes characterize particular experimental conditions . Instead, the particular structure of the coordinated expression of the entire genome characterizes a particular experiment. Funct Integr Genomics, 2000 May, 1(1), 70 - 5 An integrated web interface for large-scale characterization of sequence data; Cheung KH et al.; Large-scale genome projects require the analysis of large amounts of raw data . This analysis often involves the application of a chain of biology-based programs . Many of these programs are difficult to operate because they are non-integrated, command-line driven, and platform-dependent . The problem is compounded when the number of data files involved is large, making navigation and status-tracking difficult . To demonstrate how this problem can be addressed, we have created a platform-independent Web front end that integrates a set of programs used in a genomic project analyzing gene function by transposon mutagenesis in Saccharomyces cerevisiae . In particular, these programs help define a large number of transposon insertion events within the yeast genome, identifying both the precise site of transposon insertion as well as potential open reading frames disrupted by this insertion event . Our Web interface facilitates this analysis by performing the following tasks . Firstly, it allows each of the analysis programs to be launched against multiple directories of data files . Secondly, it allows the user to view, download, and upload files generated by the programs . Thirdly, it indicates which sets of data directories have been processed by each program . Although designed specifically to aid in this project, our interface exemplifies a general approach by which independent software programs may be integrated into an efficient protocol for large-scale genomic data processing. Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 832 - 7 Epub 2002 Jan 15. Ku86 is essential in human somatic cells; Li G et al.; Ku86 plays a key role in nonhomologous end joining in mammals . Functional inactivation in rodents of either Ku86 or Ku70, which form the heterodimeric DNA end-binding subunit of the DNA-dependent protein kinase complex, is nevertheless compatible with viability . In contrast, no human patient has been described with mutations in either Ku86 or Ku70 . This has led to the hypotheses that either these genes are performing an additional essential role(s) and/or redundant pathways exist that mask the phenotypic expression of these genes when they are mutated in humans . To address this issue, we describe here the construction of human somatic cell lines containing a targeted disruption of the Ku86 locus . Human HCT116 colon cancer cells heterozygous for Ku86 were haploinsufficient with an increase in polyploid cells, a reduction in cell proliferation, elevated p53 levels, and a slight hypersensitivity to ionizing radiation . Functional inactivation of the second Ku86 allele resulted in cells with a drastically reduced doubling time . These cells were capable of undergoing only a limited number of cell divisions, after which they underwent apoptosis . These experiments demonstrate that the Ku86 locus is essential in human somatic tissue culture cells. Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 727 - 32 Epub 2002 Jan 15. Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment; Muchowski PJ et al.; Huntington's disease is caused by the expansion of CAG repeats coding for a polyglutamine tract in the huntingtin protein . The major pathological feature found in Huntington's disease neurons is the presence of detergent-insoluble ubiquitinated inclusion bodies composed of the huntingtin protein . However, the mechanisms that underlie inclusion body formation, and the precise relationship between inclusion bodies and events that initiate toxicity, remain unclear . Here, we analyzed the effects of drugs or genetic mutations that disrupt the microtubule cytoskeleton in a Saccharomyces cerevisiae model of the aggregation of an amino-terminal polyglutamine-containing fragment of huntingtin exon 1 (HtEx1) . Treatment of yeast with drugs that disrupt microtubules resulted in less than 2% of the detergent-insoluble HtEx1 observed in mock-treated cells and prevented the formation of large juxtanuclear inclusion bodies . Disruption of microtubules also unmasked a potent glutamine length-dependent toxicity of HtEx1 under conditions where HtEx1 exists in an entirely detergent-soluble nonaggregated form . Results from the yeast model paralleled those from neuronal pheochromocytoma cells, where disruption of microtubules eliminated the formation of juxtanuclear and intranuclear inclusion bodies by HtEx1 . Our results suggest that active transport along microtubules may be required for inclusion body formation by HtEx1 and that inclusion body formation may have evolved as a cellular mechanism to promote the sequestration or clearance of soluble species of HtEx1 that are otherwise toxic to cells. J Cell Sci, 2001 Dec, 114(Pt 24), 4629 - 35 Interaction of the endoplasmic reticulum alpha 1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system; Massaad MJ et al.; The alpha1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum . The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p . A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi alpha1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Deltapep4 cells and in the vacuoles of rer1/Deltapep4 by indirect immunofluorescence . The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo . Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of beta-galactosidase activity . Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction . A weak interaction was observed between Alg5p and Rer1p . These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact . Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol. J Natl Cancer Inst, 2002 Jan 16, 94(2), 88 - 94 Cell-based assays for identification of novel double-strand break-inducing agents; Dunstan HM et al.; BACKGROUND:We are developing cell-based assays to identify anticancer agents that are selectively toxic to cells with defined mutations . As a test, we used a three-stage strategy to screen compounds from the National Cancer Institute's repository for agents that are selectively toxic to double-strand break repair-deficient yeast cells . METHODS:Compounds identified in the screen were further analyzed by use of yeast and vertebrate cell-based and in vitroassays to distinguish between topoisomerase I and II poisons . RESULTS:Of the more than 85 000 compounds screened, 126 were selectively toxic to yeast deficient in DNA double-strand break repair . Eighty-seven of these 126 compounds were structurally related to known topoisomerase poisons, and 39 were not . Twenty-eight of the 39 were characterized, and we present data for eight of the compounds . Among these eight compounds, we identified two novel topoisomerase II poisons (NSC 327929 and NSC 638432) that were equipotent to etoposide in biochemical tests and in cells, five (NSC 63599, NSC 65601, NSC 380271, NSC 651646, and NSC 668370) with topoisomerase I-dependent toxicity in yeast that induced DNA damage and toxicity in mammalian cells, and one (NSC 610898) that directly bound to DNA and induced strand breaks . CONCLUSIONS:Cell-based assays can be used to identify molecules that are selectively toxic to cells with a predetermined genetic background, including mutations in genes involved in the cell cycle and its checkpoints, for which there are currently no selectively toxic compounds. Bioconjug Chem, 2002 Jan-Feb, 13(1), 136 - 42 Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A; Mislovicova D et al.; Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups . Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa . The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates . Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6 . The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance . Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates . Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan) . In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A. Bioconjug Chem, 2002 Jan-Feb, 13(1), 97 - 103 An efficient binding chemistry for glass polynucleotide microarrays; Lee PH et al.; A variety of methods have been described for making synthetic polynucleotide microarrays . These include in situ synthesis directly on the array surface, for example, by photolithography or ink-jet printing technologies, and the application of presynthesized polynucleotides that are derivatized with various nucleophiles or electrophiles . In the latter case, a variety of surface chemistries have been developed, and several are available commercially . These chemistries must be compatible with nanoliter-scale volumes of polynucleotide reagents, which contact the array over a small portion of their surface . We reasoned that a three-dimensional polymer coating could potentially offer greater surface contact and higher binding efficiency . Here we describe a polyethylenimine-based coating chemistry that provides exceptional binding and hybridization characteristics . In our preferred process, size-fractionated polyethylenimine polymers are cross-linked onto an aminopropylsilanated glass surface in the presence of cyanuric chloride . The resulting three-dimensional coating binds polynucleotides through a mixture of covalent and noncovalent interactions as evidenced by comparisons between 5'-aminoalkyl modified and unmodified polynucleotides . Binding and hybridization comparisons are presented including analogous two-dimensional electrophilic and electrostatic chemistries. Biosci Biotechnol Biochem, 2001 Nov, 65(11), 2465 - 71 Substrate recognition mechanism of carboxypeptidase Y; Nakase H et al.; To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu)n Ala-OH (n = 1 to 6), Fmoc-(Glu)n Ala-NH2 (1 to 5), and Fmoc-Lys(Glu)3Ala-NH2 were synthesized, and kinetic parameters for these substrates were measured . Km for Fmoc-peptides significantly decreased as peptide length increased from n = 1 to n = 5 with only slight changes in kcat . Km for Fmoc-(Glu)(5,6)Ala-OH were almost the same as one for protein substrates described previously (Nakase et al., Bull . Chem . Soc . Jpn., 73, 2587-2590) . These results show that the enzyme has six subsites (S1' and S1-S5) . Each subsite affinity calculated from the Km revealed subsite properties, and from the differences of subsite affinity between pH 6.5 and 5.0, the residues in each subsite were predicted . For Fmoc-peptide amide substrates, the priorities of amidase and carboxamide peptidase activities were dependent on the substrate . It is likely that the interactions between side chains of peptide and subsites compensate for the lack of P1'-S1' interaction, so the amidase activity prevailed for Fmoc-(Glu)(3,5)Ala-NH2 . These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network. Genome Inform Ser Workshop Genome Inform, 2001, 12, 123 - 34 The potential use of SUISEKI as a protein interaction discovery tool; Blaschke C et al.; Relevant information about protein interactions is stored in textual sources . This sources are commonly used not only as archives of what is already known but also as information for generating new knowledge, particularly to pose hypothesis about new possible interactions that can be inferred from the existing ones . This task is the more creative part of scientific work in experimental systems . We present a large-scale analysis for the prediction of new interactions based on the interaction network for the ones already known and detected automatically in the literature . During the last few years it has became clear that part of the information about protein interactions could be extracted with automatic tools, even if these tools are still far from perfect and key problems such as detection of protein names are not completely solved . We have developed a integrated automatic approach, called SUISEKI (System for Information Extraction on Interactions), able to extract protein interactions from collections of Medline abstracts . Previous experiments with the system have shown that it is able to extract almost 70% of the interactions present in relatively large text corpus, with an accuracy of approximately 80% (for the best defined interactions) that makes the system usable in real scenarios, both at the level of extraction of protein names and at the level of extracting interaction between them . With the analysis of the interaction map of Saccharomyces cerevisiae we show that interactions published in the years 2000/2001 frequently correspond to proteins or genes that were already very close in the interaction network deduced from the literature published before these years and that they are often connected to the same proteins . That is, discoveries are commonly done among highly connected entities . Some biologically relevant examples illustrate how interactions described in the year 2000 could have been proposed as reasonable working hypothesis with the information previously available in the automatically extracted network of interactions. Genome Inform Ser Workshop Genome Inform, 2001, 12, 24 - 33 Minimum spanning trees for gene expression data clustering; Xu Y et al.; This paper describes a new framework for microarray gene-expression data clustering . The foundation of this framework is a minimum spanning tree (MST) representation of a set of multi-dimensional gene expression data . A key property of this representation is that each cluster of the expression data corresponds to one subtree of the MST, which rigorously converts a multi-dimensional clustering problem to a tree partitioning problem . We have demonstrated that though the inter-data relationship is greatly simplified in the MST representation, no essential information is lost for the purpose of clustering . Two key advantages in representing a set of multi-dimensional data as an MST are: (1) the simple structure of a tree facilitates efficient implementations of rigorous clustering algorithms, which otherwise are highly computationally challenging; and (2) as an MST-based clustering does not depend on detailed geometric shape of a cluster, it can overcome many of the problems faced by classical clustering algorithms . Based on the MST representation, we have developed a number of rigorous and efficient clustering algorithms, including two with guaranteed global optimality . We have implemented these algorithms as a computer software EXCAVATOR . To demonstrate its effectiveness, we have tested it on two data sets, i.e., expression data from yeast Saccharomyces cerevisiae, and Arabidopsis expression data in response to chitin elicitation. J Biol Chem, 2002 Mar 29, 277(13), 11026 - 33 Epub 2002 Jan 14. Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p; Faber KN et al.; We show that the synthesis of the N-terminal 50 amino acids of Pex3p (Pex3p(1-50)) in Hansenula polymorpha pex3 cells is associated with the formation of vesicular membrane structures . Biochemical and ultrastructural findings suggest that the nuclear membrane is the donor membrane compartment of these vesicles . These structures also contain Pex14p and can develop into functional peroxisomes after subsequent reintroduction of the full-length Pex3p protein . We discuss the significance of this finding in relation to peroxisome reintroduction, e.g . in case peroxisomes are lost due to failure in inheritance. Curr Opin Genet Dev, 2002 Feb, 12(1), 73 - 9 SWI/SNF chromatin remodeling and cancer; Klochendler-Yeivin A et al.; The SWI/SNF complex contributes to the regulation of gene expression by altering the chromatin structure . Depending on the context, it can be involved in either transcriptional activation or repression . Growing genetic and molecular evidence indicate that subunits of the SWI/SNF complex act as tumor suppressors in human and mice . Results from biochemical and transfection studies suggest also that SWI/SNF participates either in the inhibition or activation of several oncogenes and tumor suppressor genes and/or control their transcriptional activity . These activities provide molecular insight into the mechanism underlying SWI/SNF function in tumor suppression. Curr Biol, 2002 Jan 8, 12(1), R18 - 20 Membrane traffic: exocyst III--makes a family; Short B et al.; Although many factors have been implicated in the docking steps that precede vesicle fusion with a target membrane, few similarities have been found between them . New evidence suggests that at least some of these factors form related multimeric complexes that may help to explain the mechanism of vesicle docking. Yeast, 2002 Jan 30, 19(2), 99 - 114 Phylogenetic footprinting reveals multiple regulatory elements involved in control of the meiotic recombination gene, REC102; Jiao K et al.; REC102 is a meiosis-specific early exchange gene absolutely required for meiotic recombination in Saccharomyces cerevisiae . Sequence analysis of REC102 indicates that there are multiple potential regulatory elements in its promoter region, and a possible regulatory element in the coding region . This suggests that the regulation of REC102 may be complex and may include elements not yet reported in other meiotic genes . To identify potential cis-regulatory elements, phylogenetic footprinting analysis was used . REC102 homologues were cloned from other two Saccharomyces spp . and sequence comparison among the three species defined evolutionarily conserved elements . Deletion analysis demonstrated that the early meiotic gene regulatory element URS1 was necessary but not sufficient for proper regulation of REC102 . Upstream elements, including the binding sites for Gcr1p, Yap1p, Rap1p and several novel conserved sequences, are also required for the normal regulation of REC102 as well as a Rap1p binding site located in the coding region . The data in this paper support the use of phylogenetic comparisions as a method for determining important sequences in complex promoters . Nat Cell Biol, 2002 Feb, 4(2), 117 - 23 A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies; Reggiori F et al.; Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole . Sorting of some, although not all, proteins into these structures is mediated by ubiquitination . We have identified a transmembrane ubiquitin ligase, Tul1, that is resident in the Golgi apparatus and is required for the ubiquitination of proteins with polar TMDs, including vacuolar proteins such as carboxypeptidase S . We suggest that Tul1 provides quality control, identifying misfolded membrane proteins and marking them for transport to endosomes and degradation in the vacuole. Plant Physiol, 2002 Jan, 128(1), 165 - 72 Effects of phosphorylation on phosphoenolpyruvate carboxykinase from the C4 plant Guinea grass; Walker RP et al.; In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves . To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves . The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms . In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate . The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations . Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass . Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity . Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves . There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation. J Biol Chem, 2002 Mar 22, 277(12), 9929 - 35 Epub 2002 Jan 11. Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress; Ayene IS et al.; Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative pentose phosphate cycle, regulates the NADPH/NADP(+) ratio in eukaryotic cells . G6PD deficiency is one of the most common mutations in humans and is known to cause health problems for hundreds of millions worldwide . Although it is known that decreased G6PD functionality can result in increased susceptibility to oxidative stress, the molecular targets of this stress are not known . Using a Chinese hamster ovary G6PD-null mutant, we previously demonstrated that exposure to a thiol-specific oxidant, hydroxyethyldisulfide, caused enhanced radiation sensitivity and an inability to repair DNA double strand breaks . We now demonstrate a molecular mechanism for these observations: the direct inhibition of DNA end binding activity of the Ku heterodimer, a DNA repair protein, by oxidation of its cysteine residues . Inhibition of Ku DNA end binding was found to be reversible by treatment of the nuclear extract with dithiothreitol, suggesting that the homeostatic regulation of reduced cysteine residues in Ku is a critical function of G6PD and the oxidative pentose cycle . In summary, we have discovered a new layer of DNA damage repair, that of the functional maintenance of repair proteins themselves . In view of the rapidly escalating number of roles ascribed to Ku, these results may have widespread ramifications. J Biol Chem, 2002 Apr 5, 277(14), 12190 - 9 Epub 2002 Jan 10. Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein; Lindsay AJ et al.; Rab4 and Rab11 are small GTPases belonging to the Ras superfamily . They both function as regulators along the receptor recycling pathway . We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11 . We name this protein Rab coupling protein (RCP) . RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested . It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor . Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment . Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays . RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family. Am J Pathol, 2002 Jan, 160(1), 247 - 54 Potent mitogenicity of the RET/PTC3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma; Basolo F et al.; The tall-cell variant (TCV) of papillary thyroid carcinoma (PTC), characterized by tall cells bearing an oxyphilic cytoplasm, is more clinically aggressive than conventional PTC . RET tyrosine kinase rearrangements, which represent the most frequent genetic alteration in PTC, lead to the recombination of RET with heterologous genes to generate chimeric RET/PTC oncogenes . RET/PTC1 and RET/PTC3 are the most prevalent variants . We have found RET rearrangements in 35.8% of TCV (14 of 39 cases) . Whereas the prevalences of RET/PTC1 and RET/PTC3 were almost equal in classic and follicular PTC, all of the TCV-positive cases expressed the RET/PTC3 rearrangement . These findings prompted us to compare RET/PTC3 and RET/PTC1 in an in vitro thyroid model system . We have expressed the two oncogenes in PC Cl 3 rat thyroid epithelial cells and found that RET/PTC3 is endowed with a strikingly more potent mitogenic effect than RET/PTC1 . Mechanistically, this difference correlated with an increased signaling activity of RET/PTC3 . In conclusion, we postulate that the correlation between the RET/PTC rearrangement type and the aggressiveness of human PTC is related to the efficiency with which the oncogene subtype delivers mitogenic signals to thyroid cells. J Theor Biol, 2002 Jan 7, 214(1), 85 - 97 Analysis of the kinetic and equilibrium binding of Ku protein to DNA; Taghva A et al.; The loading of Ku onto a DNA end in a double-strand DNA break is thought to be one of the first steps in the non-homologous DNA end joining (NHEJ) pathway, giving it an essential role in the maintenance of genomic integrity . The binding of Ku to DNA is complicated since DNA can accommodate multiple Ku subunits, which can translocate on the DNA strand . Furthermore, Ku may exhibit cooperativity in the loading process . Therefore, simple one- to-one kinetic models are unable to adequately simulate the process . However, through the use of computer simulation and curve-fitting, we are able to provide a comprehensive mechanistic model and rate constants that closely approximate experimental data for DNA molecules that bind one, two, and three Ku molecules under both kinetic and equilibrium conditions . The model obtains a best fit with Ku having a roughly seven-fold preference to bind to DNA ends rather than internal positions and is consistent with Ku having a strong preference of which face of the protein loads onto the DNA end . Plant Mol Biol, 2001 Dec, 47(6), 771 - 83 Identification of a S-ribonuclease-binding protein in Petunia hybrida; Sims TL et al.; To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein . These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease . Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific . Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain . Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions . Together, these results suggest that PhSBPI may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases. Mol Cell Biol, 2002 Feb, 22(3), 835 - 48 Role of the Sin3-histone deacetylase complex in growth regulation by the candidate tumor suppressor p33(ING1); Kuzmichev A et al.; Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2 . Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure . We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18 . SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD . SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes . We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein . Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex . The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30 . The N-terminal domain of p33 is present in several uncharacterized human proteins . We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain. Mol Cell Biol, 2002 Feb, 22(3), 737 - 49 Direct activation of mitogen-activated protein kinase kinase kinase MEKK1 by the Ste20p homologue GCK and the adapter protein TRAF2; Chadee DN et al.; Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues . The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood . Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) . These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive . Here we show that endogenous GCK and MEKK1 associate in vivo . In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1 . The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not . Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation . Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo . These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins. Mol Cell Biol, 2002 Feb, 22(3), 693 - 703 Histone-dependent association of Tup1-Ssn6 with repressed genes in vivo; Davie JK et al.; The Tup1-Ssn6 complex regulates diverse classes of genes in Saccharomyces cerevisiae and serves as a model for corepressor functions in many organisms . Tup1-Ssn6 does not directly bind DNA but is brought to target genes through interactions with sequence-specific DNA binding factors . Full repression by Tup1-Ssn6 appears to require interactions with both the histone tails and components of the general transcription machinery, although the relative contribution of these two pathways is not clear . Here, we map Tup1 locations on two classes of Tup1-Ssn6-regulated genes in vivo via chromatin immunoprecipitations . Distinct profiles of Tup1 are observed on a cell-specific genes and DNA damage-inducible genes, suggesting that alternate repressive architectures may be created on different classes of repressed genes . In both cases, decreases in acetylation of histone H3 colocalize with Tup1 . Strikingly, although loss of the Srb10 mediator protein had no effect on Tup1 localization, both histone tail mutations and histone deacetylase mutations crippled the association of Tup1 with target loci . Together with previous findings that Tup1-Ssn6 physically associates with histone deacetylase activities, these results indicate that the repressor complex alters histone modification states to facilitate interactions with histones and that these interactions are required to maintain a stable repressive state. Eur J Biochem, 2002 Jan, 269(1), 128 - 38 Human and Drosophila UDP-galactose transporters transport UDP-N-acetylgalactosamine in addition to UDP-galactose; Segawa H et al.; A putative Drosophila nucleotide sugar transporter was characterized and shown to be the Drosophila homologue of the human UDP-Gal transporter (hUGT) . When the Drosophila melanogaster UDP-Gal transporter (DmUGT) was expressed in mammalian cells, the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport deficiency of Lec8 cells but not the CMP-Sia transport deficiency of Lec2 cells . DmUGT and hUGT were expressed in Saccharomyces cerevisiae cells in functionally active forms . Using microsomal vesicles isolated from Saccharomyces cerevisiae expressing these transporters, we unexpectedly found that both hUGT and DmUGT could transport UDP-GalNAc as well as UDP-Gal . When amino-acid residues that are conserved among human, murine, fission yeast and Drosophila UGTs, but are distinct from corresponding ones conserved among CMP-Sia transporters (CSTs), were substituted by those found in CST, the mutant transporters were still active in transporting UDP-Gal . One of these mutants in which Asn47 was substituted by Ala showed aberrant intracellular distribution with concomitant destabilization of the protein product . However, this mutation was suppressed by an Ile51 to Thr second-site mutation . Both residues were localized within the first transmembrane helix, suggesting that the structure of the helix contributes to the stabilization and substrate recognition of the UGT molecule. Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 333 - 8 Amino acid runs in eukaryotic proteomes and disease associations; Karlin S et al.; We present a comparative proteome analysis of the five complete eukaryotic genomes (human, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Arabidopsis thaliana), focusing on individual and multiple amino acid runs, charge and hydrophobic runs . We found that human proteins with multiple long runs are often associated with diseases; these include long glutamine runs that induce neurological disorders, various cancers, categories of leukemias (mostly involving chromosomal translocations), and an abundance of Ca(2 +) and K(+) channel proteins . Many human proteins with multiple runs function in development and/or transcription regulation and are Drosophila homeotic homologs . A large number of these proteins are expressed in the nervous system . More than 80% of Drosophila proteins with multiple runs seem to function in transcription regulation . The most frequent amino acid runs in Drosophila sequences occur for glutamine, alanine, and serine, whereas human sequences highlight glutamate, proline, and leucine . The most frequent runs in yeast are of serine, glutamine, and acidic residues . Compared with the other eukaryotic proteomes, amino acid runs are significantly more abundant in the fly . This finding might be interpreted in terms of innate differences in DNA-replication processes, repair mechanisms, DNA-modification systems, and mutational biases . There are striking differences in amino acid runs for glutamine, asparagine, and leucine among the five proteomes. EMBO J, 2002 Jan 15, 21(1-2), 181 - 93 Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation; Janke C et al.; We show here that Ask1p, Dad2p, Spc19p and Spc34p are subunits of the budding yeast Duo1p-Dam1p- Dad1p complex, which associate with kinetochores and localize along metaphase and anaphase spindles . Analysis of spc34-3 cells revealed three novel functions of the Duo1-Dam1p-Dad1p subunit Spc34p . First, SPC34 is required to establish biorientation of sister kinetochores . Secondly, SPC34 is essential to maintain biorientation . Thirdly, SPC34 is necessary to maintain an anaphase spindle independently of chromosome segregation . Moreover, we show that in spc34-3 cells, sister centromeres preferentially associate with the pre-existing, old spindle pole body (SPB) . A similar preferential attachment of sister centromeres to the old SPB occurs in cells depleted of the cohesin Scc1p, a protein with a known role in facilitating biorientation . Thus, the two SPBs are not equally active in early S phase . We suggest that not only in spc34-3 and Deltascc1 cells but also in wild-type cells, sister centromeres bind after replication preferentially to microtubules organized by the old SPB . Monopolar attached sister centromeres are resolved to bipolar attachment in wild-type cells but persist in spc34-3 cells. EMBO J, 2002 Jan 15, 21(1-2), 175 - 80 A role for RAD54B in homologous recombination in human cells; Miyagawa K et al.; In human somatic cells, homologous recombination is a rare event . To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells . Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells . Here, we report that RAD54B plays a critical role in targeted integration in human cells . Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency . Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells . Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54 . In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54 . Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans. EMBO J, 2002 Jan 15, 21(1-2), 12 - 21 Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants; Hattendorf DA et al.; AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes . AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis . Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis . Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state . In vitro, reducing k(cat) at NBD2 significantly alters the steady-state kinetic behavior of NBD1 . Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2 . In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 function. Cancer Res, 2002 Jan 1, 62(1), 67 - 74 Deregulation of polyamine biosynthesis alters intrinsic histone acetyltransferase and deacetylase activities in murine skin and tumors; Hobbs CA et al.; The essential requirement for polyamines for normal cell growth and differentiation may be partly attributed to their influence on gene expression, a process regulated by the acetylation state of nucleosomal histones . We used transgenic mice to examine the effects of constitutive expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on histone acetylation in epithelial cells in skin . As compared with the skin of normal littermate mice, both intrinsic histone acetyltransferase (HAT) and deacetylase activities are elevated in ODC transgenic skin . Skin tumors that form spontaneously in ODC/Ras double transgenic mice exhibit exceptionally high HAT activity having a distinct specificity for Lys-12 in the tail domain of histone H4, which may have implications for gene transcription . However, acetylation of histones by HAT enzymes was impeded in cultured ODC transgenic keratinocytes, and there were only modest changes in levels of acetylated histones in the skin of ODC transgenic mice . Treatment with the ODC enzyme inhibitor alpha-difluoromethylornithine, which results in regression of ODC/Ras tumors, reverses the effects on HAT and deacetylase enzyme function, implicating polyamine biosynthesis in the regulation of histone acetylation . Polyamines do not directly stimulate the enzymatic activity of either p300 or p300/CREB-binding protein (CBP)-associated factor, members of two distinct classes of HAT enzymes, implying that the elevated CBP/p300-associated HAT activity detected in ODC transgenic skin is attributable to indirect influence of polyamines . These results suggest that multiple mechanisms exist by which endogenous polyamines influence chromatin in mammals . Furthermore, they suggest that the elevated polyamine levels inherent in many solid tumors alter chromatin structure, likely affecting gene expression and promoting the neoplastic process. Dev Cell, 2002 Jan, 2(1), 9 - 19 Model organisms as a guide to mammalian aging; Tissenbaum HA et al.; Recent studies on aging in model systems such as yeast and roundworms have revealed conserved regulation of the process in response to nutrient availability and specific genes that appear to mediate this regulation . Here we review these findings with a focus on the nematode Caenorhabditis elegans and the budding yeast Saccharomyces cerevisiae and highlight general features of the regulation of aging that may have implications for mammals. Oncogene, 2001 Dec 6, 20(56), 8116 - 24 Crosstalk between Myc and activating transcription factor 2 (ATF2): Myc prolongs the half-life and induces phosphorylation of ATF2; Miethe J et al.; Myc is a key regulator of cell growth, differentiation and apoptosis, and affects cell fate decisions by activating as well as by inhibiting the expression of cellular genes . Myc is a member of the basic region-helix-loop-helix-leucine zipper (b-HLH-Zip) class of transcription factors, which heterodimerizes with the Max protein and recognizes a consensus Myc binding motif . Stimulation of gene expression by Myc is thought to be mediated by direct binding of Myc-Max heterodimers to specific target genes . So far, only a few genes have been identified as direct binding targets of Myc, raising the possibility that Myc affects gene expression also by indirect mechanisms . In this work we present evidence that v-Myc encoded by the avian retrovirus MC29 stimulates activating transcription factor 2 (ATF2)-dependent transcription . Analysis of the effect of Myc on ATF2 shows that v-Myc prolongs the half-life of ATF2 and induces the phosphorylation of N-terminal sites of ATF2 (Thr-69 and Thr-71) which have previously been identified as the target sites of stress-activated protein kinases and implicated in the regulation of ATF2 activity . Taken together, our results suggest that v-Myc can affect gene expression indirectly by modulating the activity of ATF2. Eur J Hum Genet, 2001 Nov, 9(11), 823 - 8 Analysis of TSC2 stop codon variants found in tuberous sclerosis patients; Goedbloed MA et al.; Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations to the TSC1 and TSC2 tumour suppressor genes . We detected two sequence changes involving the TSC2 stop codon and investigated the effects of these changes on the expression of tuberin, the TSC2 gene product, and on the binding between tuberin and the TSC1 gene product, hamartin . While elongation of the tuberin open reading frame by 17 amino acids did not interfere with tuberin-hamartin binding, a longer extension prevented this interaction . Our data illustrate how functional protein assays can assist in the verification and characterisation of disease-causing mutations. Nat Cell Biol, 2001 Dec, 3(12), 1086 - 91 Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly; Hetzer M et al.; Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized . NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion . Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly . Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion . Subsequent NE growth involves a p97-p47 complex . This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation. J Cell Biol, 2002 Jan 7, 156(1), 35 - 9 Epub 2002 Jan 07. Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length; Schott DH et al.; Myosins are molecular motors that exert force against actin filaments . One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells . However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs . Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis . The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene . Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model. J Biol Chem, 2002 Mar 15, 277(11), 9342 - 50 Epub 2002 Jan 07. Inhibition of transforming growth factor beta signaling and Smad-dependent activation of transcription by the Latent Membrane Protein 1 of Epstein-Barr virus; Prokova V et al.; Inhibition of transforming growth factor beta (TGFbeta) signaling by the Epstein-Barr virus Latent Membrane Protein 1 (LMP1) may account, at least in part, for the oncogenic activity of LMP1 . We found that LMP1 is a potent inhibitor of TGFbeta signaling and Smad-dependent activation of transcription in 293 epithelial cells and COS-7 fibroblasts . LMP1 strongly inhibited the uninduced and the Smad-inducible activity of the promoters of the human p21/WAF1/Cip1 gene and the mouse Smad7 gene . Inhibition of TGFbeta signaling and Smad-dependent activation of transcription by LMP1 was greatly reduced by deletion of both C-terminal activating regions 1 and 2 of LMP1 as well as by overexpression of a non-degradable form of IkappaB . In contrast, specific inhibitors of p38 kinase or MEK kinase did not reverse the inhibitory activity of LMP1 . TGFbeta signaling was enhanced by overexpression of dominant negative forms of the LMP1 effectors TRAF2, NIK, and IKKbeta and was abolished by overexpression of p65/RelA or a p50/p65 fusion protein . Deletion of the transactivation domain of p65 abolished its inhibitory activity . Immunoblotting and immunofluorescence microscopy indicated that suppression of TGFbeta signaling and Smad transcriptional activity by LMP1 was not due to Smad degradation or cytoplasmic retention suggesting that LMP1 affects the nuclear function of Smad proteins . Our data are consistent with an essential role of NF-kappaB activation by LMP1 in the inhibition of TGFbeta signaling and Smad-mediated transcriptional responses. Gastroenterology, 2002 Jan, 122(1), 211 - 9 Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system; Trojan J et al.; BACKGROUND & AIMS: Germline mutations in the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 are associated with susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC) . Because a significant proportion of hMLH1 mutations are missense, the assessment of their pathogenic role may be difficult . To date, functional analysis of missense mutations has been performed primarily in Saccharomyces cerevisiae . The aim of this study was to examine the biochemical properties of hMLH1 protein variants in a human expression system . METHODS: The HNPCC-related hMLH1 mutations T117M, V185G, R217C, G244D, R265C, V326A, and K618T, the polymorphisms I219V and R265H, and a hMLH1 splicing variant lacking exon 9 and 10 (hMLH1 Delta 9/10) were cloned . On transfection of these constructs into human 293T cells, which do not express hMLH1 because of promoter hypermethylation, the hMLH1 protein variants were analyzed by Western blotting and in a MMR assay . RESULTS: Transfection was successful for all hMLH1 constructs . As anticipated, the mutations K618T and T117M, which affect the highly conserved domains of hMLH1 that are necessary for interaction with hPMS2 or for adenosine triphosphate (ATP) binding, respectively, affected protein stability or its ability to complement MMR-deficient 293T-cell extracts . The V185G, G244D, and Delta 9/10 variants were also unable to complement MMR in 293T cells, whereas hMLH1 proteins carrying the I219V, R265H, R265C, R217C, and V326A mutations were MMR competent . CONCLUSIONS: These data show that the pathogenic role of hMLH1 missense mutations and splicing variants can be assessed by analyzing the biochemical properties of their protein products in a homologous expression system. Biochemistry, 2002 Jan 15, 41(2), 579 - 87 Eukaryotic initiation factor 2 alpha subunit associates with TGF beta receptors and 14-3-3 epsilon and acts as a modulator of the TGF beta response; McGonigle S et al.; Schistosoma mansoni receptor kinase 1 (SmRK1) is a divergent member of the TGF beta receptor family . Intracellular proteins that associate with these receptors are likely to play an important role in signaling . 14-3-3 epsilon is a previously described cytoplasmic protein, which associates with both SmRK1 and the human type I TGF beta receptor (T beta RI); overexpression of 14-3-3 epsilon leads to enhanced TGF beta-mediated signaling by T beta RI . We now describe the identification of S . mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2 alpha), through its interaction with SmRK1 in a yeast two-hybrid assay . S . mansoni eIF2 alpha also interacts with human TGF beta