Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 1064 - 9 The tomato Blind gene encodes a MYB transcription factor that controls the formation of lateral meristems; Schmitz G et al.; The multitude of forms observed in flowering plants is largely because of their ability to establish new axes of growth during postembryonic development . This process is initiated by the formation of secondary meristems that develop into vegetative or reproductive branches . In the blind and torosa mutants of tomato, initiation of lateral meristems is blocked during shoot and inflorescence development, leading to a strong reduction in the number of lateral axes . In this study, it is shown that blind and torosa are allelic . The Blind gene has been isolated by positional cloning, and it was found that the mutant phenotype is caused by a loss of function of an R2R3 class Myb gene . RNA interference-induced blind phenocopies confirmed the identity of the isolated gene . Double mutant analysis shows that Blind acts in a novel pathway different from the one to which the previously identified Lateral suppressor gene belongs . The findings reported add a new class of transcription factors to the group of genes controlling lateral meristem initiation and reveal a previously uncharacterized function of R2R3 Myb genes. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1347 - 52 Epub 2002 Jan 22. Analysis of Cdc6 function in the assembly of mammalian prereplication complexes; Cook JG et al.; Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex . Although considerable insight has been gained from in vitro studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking . We have made use of adenoviral vectors to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function . We demonstrate that Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low . Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells . Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6 . Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication . Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication . Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6. J Pharmacol Exp Ther, 2002 Feb, 300(2), 399 - 407 Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate; Li XQ et al.; Amodiaquine (AQ) metabolism to N-desethylamodiaquine (DEAQ) is the principal route of disposition in humans . Using human liver microsomes and two sets of recombinant human cytochrome P450 isoforms (from lymphoblastoids and yeast) we performed studies to identify the CYP isoform(s) involved in the metabolism of AQ . CYP2C8 was the main hepatic isoform that cleared AQ and catalyzed the formation of DEAQ . The extrahepatic P450s, 1A1 and 1B1, also cleared AQ and catalyzed the formation of an unknown metabolite M2 . The K(m) and V(max) values for AQ N-desethylation were 1.2 microM and 2.6 pmol/min/pmol of CYP2C8 for recombinant CYP2C8, and 2.4 microM and 1462 pmol/min/mg of protein for human liver microsomes (HLMs), respectively . Relative contribution of CYP2C8 in the formation of DEAQ was estimated at 100% using the relative activity factor method . Correlation analyses between AQ metabolism and the activities of eight hepatic P450s were made on 10 different HLM samples . Both the formation of DEAQ and the clearance of AQ showed excellent correlations (r(2) = 0.98 and 0.95) with 6alpha-hydroxylation of paclitaxel, a marker substrate for CYP2C8 . The inhibition of DEAQ formation by quercetin was competitive with K(i) values of 1.96 for CYP2C8 and 1.56 microM for HLMs . Docking of AQ into the active site homology models of the CYP2C isoforms showed favorable interactions with CYP2C8, which supported the likelihood of an N-desethylation reaction . These data show that CYP2C8 is the main hepatic isoform responsible for the metabolism of AQ . The specificity, high affinity, and high turnover make AQ desethylation an excellent marker reaction for CYP2C8 activity. J Biol Chem, 2002 Apr 5, 277(14), 12359 - 63 Epub 2002 Jan 22. Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95; Choi J et al.; Stargazin is the first transmembrane protein known to associate with AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins . However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated . Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif . The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA) . Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo . Stargazin phosphorylation is enhanced by the catalytic subunit of PKA . Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitro coimmunoprecipitation, and coclustering between stargazin and PSD-95 . Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain . These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs. J Biol Chem, 2002 Apr 5, 277(14), 12351 - 8 Epub 2002 Jan 22. Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST); Lee KK et al.; Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis . However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization . In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner . Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import . When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST . Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus . Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus . However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation . In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage . Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage . Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology. J Biol Chem, 2002 May 3, 277(18), 16179 - 88 Epub 2002 Jan 22. Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes; Motz M et al.; Thermostable DNA polymerases are an important tool in molecular biology . To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus . To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points . This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors . Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors . In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue . This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA . Indeed, addition of A . fulgidus PCNA resulted in marked stimulation of PCR product generation . The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase . Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA . This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity. J Biol Chem, 2002 Mar 15, 277(11), 8771 - 4 Epub 2002 Jan 22. A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase; Cao H et al.; Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl . To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions . A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl . We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk) . TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation . In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells . Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae . This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae. Am J Physiol Gastrointest Liver Physiol, 2002 Feb, 282(2), G375 - 81 Cloning of human agmatinase . An alternate path for polyamine synthesis induced in liver by hepatitis B virus; Mistry SK et al.; Agmatinase, which hydrolyzes agmatine to putrescine and urea, not only represents a potentially important mechanism for regulating the biological effects of agmatine in mammalian cells but also represents an alternative to ornithine decarboxylase for polyamine biosynthesis . We have isolated a full-length cDNA encoding human agmatinase whose function was confirmed by complementation in yeast . The single-copy human agmatinase gene located on chromosome 1 encodes a 352-residue protein with a putative mitochondrial targeting sequence at the NH(3)-terminus . Human agmatinase has about 30% identity to bacterial agmatinases and <20% identity to mammalian arginases . Residues required for binding of Mn(2+) at the active site in bacterial agmatinase and other members of the arginase superfamily are fully conserved in human agmatinase . Agmatinase mRNA is most abundant in human liver and kidney but also is expressed in several other tissues, including skeletal muscle and brain . Its expression in human liver is induced during hepatitis B virus infection, suggesting that agmatinase may play a role in the pathophysiology of this disease. Mol Cell, 2002 Jan, 9(1), 45 - 57 Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR; Fischle W et al.; Histone deacetylases (HDACs) play a key role in regulating eukaryotic gene expression . The HDAC domain, homologous to the yeast repressors RPD3 and HDA1, is considered necessary and sufficient for enzymatic activity . Here, we show that the catalytic domain of HDAC4 interacts with HDAC3 via the transcriptional corepressor N-CoR/SMRT . All experimental conditions leading to the suppression of HDAC4 binding to SMRT/N-CoR and to HDAC3 result in the loss of enzymatic activity associated with HDAC4 . In vitro reconstitution experiments indicate that HDAC4 and other class II HDACs are inactive in the context of the SMRT/N-CoR-HDAC3 complex and do not contribute to its enzymatic activity . These observations indicate that class II HDACs regulate transcription by bridging the enzymatically active SMRT/N-CoR-HDAC3 complex and select transcription factors independently of any intrinsic HDAC activity. Mol Cells, 2001 Dec 31, 12(3), 329 - 35 In vitro function of S rnases in Lycopersicon peruvianum; Kim MH et al.; S RNases are products of the S locus that are expressed in the stylar tissue of Lycopersicon peruvianum with the gametophytic self-incompatibility (SI) system . Two S RNases (S12 and Sa) with RNase activity from the S12Sa genotype of L . peruvianum were purified using gel filtration and cation-exchange chromatography . The molecular masses of the two RNases, S12 and Sa, were 21 and 23.1 kDa, respectively . The specific activities of S12 and Sa for torula yeast rRNA as a substrate were 8,500 and 6,000 units/ml, respectively . Of various reagents tested for RNase activities, ZnSO4 and CuSO4 were found to remarkably reduce its activity . The growth of S12Sa pollen was inhibited when it was cultured in a pollen germination medium that contained the purified S12 RNase . The result suggested that the S RNase was either a probable inhibitor of pollen growth or controlled pollen growth . Additionally, 512Sa pollens germinated well in vitro in a germination medium that contained S12 RNase in the presence of ZnSO4 and CuSO4 . Our finding suggests that the treatment of S RNase with its inhibitor destroys the SI ability on an in vitro self-pollen growth test. Mol Cells, 2001 Dec 31, 12(3), 304 - 12 A novel leucine-rich repeat protein (LRR-1): potential involvement in 4-1BB-mediated signal transduction; Jang LK et al.; 4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is induced on CD4+ and CD8+ T cells upon engagement of the T cell receptor (TCR)/ CD3 complex with the antigen bound to MHC . 4-1BB plays an important role in transmitting costimulatory signal during T cell activation . However, 4-1BB-mediatded signal transduction pathways have remained elusive . We conducted the yeast two-hybrid screening to identify intracellular signaling molecules that associate with 4-1BB . A novel leucine-rich repeat (LRR)-containing protein, herein named LRR-1, was found to specifically interact with the cytoplasmic domain of 4-1BB . Overexpression of LRR-1 suppressed the activation of NF-KB induced by 4-1BB or TNF receptor-associated factor (TRAF) 2 . In addition, LRR-1 down-regulated JNK1 activity was induced by 4-1BB . These results indicate that LRR-1 negatively regulates the 4-1BB-mediated signaling cascades which result in the activation of NF-kappaB and JNK1. Mol Cells, 2001 Dec 31, 12(3), 298 - 303 Mapping of the interaction sites between apoptosis linked gene ALG-2 and HEED; Lee KH et al.; The apoptosis linked gene (ALG-2) is a 22 kDa Ca2+-binding protein of the penta EF-hand motif family . ALG-2 was discovered in a "death trap" assay using T-cell receptor-mediated apoptosis . Depletion of ALG-2 using an anti-sense ALG-2 construct inhibits apoptosis that is induced by several stimuli, such as staurosporin, dexamethason, Fas, and glucocorticoid . The Ca2+-dependent function of ALG-2 is consistent with the observation that the cytoplasmic Ca2+ level is elevated in apoptotic cells . We found that ALG-2 interacted specifically with the carboxy-terminal region of HEED using a yeast two-hybrid assay system . The mutants of HEED were constructed by deleting five WD repeat motifs one by one from the C-terminus of the protein . These mutants of ALG-2 were made by combining the EF hand Ca2+ binding motifs in various ways . Mapping of the interaction sites, using each of the mutants, revealed that the interaction between HEED and a third EF-hand motif of ALG-2 was stronger than the other combination. Ophthalmic Genet, 2001 Dec, 22(4), 207 - 23 Neuronal cell death in the visual cortex is a prominent feature of the X-linked recessive mitochondrial deafness-dystonia syndrome caused by mutations in the TIMM8a gene; Tranebjaerg L et al.; The Mohr-Tranebjaerg syndrome (MIM 304700) and the Jensen syndrome (MIM 311150) were previously reported as separate X-linked recessive deafness syndromes associated with progressive visual deterioration, dystonia, dementia, and psychiatric abnormalities . In the most extensively studied Norwegian family, the Mohr-Tranebjaerg syndrome was reported to be caused by a one-basepair deletion (151delT) in the deafness/dystonia peptide (DDP) gene at Xq22 . This gene has been renamed TIMM8a . We identified a stop mutation (E24X) in the TIMM8a gene segregating with the disease in the original Danish family with the Jensen syndrome, which confirms that the two disorders are allelic conditions . We also report abnormal VEP examinations and neuropathological abnormalities in affected males from the two unrelated families with different mutations . The findings included neuronal cell loss in the optic nerve, retina, striate cortex, basal ganglia, and dorsal roots of the spinal cord . The demonstration of mitochondrial abnormalities in skeletal muscle biopsies in some patients is compatible with the suggestion from recent research that the TIMM8a protein is the human counterpart of an intermembrane mitochondrial transport protein, Tim8p, recently characterized in yeast . The clinical and neuropathological abnormalities associated with mutations in the TIMM8a gene support that this X-linked deafness-dystonia-optic neuropathy syndrome is an example of progressive neurodegeneration due to mutations in a nuclear gene necessary for some, yet unknown mitochondrial transport function . We recommend sequencing the TIMM8a gene, thorough ophthalmological examination, and measuring visual evoked potentials in clinically suspected male patients with either progressive hearing impairment, dystonia, or visual disability in order to establish an early diagnosis and provide appropriate genetic counselling. Biochem J, 2002 Feb 1, 361(Pt 3), 443 - 50 Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel); Hruska-Hageman AM et al.; Neuronal members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family of cation channels include the mammalian brain Na(+) channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC) . Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli . Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related alphaENaC or betaENaC . In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1 . Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC . In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1 . We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization . Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses . These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain . This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems. J Cell Sci, 2002 Jan 1, 115(Pt 1), 207 - 16 DNA damage-dependent interaction of the nuclear matrix protein C1D with Translin-associated factor X (TRAX); Erdemir T et al.; The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination . C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon gamma-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo . In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX . Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus . We show that C1D and TRAX interact specifically in both yeast and mammalian cells . Interestingly, however, interaction of these two proteins in mammalian cells only occur following gamma-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX . Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization . These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation. J Biol Chem, 2002 Mar 29, 277(13), 11375 - 84 Epub 2002 Jan 18. bcn-1 Element-dependent activation of the laminin gamma 1 chain gene by the cooperative action of transcription factor E3 (TFE3) and Smad proteins; Kawata Y et al.; Laminin is a major component of the extracellular matrix . The laminin gamma1 chain is the least variant component of the laminin heterotrimeric assembly . The laminin gamma1 chain gene (LAMC1) expression is induced by several factors, including transforming growth factor-beta (TGF-beta) . LAMC1 promoter contains a highly conserved transcriptional element, bcn-1 . We screened cDNA libraries with the yeast one-hybrid system to identify transcriptional factors that are recognized by the bcn-1 motif . Using this strategy we isolated the basic helix-loop-helix/leucine zipper (bHLHzip) E-box-binding transcription factor, TFE3 . Until now, the E-box was the only element known to recruit the bHLHzip transcription factors . Although the bcn-1 element only remotely resembles the E-box sequence, we show that TFE3 binds and activates the bcn-1 element . TFE3 cooperates with Smad proteins in the activation of the LAMC1 promoter in cells, an effect that is critically dependent not only on the bcn-1 element but also on one of the Smad-binding elements (SBE) . The cooperative induction of the LAMC1 promoter and the endogenous LAMC1 gene by TFE3 and Smad3 is augmented by the TGF-beta signaling pathway . Thus, the bcn-1 is a novel TFE3-dependent TGF-beta target element that regulates LAMC1 gene expression. Zhong Yao Cai, 2001 Sep, 24(9), 664 - 5 {Study on anti-inflammatory, analgesic and antipyretic effects of bagmaking tea of sanyaku}; Zhong X et al.; OBJECTIVE: To observe anti-inflammatory, analgesic and antipyretic effects of the Bagmaking Tea of Sanyaku in rats . METHODS: mouse torsion modle induced by glacial acetic acid, mouse auricle swelling model induced by xylene and rat fever model induced by baker yeast were used . RESULTS: Bagmaking Tea of Sanyaku could inhibit mouse torsion action, mouse auricle swelling and rat fever . CONCLUSION: Bagmaking Tea of Sanyaku possessed anti-inflammatory, analgesic and antipyretic effects. J Biol Chem, 2002 Mar 29, 277(13), 11156 - 64 Epub 2002 Jan 17. The interaction of Pax5 (BSAP) with Daxx can result in transcriptional activation in B cells; Emelyanov AV et al.; Pax5 (BSAP) is essential for B cell development and acts both as a transcriptional activator and a repressor . Using a yeast two-hybrid assay to identify potential coregulators of Pax5, we identified Daxx, a protein that is highly conserved, ubiquitously expressed, and essential for embryonic mouse development . The interaction between Pax5 and Daxx involves the partial homeodomain of Pax5 and the C-terminal fragment of Daxx . A component of promyelocytic leukemia protein nuclear bodies, Daxx has been implicated in apoptosis and characterized as a transcriptional corepressor . Upon transient transfection assay of Daxx in B cells expressing endogenous Daxx and Pax5, we observed not only transcriptional corepression but also, unexpectedly, coactivation in M12.4.1 and A20 mouse B cell lines . Pax5 domains required for coactivation were identified using 293T cells . Coactivation apparently involves recruitment of the CREB binding protein (CBP), because we precipitated complexes containing Pax5, Daxx, and CBP in B cell lines . These data suggest that Daxx can affect Pax5's roles as an activator or repressor in B cells and describe a role for Daxx as a transcriptional coactivator. J Biol Chem, 2002 Mar 22, 277(12), 10323 - 31 Epub 2002 Jan 17. The non-ankyrin C terminus of Ikappa Balpha physically interacts with p53 in vivo and dissociates in response to apoptotic stress, hypoxia, DNA damage, and transforming growth factor-beta 1-mediated growth suppression; Chang NS; Transforming growth factor beta (TGF-beta1) suppresses the growth of mink lung Mv1Lu epithelial cells, whereas testicular hyaluronidase abolishes the growth inhibition . Exposure of Mv1Lu cells to TGF-beta1 rapidly resulted in down-regulation of cytosolic IkappaBalpha and hyaluronidase prevented this effect, suggesting a possible role of IkappaBalpha in the growth regulation . Ectopic expression of wild-type and dominant negative IkappaBalpha prevented TGF-beta1-mediated growth suppression . Nonetheless, the blocking effect of IkappaBalpha is not related to regulation of NF-kappaB function by its N-terminal ankyrin-repeat region (amino acids 1-243) . Removal of the PEST (proline-glutamic acid-serine-threonine) domain-containing C terminus (amino acids 244-314) abolished the IkappaBalpha function, and the C terminus alone blocked the TGF-beta1 growth-inhibitory effect . Co-immunoprecipitation by anti-p53 antibody using Mv1Lu and other types of cells, as well as rat liver and spleen, revealed that a portion of cytosolic IkappaBalpha physically interacted with p53 . In contrast, Mdm2, an inhibitor of p53, was barely detectable in the immunoprecipitates . The cytosolic p53 x IkappaBalpha complex rapidly dissociated in response to apoptotic stress, etoposide- and UV-mediated DNA damage, hypoxia, and TGF-beta1-mediated growth suppression . Also, a rapid increase in the formation of the nuclear p53 x IkappaBalpha complex was observed during exposure to etoposide and UV . In contrast, TGF-beta1-mediated promotion of fibroblast growth failed to mediate p53 x IkappaBalpha dissociation . Mapping by yeast two-hybrid showed that the non-ankyrin C terminus of IkappaBalpha physically interacted with the proline-rich region and a phosphorylation site, serine 46, in p53 . Deletion of serine 46 or alteration of serine 46 to glycine abolished the p53 x IkappaBalpha interaction . Alteration to threonine retained the binding interaction, suggesting that serine 46 phosphorylation is involved in the p53 x IkappaBalpha complex formation . Functionally, enhancement of p53 apoptosis was observed when p53 and IkappaBalpha were transiently co-expressed in cells . Together, the IkappaBalpha x p53 complex plays an important role in responses involving growth regulation, apoptosis, and hypoxic stress. Genes Dev, 2002 Jan 15, 16(2), 198 - 208 Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin; Zou L et al.; Cells respond to DNA damage by activating a network of signaling pathways that control cell cycle progression and DNA repair . Genetic studies in yeast suggested that several checkpoint proteins, including the RFC-related Rad17 protein, and the PCNA-related Rad1-Rad9-Hus1 protein complex might function as sensors of DNA damage . In this study, we show that the human Rad17 protein recruits the Rad9 protein complex onto chromatin after damage . Rad17 binds to chromatin prior to damage and is phosphorylated by ATR on chromatin after damage but Rad17's phosphorylation is not required for Rad9 loading onto chromatin . The chromatin associations of Rad17 and ATR are largely independent, which suggests that they localize to DNA damage independently . Furthermore, the phosphorylation of Rad17 requires Hus1, suggesting that the Rad1-Rad9-Hus1 complex recruited by Rad17 enables ATR to recognize its substrates . Our data are consistent with a model in which multiple checkpoint protein complexes localize to sites of DNA damage independently and interact to trigger the checkpoint-signaling cascade. Biochem Biophys Res Commun, 2002 Jan 25, 290(3), 1030 - 6 5T4 interacts with TIP-2/GIPC, a PDZ protein, with implications for metastasis; Awan A et al.; Overexpression of the 5T4 transmembrane glycoprotein can have marked effects on both the actin cytoskeleton and cell migration . Using a yeast two-hybrid approach, we describe a novel interaction between 5T4 and TIP-2/GIPC, a cytoplasmic interacting protein containing a PDZ domain . The cytoplasmic tail of 5T4 contains a class I PDZ-binding motif (Ser-Asp-Val) and we demonstrate that this region, in particular the terminal valine, is required for 5T4 interaction with TIP-2/GIPC . HeLa cells expressing hemagglutinin-tagged TIP-2/GIPC (HA-TIP-2/GIPC) have an altered distribution of endogenous 5T4, which colocalizes with HA-TIP-2/GIPC, thus supporting an interaction . Furthermore, TIP-2/GIPC can be coimmunoprecipitated with 5T4 from HeLa cell lysates . Identification of the 5T4 and TIP-2/GIPC interaction provides the first link between 5T4 and the actin cytoskeleton . Since other proteins, like 5T4, associate with TIP-2/GIPC and are linked with cancer, we explore the possibility that TIP-2/GIPC may be a common factor involved in the cancer process. Med Mycol, 2001 Dec, 39(6), 517 - 21 Molecular cloning of Aspergillus fumigatus CgrA, the ortholog of a conserved fungal nucleolar protein; Boettner et al.; In this report we describe the cloning of cgrA, the Aspergillus fumigatus ortholog of the yeast nucleolar protein Cgr1p . The cgrA complementary DNA (cDNA) contains a single open reading frame that would encode a protein of 114 amino acids that has 42% sequence identity to yeast Cgrlp . Heterologous expression of a green fluorescent protein (GFP)-tagged A . fumigatus cgrA gene demonstrated that the CgrA protein could localize to the yeast nucleolus . Moreover, the cgrA cDNA complemented the growth deficiency caused by inducible depletion of intracellular Cgr1p levels in yeast . These results support an orthologous relationship between the CgrA and Cgr1 proteins, and open the way for future studies into the potential value of nucleolar proteins as antifungal targets. Med Mycol, 2001 Dec, 39(6), 483 - 5 Candida dubliniensis fungemia in a solid organ transplant patient: case report and review of the literature; Gottlieb GS et al.; We report a case of Candida dubliniensis fungemia in a solid organ transplant patient, which, to our knowledge is the first such case in this patient population . C . dubliniensis is a recently described, emerging fungal pathogen, thus far, found in AIDS and a limited number of other immunosuppressed patients . It is of interest and concern because it can be misidentified as C . albicans and it may be resistant to azole antifungal agents . This case illustrates the need to be aware of emerging pathogens in new host populations and that new techniques used to identify yeast species may provide more accurate identification. IUBMB Life, 2001 Sep-Nov, 52(3-5), 93 - 100 Topology of the mitochondrial inner membrane: dynamics and bioenergetic implications; Mannella CA et al.; Electron tomography indicates that the mitochondrial inner membrane is not normally comprised of baffle-like folds as depicted in textbooks . In actuality, this membrane is pleomorphic, with narrow tubular regions connecting the internal compartments (cristae) to each other and to the membrane periphery . The membrane topologies observed in condensed (matrix contracted) and orthodox (matrix expanded) mitochondria cannot be interconverted by passive folding and unfolding . Instead, transitions between these morphological states likely involve membrane fusion and fission . Formation of tubular junctions in the inner membrane appears to be energetically favored, because they form spontaneously in yeast mitochondria following large-amplitude swelling and recontraction . However, aberrant, unattached, vesicular cristae are also observed in these mitochondria, suggesting that formation of cristae junctions depends on factors (such as the distribution of key proteins and/or lipids) that are disrupted during extreme swelling . Computer modeling studies using the "Virtual Cell" program suggest that the shape of the inner membrane can influence mitochondrial function . Simulations indicate that narrow cristae junctions restrict diffusion between intracristal and external compartments, causing depletion of ADP and decreased ATP output inside the cristae. IUBMB Life, 2001 Sep-Nov, 52(3-5), 143 - 52 Regulation of cytochrome c oxidase by adenylic nucleotides . Is oxidative phosphorylation feedback regulated by its end-products? Beauvoit B, Rigoulet M. Cytochrome c oxidase, which catalyzes an irreversible step of the respiratory chain, is one of the rate-controlling steps of oxidative phosphorylation on isolated mitochondria . The rate of electron transfer through the complex is primarily controlled by the associated thermodynamic forces, i.e., the span in redox potential between oxygen and cytochrome c and the protonmotive force . However, the electron flux also depends on the various kinetic effectors, including adenylic nucleotides . Although the number of binding sites for ATP and ADP on cytochrome oxidase is still a matter of debate, experiments performed on the solubilized and reconstituted enzyme provide strong functional evidence that the mammalian cytochrome c oxidase binds adenylic nucleotides on both sides of the inner membrane . These effects include modification in cytochrome c affinity, allosteric inhibition and changes in proton pumping efficiency . Immunological studies have pointed out the role of subunit IV and that of an ATP-binding protein, subunit VIa, in these kinetic regulations . In yeast, the role of the nuclear-encoded subunits in assembly and regulation of the cytochrome c oxidase has been further substantiated by using gene-disruption analysis . Using a subunit VIa-null mutant, the consequences of the ATP regulation on oxidative phosphorylation have been further investigated on isolated mitochondria . Taken together, the data demonstrate that there are multiple regulating sites for ATP on the yeast cytochrome oxidase with respect to the location (matrix versus cytosolic side), kinetic effect (activation versus inhibition) and consequence on the flow-force relationships . The question is therefore raised as to the physiological meaning of such feedback regulation of the respiratory chain by ATP in the control and regulation of cellular energy metabolism. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 491 - 3 {Progress in proteomics}; Zhen Z; Proteomics, the large-scale analysis of proteins, will contribute greatly to our understanding of gene function in the post-genome era . Proteomics uses a combination of sophisticated techniques including two-dimensional(2D) gel electrophoresis, mass spectrometry, yeast two-hybrid system, bioinformatics, etc . to characterize, to quantify, and to analyze cellular proteins in a global way . The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new therapeutic targets . This review aims to explain the definition of proteomics and then to outline proteomic techniques . Finally, applications to the study of human disease are briefly discussed. Nature, 2002 Jan 17, 415(6869), 339 - 43 Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase; Minokoshi Y et al.; Leptin is a hormone secreted by adipocytes that plays a pivotal role in regulating food intake, energy expenditure and neuroendocrine function . Leptin stimulates the oxidation of fatty acids and the uptake of glucose, and prevents the accumulation of lipids in nonadipose tissues, which can lead to functional impairments known as "lipotoxicity" . The signalling pathways that mediate the metabolic effects of leptin remain undefined . The 5'-AMP-activated protein kinase (AMPK) potently stimulates fatty-acid oxidation in muscle by inhibiting the activity of acetyl coenzyme A carboxylase (ACC) . AMPK is a heterotrimeric enzyme that is conserved from yeast to humans and functions as a 'fuel gauge' to monitor the status of cellular energy . Here we show that leptin selectively stimulates phosphorylation and activation of the alpha2 catalytic subunit of AMPK (alpha2 AMPK) in skeletal muscle, thus establishing a previously unknown signalling pathway for leptin . Early activation of AMPK occurs by leptin acting directly on muscle, whereas later activation depends on leptin functioning through the hypothalamic-sympathetic nervous system axis . In parallel with its activation of AMPK, leptin suppresses the activity of ACC, thereby stimulating the oxidation of fatty acids in muscle . Blocking AMPK activation inhibits the phosphorylation of ACC stimulated by leptin . Our data identify AMPK as a principal mediator of the effects of leptin on fatty-acid metabolism in muscle. Infect Immun, 2002 Feb, 70(2), 692 - 701 Improved immunogenicity and efficacy of the recombinant 19-kilodalton merozoite surface protein 1 by the addition of oligodeoxynucleotide and aluminum hydroxide gel in a murine malaria vaccine model; Near KA et al.; Vaccination of mice with yeast-secreted Plasmodium yoelii-derived 19-kilodalton merozoite surface protein 1 (yMSP1(19)) has been shown to afford protection from challenge with a lethal strain of P . yoelii . Sterile immunity can be achieved when MSP1(19) is emulsified in Freund adjuvant but not when it is adsorbed to aluminum hydroxide gel (alum) . Because complete Freund adjuvant is not an acceptable adjuvant for use in humans, alternative adjuvants must be identified for formulating MSP1(19) as a vaccine for use in humans . To determine whether oligodeoxynucleotides with CpG motifs (ODN), reported to be a powerful new class of adjuvants, could enhance the immunogenicity of yMSP1(19), C57BL/6 mice were vaccinated either with yMSP1(19) formulated with Freund adjuvant, with alum, or with ODN plus alum and challenged intravenously with P . yoelii 17XL asexual blood-stage parasites . Adsorption of immunogen and adjuvant to alum was optimized by adjusting buffer (phosphate versus acetate) and pH . We found that the adjuvant combination of ODN plus alum with yMSP1(19), injected intraperitoneally (i.p.), increased immunoglobulin G (IgG) yMSP1(19)-specific antibody production 12-fold over Freund adjuvant given i.p., 3-fold over Freund adjuvant given subcutaneously (s.c.), 300-fold over alum given i.p., and 48-fold over alum given s.c . The predominant antibody isotype in the group receiving alum-ODN-yMSP1(19) was IgG1 . Increased antibody levels correlated to protection from a challenge with P . yoelii 17XL . Supernatant cytokine levels of gamma interferon in yMSP1(19)-stimulated splenocytes were dramatically elevated in the alum-ODN-yMSP1(19) group . Interleukin-10 (IL-10) levels were also elevated; however, no IL-5 was detected . The cytokine profile, as well as the predominant IgG1 antibody isotype, suggests the protective immune response was a mixed Th1/Th2 response. Toxicol Pathol, 2001 Nov-Dec, 29(6), 673 - 6 Mutant models of prolonged life span; Mahler JF; Aging is an important biological process that affects all creatures . For humans, age-related diseases and the question of why we age and die also have tremendous social and philosophical impact . We can therefore expect that models to study mechanisms of the aging process will always attract much interest . Until recently, the mutant model approach to study molecular mechanisms of aging has been limited to lower animals such as yeast, worms, and flies . However, given the current power of genetic technology in mammals, we can expect that phenotypes of prolonged life span will increasingly be seen in mice and subject to evaluation by pathologists . A brief review of current models is presented. J Med Virol, 2002 Mar, 66(3), 304 - 11 Characterization of a human monoclonal antibody obtained after immunization with plasma vaccine and a booster with recombinant-DNA hepatitis B vaccine; Heijtink RA et al.; A human monoclonal antibody type IgG4, designated 1Ff4, was obtained by Epstein Barr virus transformation of peripheral blood lymphocytes from a hepatitis B vaccinee (HB-VAX: plasma-derived vaccine) after one boost of yeast recombinant DNA derived vaccine (Engerix-B) . 1Ff4 binds preferentially to HBsAg/adw(2) and HBsAg/ayw(1) . In binding experiments, it competes with antibodies induced by vaccination with HB-VAX-DNA (yeast recombinant) and HB-VAX (plasma-derived vaccine) . 1Ff4 competes in part with a monoclonal antibody for the w/r region . Partial inhibition of binding of HBsAg/adw(2) to solid phase anti-HBs was detected, resembling inhibition obtained using other human monoclonal specific for the "a"-loop . 1Ff4 does not bind to linear peptides covering the two "a"-loops or to an adw(2)/G145R mutant, its binding to wild type HBsAg strongly depends on the presence of disulphide bonds . In a large series of HBsAg-positive samples from an endemic area, 1Ff4 antibodies were successfully used to discriminate between an adw(2) and an adrq+ strain . The characterisation of 1Ff4 and other human monoclonal anti-HBs antibodies may help to understand the fine specificity of protective antibodies elicited by immunization . Funct Integr Genomics, 2001 Mar, 1(4), 256 - 68 Mining functional information associated with expression arrays; Blaschke C et al.; Deciphering the networks of interactions between molecules in biological systems has gained momentum with the monitoring of gene expression patterns at the genomic scale . Expression array experiments provide vast amounts of experimental data about these networks, the analysis of which requires new computational methods . In particular, issues related to the extraction of biological information are key for the end users . We propose here a strategy, implemented in a system called GEISHA (gene expression information system for human analysis) and able to detect biological terms significantly associated to different gene expression clusters by mining collections of Medline abstracts . GEISHA is based on a comparison of the frequency of abstracts linked to different gene clusters and containing a given term . Interpretation by the end user of the biological meaning of the terms is facilitated by embedding them in the corresponding significant sentences and abstracts and by establishing relations with other, equally significant terms . The information provided by GEISHA for the available yeast expression data compares favorably with the functional annotations provided by human experts, demonstrating the potential value of GEISHA as an assistant for the analysis of expression array experiments. Funct Integr Genomics, 2000 May, 1(1), 2 - 11 Weeding out the genes: the Arabidopsis genome project; Martienssen RA; The Arabidopsis genome sequence is scheduled for completion at the end of this year (December 2000) . It will be the first higher plant genome to be sequenced, and will allow a detailed comparison with bacterial, yeast and animal genomes . Already, two of the five chromosomes have been sequenced, and we have had our first glimpse of higher eukaryotic centromeres, and the structure of heterochromatin . The implications for understanding plant gene function, genome structure and genome organization are profound . In this review, the lessons learned for future genome projects are reviewed as well as a summary of the initial findings in Arabidopsis. Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 780 - 5 Epub 2002 Jan 15. Genomic structure and functional control of the Dlx3-7 bigene cluster; Sumiyama K et al.; The Dlx genes are involved in early vertebrate morphogenesis, notably of the head . The six Dlx genes of mammals are arranged in three convergently transcribed bigene clusters . In this study, we examine the regulation of the Dlx3-7 cluster of the mouse . We obtained and sequenced human and mouse P1 clones covering the entire Dlx3-7 cluster . Comparative analysis of the human and mouse sequences revealed several highly conserved noncoding regions within 30 kb of the Dlx3-7-coding regions . These conserved elements were located both 5' of the coding exons of each gene and in the intergenic region 3' of the exons, suggesting that some enhancers might be shared between genes . We also found that the protein sequence of Dlx7 is evolving more rapidly than that of Dlx3 . We conducted a functional study of the 79-kb mouse genomic clone to locate cis-element activity able to reproduce the endogenous expression pattern by using transgenic mice . We inserted a lacZ reporter gene into the first exon of the Dlx3 gene by using homologous recombination in yeast . Strong lacZ expression in embryonic (E) stage E9.5 and E10.5 mouse embryos was found in the limb buds and first and second visceral arches, consistent with the endogenous Dlx3 expression pattern . This result shows that the 79-kb region contains the major cis-elements required to direct the endogenous expression of Dlx3 at stage E10.5 . To test for enhancer location, we divided the construct in the mid-intergenic region and injected the Dlx3 gene portion . This shortened fragment lacking Dlx7-flanking sequences is able to drive expression in the limb buds but not in the visceral arches . This observation is consistent with a cis-regulatory enhancer-sharing model within the Dlx bigene cluster. J Cell Sci, 2001 Dec, 114(Pt 24), 4435 - 45 Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy; Ostlund C et al.; Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene, which encodes lamin A and lamin C . Mutations in this gene also give rise to limb girdle muscular dystrophy type 1B, dilated cardiomyopathy with atrioventricular conduction defect and Dunnigan-type partial lipodystrophy . The properties of the mutant lamins that cause muscular dystrophy, lipodystrophy and dilated cardiomyopathy are not known . We transfected C2C12 myoblasts with cDNA encoding wild-type lamin A and 15 mutant forms found in patients affected by these diseases . Immunofluorescence microscopy showed that four mutants, N195K, E358K, M371K and R386K, could have a dramatically aberrant localization, with decreased nuclear rim staining and formation of intranuclear foci . The distributions of endogenous lamin A/C, lamin B1 and lamin B2 were also altered in cells expressing these four mutants and three of them caused a loss of emerin from the nuclear envelope . In the yeast two-hybrid assay, the 15 lamin A mutants studied interacted with themselves and with wild-type lamin A and lamin B1 . Pulse-chase experiments showed no decrease in the stability of several representative lamin A mutants compared with wild-type . These results indicate that some lamin A mutants causing disease can be aberrantly localized, partially disrupt the endogenous lamina and alter emerin localization, whereas others localize normally in transfected cells. J Cell Sci, 2001 Dec, 114(Pt 24), 4385 - 95 Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells; Taylor SS et al.; BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly . Two related human protein kinases, Bub1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle . However, how the activities of Bub1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known . To investigate how spindle events regulate Bub1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins . In prometaphase cells, both kinases colocalise to the same domain of the kinetochore . However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of Bub1 is often asymmetrical . This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker Bub1 staining is typically closer to the nearest spindle pole . In addition, a 30 minute nocodazole treatment dramatically increases the amount of Bub1 localising to kinetochores but has little effect on BubR1 . Furthermore, Bub1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment . Thus, these observations suggest that Bub1 localisation is sensitive to changes in both tension and microtubule attachment . Consistent with this, we also show that Bub1 is rapidly phosphorylated following brief treatments with nocodazole or taxol . In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect . Although these observations indicate that Bub1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis . We suggest therefore that Bub1 and BubR1 may integrate different 'spindle assembly signals' into a single signal which can then be interpreted by downstream cell cycle regulators. J Cell Sci, 2001 Dec, 114(Pt 24), 4371 - 84 The S . pombe aurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation; Petersen J et al.; Metazoans contain three aurora-related kinases . Aurora A is required for spindle formation while aurora B is required for chromosome condensation and cytokinesis . Less is known about the function of aurora C . S . pombe contains a single aurora-related kinase, Ark1 . Although Ark1 protein levels remained constant as cells progressed through the mitotic cell cycle, its distribution altered during mitosis and meiosis . Throughout G2 Ark1 was concentrated in one to three nuclear foci that were not associated with the spindle pole body/centromere complex . Following commitment to mitosis Ark1 associated with chromatin and was particularly concentrated at several sites including kinetochores/centromeres . Kinetochore/centromere association diminished during anaphase A, after which it was distributed along the spindle . The protein became restricted to a small central zone that transiently enlarged as the spindle extended . As in many other systems mitotic fission yeast cells exhibit a much greater degree of phosphorylation of serine 10 of histone H3 than interphase cells . A number of studies have linked this modification with chromosome condensation . Ark1 immuno-precipitates phosphorylated serine 10 of histone H3 in vitro . This activity was highest in mitotic extracts . The absence of the histone H3 phospho-serine 10 epitope from mitotic cells in which the ark1(+) gene had been deleted (ark1.Delta1); the inability of these cells to resolve their chromosomes during anaphase and the co-localisation of this phospho-epitope with Ark1 early in mitosis, all suggest that Ark1 phosphorylates serine 10 of histone H3 in vivo . ark1.Delta1 cells also exhibited a reduction in kinetochore activity and a minor defect in spindle formation . Thus the enzyme activity, localisation and phenotype arising from our manipulations of this single fission yeast aurora kinase family member suggest that this single kinase is executing functions that are separately implemented by distinct aurora A and aurora B kinases in higher systems. Oncogene, 2002 Jan 3, 21(1), 148 - 52 Molecular cloning of ESET, a novel histone H3-specific methyltransferase that interacts with ERG transcription factor; Yang L et al.; The ets-related gene erg encodes a transcription factor that is implicated in the control of cell growth and differentiation . To identify interacting partners of ERG, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the N-terminal region of ERG as a bait . We isolated a 4.6 kb full-length mouse cDNA encoding a 1307-amino acid protein migrating as a 180 kD band, which was termed ESET (ERG-associated protein with SET domain) . ESET is 92% identical to the human protein SETDB1 (SET domain, bifurcated 1) . The interaction between ESET and ERG was supported by in vitro pull-down using glutathione S-transferase (GST) fusion protein, by transfection and co-immunoprecipitation experiments, and by association of endogenous SETDB1 with ERG . Since ESET possesses evolutionarily conserved SET, preSET, and postSET domains implicated in histone methylation, we tested the ability of ESET to methylate core histones . The results of these studies demonstrated that ESET is a histone H3-specific methyltransferase, and that mutations within ESET abolished its methyltransferase activity . Together, these findings raise the possibility that transcription factor ERG may participate in transcriptional regulation through ESET-mediated histone methylation. J Biol Chem, 2002 Apr 5, 277(14), 12437 - 45 Epub 2002 Jan 14. Association of Bcr-Abl with the proto-oncogene Vav is implicated in activation of the Rac-1 pathway; Bassermann F et al.; Vav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization . The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis . Previous studies by Matsuguchi et al . (Matsuguchi, T., Inhorn, R . C., Carlesso, N., Xu, G., Druker, B., and Griffin, J . D . (1995) EMBO J . 14, 257-265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins . Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo . Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl . Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction . Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominant negative construct . Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow . These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus . Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1. J Biol Chem, 2002 Mar 22, 277(12), 10512 - 22 Epub 2002 Jan 14. Protein binding and functional characterization of plakophilin 2 . Evidence for its diverse roles in desmosomes and beta -catenin signaling; Chen X et al.; Plakophilins are a subfamily of p120-related arm-repeat proteins that can be found in both desmosomes and the nucleus . Among the three known plakophilin members, plakophilin 1 has been linked to a genetic skin disorder and shown to play important roles in desmosome assembly and organization . However, little is known about the binding partners and functions of the most widely expressed member, plakophilin 2 . To better understand the cellular functions of plakophilin 2, we have examined its protein interactions with other junctional molecules using co-immunoprecipitation and yeast two-hybrid assays . Here we show that plakophilin 2 can interact directly with several desmosomal components, including desmoplakin, plakoglobin, desmoglein 1 and 2, and desmocollin 1a and 2a . The head domain of plakophilin 2 is critical for most of these interactions and is sufficient to direct plakophilin 2 to cell borders . In addition, plakophilin 2 is less efficient than plakophilin 1 in localizing to the nucleus and enhancing the recruitment of excess desmoplakin to cell borders in transiently transfected COS cells . Furthermore, plakophilin 2 is able to associate with beta-catenin through its head domain, and the expression of plakophilin 2 in SW480 cells up-regulates the endogenous beta-catenin/T cell factor-signaling activity . This up-regulation by plakophilin 2 is abolished by ectopic expression of E-cadherin, suggesting that these proteins compete for the same pool of signaling active beta-catenin . Our results demonstrate that plakophilin 2 interacts with a broader repertoire of desmosomal components than plakophilin 1 and provide new insight into the possible roles of plakophilin 2 in regulating the signaling activity of beta-catenin. Comp Biochem Physiol C Toxicol Pharmacol, 2000 Mar, 125(3), 325 - 32 Cloning and sequencing of cDNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus; Syring RA et al.; Metallothioneins (MTs) are cysteine-rich metal-binding proteins found in micro-organisms, plants and all invertebrate and vertebrate animals . Unicellular eukaryotes such as yeast have a copper-MT whose synthesis is induced by a copper-activated transcription factor . Most higher organisms have two major cadmium/zinc MT isoforms, whose synthesis is controlled by a zinc-activated transcription factor . The blue crab, Callinectes sapidus, has two cadmium-inducible isoforms, CdMT-I and CdMT-II, and a third isoform, CuMT-II, which is induced by copper, but not by cadmium . The cDNA sequence of the copper-specific MT, along with those of the two CdMTs, was determined utilizing 3' and 5' rapid amplification of cDNA ends (RACE) . CuMT-II cDNA encodes a 63 amino acid protein containing 21 cysteine residues . CdMT-I and CdMT-II cDNA encode a 58 and 57 amino acid protein, respectively, each with 18 cysteines . Molecular phylogeny analysis shows that the CdMT isoforms cluster with other crustacean CdMTs, whereas the copper-specific MT is more closely related to mollusk MTs . CuMT-II shows considerable homology to a copper-specific, non-cadmium inducible, MT from the snail, Helix pomatia . The presence of copper-specific MTs in mollusks and crustaceans, both of which are dependent on hemocyanin for oxygen transport, suggests that CuMT-II is involved in copper homeostasis associated with the synthesis and degradation of hemocyanin. Curr Biol, 2002 Jan 8, 12(1), R6 - 8 Cell polarity: following formin function; Sawin KE; Mutation of a novel fission yeast formin, for3p, leads to marked changes in both the actin and microtubule cytoskeleton, as well as a surprising asymmetric pattern of cell growth . At the same time, new work in budding yeast implicates formins directly in actin filament assembly. J Environ Qual, 2001 Nov-Dec, 30(6), 2077 - 80 Persistence of estrogenic hormones in agricultural soils: II . 17Alpha-ethynylestradiol; Colucci MS et al.; The persistence of 17alpha-ethynylestradiol in agricultural soil was established in laboratory microcosm studies . The hormone was rapidly dissipated in loam, sandy loam, and silt loam soils under a range of moisture and temperature conditions . Dissipation of 17alpha-ethynylestradiol correlated closely with removal of total estrogenicity determined with a recombinant yeast bioassay, indicating that extractable estrogenic transformation products did not accumulate . The stability of 17alpha-ethynylestradiol in sterile soil, decreased removal in the absence of oxygen, and the response of dissipation kinetics to variation in temperature and moisture suggested that the removal was microbially mediated . We conclude that 17alpha-ethynylestradiol is rapidly dissipated in agricultural soils under a range of conditions typical of a temperate growing season. J Environ Qual, 2001 Nov-Dec, 30(6), 2070 - 6 Persistence of estrogenic hormones in agricultural soils: I . 17Beta-estradiol and estrone; Colucci MS et al.; The persistence and pathways of dissipation of 17beta-estradiol and estrone in soil were established in laboratory microcosm incubations . {4-14C}-17beta-Estradiol dissipation and mineralization rates were determined over a range of temperatures and moistures, and this compound was rapidly removed in soil conditions typical of a temperate growing season . 17beta-Estradiol was oxidized to estrone in both autoclaved and nonsterile loam, silt loam, and sandy loam soils, suggesting an abiological transformation . In contrast, estrone was stable in autoclaved soil, suggesting that its removal was microbially mediated . Both {4-14C}-17beta-estradiol and {4-14C}-estrone formed non-extractable residues, and soil-bound residues were only slowly mineralized, suggesting that their bioavailability was low . Determination of total estrogenicity in soil extracts by means of a recombinant yeast assay indicated that there were no other estrogenic compounds produced during 17beta-estradiol dissipation, and that total estrogenicity was rapidly dissipated below the detection limit . We suggest that environmental studies evaluating the movement and persistence of estrogenic hormones from animal wastes should include estrone in their analyses. J Biol Chem, 2002 Mar 29, 277(13), 10783 - 8 Epub 2002 Jan 11. Evidence for a direct interaction between the tumor suppressor serpin, maspin, and types I and III collagen; Blacque OE et al.; Maspin (mammary serine protease inhibitor) was originally identified as a tumor suppressor protein in human breast epithelial cells and is a member of the serine proteases inhibitor (serpin) superfamily . It inhibits tumor cell motility and angiogenesis, and although predominantly cytoplasmic, it is also localized to the cell surface . In this study we have investigated the use of the yeast two-hybrid interaction trap to identify novel maspin targets . A target human fibroblast cDNA library was screened, and the alpha-2 chain of type I collagen was identified as a potential interactant . Binding studies with isolated proteins showed interaction between recombinant maspin and types I and III collagen but not other collagen subtypes, a profile strikingly similar to mouse pigment epithelium-derived factor (caspin), which is similarly down-regulated in murine adenocarcinoma tumors and is a potent inhibitor of angiogenesis . Kinetic analysis using an IAsys resonant mirror biosensor determined the dissociation constant of maspin for collagen type I to be 0.63 microm . Further two-hybrid interactions with maspin truncation constructs suggest that collagen binding is localized to amino acids 84-112 of maspin, which aligns with the collagen-binding region of colligin . A direct interaction between exogenous or cell surface maspin and extracellular matrix collagen may contribute to a cell adhesion role in the prevention of tumor cell migration and angiogenesis. J Biol Chem, 2002 Mar 22, 277(12), 9952 - 7 Epub 2002 Jan 11. Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner; Christie GR et al.; Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway . Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells . Small ( approximately 48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70(S6K) and its substrate ribosomal protein S6 . This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway . Alcohols of active amino acids substituted for amino acids with lower efficiency . Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter . Amino acid-induced, rapamycin-sensitive activation of p70(S6K) was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor . In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70(S6K). J Biol Chem, 2002 Mar 8, 277(10), 8255 - 9 Epub 2002 Jan 11. Activation of p53 by protein inhibitor of activated Stat1 (PIAS1); Megidish T et al.; The tumor suppressor protein p53 functions as a transcriptional factor that activates genes controlling cell cycle arrest and apoptosis . Here, we report that protein inhibitor of activated Stat1 (PIAS1) interacts with the tetramerization and C-terminal regulatory domains of p53 in yeast two-hybrid analyses . Endogenous PIAS1 is also associated with endogenous p53 in mammalian cells . Ectopic expression of PIAS1 activates p53-mediated expression in mouse embryonic fibroblast cells (p53(-/-)) as well as a variety of other cell lines . Furthermore, ectopic expression of PIAS1 induces p53-mediated expression of cyclin-dependent kinase inhibitor p21 and G(1) arrest of the cell cycle in H1299 cells . In addition, a PIAS1 mutant without the RING-finger domain required for sumoylation could still activate p53-mediated gene expression, indicating that activation of p53 by PIAS1 does not require the RING-finger domain . Taken together, our results suggest that PIAS1 is a novel activator of p53. Bioseparation, 2001, 10(1-3), 1 - 6 Effects of adsorbent properties on zone spreading in expanded bed chromatography; Yamamoto S et al.; The mixing performance as well as the adsorption performance in expanded bed chromatography (EBC) was investigated by using various types of adsorption media (average particle size = 100-700 microm, density = 1100-1700 kg/m3, base matrix = hydroxyapatite, styrene-divinylbenzene, cross-linked agarose) . The scale down study with 0.8 cm diameter columns was also attempted . Pulse response curves were measured with vitamin B12 as a tracer {Residence time distribution RTD experiments}, and the HETP (height equivalent to a theoretical plate or plate height) values were calculated from the peak variance and the peak retention time . The HETP values for different types of packing media tested showed very similar values (0.5-1.0 cm), which did not depend on the flow-rate or the column diameter (0.8-2.6 cm) . Dynamic binding capacity (DBC) values of lactic acid on a Dowex anion-exchange resin were determined from breakthrough curve (BTC) measurements for both EBC and fixed bed chromatography (FBC) . The DBC values for EBC were similar to those for FBC . When the liquid feed contained insoluble particles (yeast cells) the degree of mixing increased . However, the contribution of the mixing to the total spreading of BTCs for EBC was usually small so that this increase in the mixing did not affect the adsorption performance or the DBC values significantly. J Biol Chem, 2002 Mar 22, 277(12), 10506 - 11 Epub 2002 Jan 10. Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes; DeMarco SJ et al.; Spatial and temporal regulation of Ca(2+) signaling require the assembly of multiprotein complexes linking molecules involved in Ca(2+) influx, sensing, buffering, and extrusion . Recent evidence indicates that plasma membrane Ca(2+) ATPases (PMCAs) participate in the control of local Ca(2+) fluxes, but the mechanism of multiprotein complex formation of specific PMCAs is poorly understood . Using the PMCA2b COOH-terminal tail as bait in a yeast two-hybrid screen, we identified the PSD-95, Dlg, ZO-1 (PDZ) domain-containing Na(+)/H(+) exchanger regulatory factor-2 (NHERF2) as an interacting partner . Protein pull-down and coimmunoprecipitation experiments using recombinant PMCA2b and PMCA4b as well as NHERF1 and NHERF2 showed that the interaction of PMCA2b with NHERF2 was specific and selective . PMCA4b did not interact with either of the NHERFs, and PMCA2b selectively preferred NHERF2 over NHERF1 . Green fluorescent protein-tagged PMCA2b was expressed at the apical membrane in Madin-Darby canine kidney epithelial cells, where it colocalized with apically targeted NHERF2 . Our study identifies NHERF2 as the first specific PDZ partner for PMCA2b not shared with PMCA4b, and demonstrates that PMCA splice forms differing only minimally in their COOH-terminal residues interact with unique PDZ proteins . NHERFs have been implicated in the targeting, retention and regulation of membrane proteins including the beta(2)-adrenergic receptor, cystic fibrosis transmembrane conductance regulator, and Trp4 Ca(2+) channel, and NHERF2 is now shown to also interact with PMCA2b . This interaction may allow the functional assembly of PMCA2b in a multiprotein Ca(2+) signaling complex, facilitating integrated cross-talk between local Ca(2+) influx and efflux. J Biol Chem, 2002 Mar 22, 277(12), 10273 - 82 Epub 2002 Jan 10. Regulation of synaptophysin degradation by mammalian homologues of seven in absentia; Wheeler TC et al.; Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm . Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved . Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia . We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association . Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes . In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin . Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway . Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin. FEMS Microbiol Lett, 2002 Jan 2, 206(1), 45 - 50 Presence of organic sources of nitrogen is critical for filament formation and pH-dependent morphogenesis in Yarrowia lipolytica; Szabo R et al.; Yeast dimorphism is an attractive model for the study of cell morphogenesis and differentiation . The non-conventional yeast Yarrowia lipolytica was chosen to characterise the regulation of dimorphic transition by extracellular pH and by the presence of organic sources of nitrogen . Organic nitrogen sources appear to be required for the morphogenic effect of pH . Two sets of mutants defective in either pH-dependent or nitrogen source-dependent signalling pathway were analysed . The results suggest that the latter but not the former is required for both normal filament formation on solid medium and pH-dependent dimorphic behaviour of Y . lipolytica in liquid medium . We propose that in this organism pH affects the formation of hyphae indirectly by modulation of availability and/or utilisation of transportable sources of nitrogen. Biochem Biophys Res Commun, 2002 Jan 18, 290(2), 783 - 9 Molecular cloning and functional characterization of mouse coactosin-like protein; Doucet J et al.; Coactosin was first isolated from Dictyostelium discoideum and, as reported, human coactosin-like protein (CLP) was identified in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait . A mouse CLP (mCLP) cDNA clone was identified among EMBL/GenBank EST sequences . The derived amino acid sequence (142 residues) was 95.1% identical with human CLP . Here, we also show that mCLP interacts with actin and 5LO in the two-hybrid system . High-speed cosedimentation assays and GST-binding assays confirmed these protein interactions . In chemical cross-linking experiments, one molecule of mCLP was covalently linked to either one subunit of actin or one molecule of 5LO . The mCLP-F-actin and mCLP-5LO associations were pH-insensitive and Ca(2+)-independent . However, association with actin was best observed at low salt concentrations, while association with 5LO was favored by salt, indicating different binding characteristics. Plant Mol Biol, 2001 Dec, 47(6), 693 - 701 Plant callose synthase complexes; Verma DP et al.; Synthesis of callose (beta-1,3-glucan) in plants has been a topic of much debate over the past several decades . Callose synthase could not be purified to homogeneity and most partially purified cellulose synthase preparations yielded beta-1,3-glucan in vitro, leading to the interpretation that cellulose synthase might be able to synthesize callose . While a rapid progress has been made on the genes involved in cellulose synthesis in the past five years, identification of genes for callose synthases has proven difficult because cognate genes had not been identified in other organisms . An Arabidopsis gene encoding a putative cell plate-specific callose synthase catalytic subunit (CalS1) was recently cloned . CalS1 shares high sequence homology with the well-characterized yeast beta-1,3-glucan synthase and transgenic plant cells over-expressing CalS1 display higher callose synthase activity and accumulate more callose . The callose synthase complex exists in at least two distinct forms in different tissues and interacts with phragmoplastin . UDP-glucose transferase, Rop1 and, possibly, annexin . There are 12 CalS isozymes in Arabidopsis, and each may be tissue-specific and/or regulated under different physiological conditions responding to biotic and abiotic stresses. Genetika, 2001 Dec, 37(12), 1621 - 31 {Genetic screening of meiotic mutations in mosaic clones of the Drosophila melanogaster female germline}; Fedorova SA et al.; A method of screening for meiotic mutations based on genetic analysis of chromosome disjunction in germline mosaic clones of females homozygous for potential mutations is proposed . The clones are obtained at high frequency due to the use of the transgenic FLP/FRT system of mitotic recombination . This system permits obtaining homozygous clones in the first generation after mutagenesis, whereas the cultures are set up after selection for potential meiotic mutations . This significantly enhances, the efficiency of screening by the elimination of the limiting stage . Using this method, the following mutations were revealed in the 3L arm of Drosophila: ff6 leading to disturbed centriole disjunction, which results in appearance of multi-tail spermatids and three-pole spindles during male meiosis; ff3 leading to the formation of chromosome bridges in anaphase and telophase, chromosome nondisjunction, and premature chromatin condensation after metaphase; embryonic lethal ff29, with disturbed coordination between nuclear and centrosome cycles during syncytial cleavage; and a series of other mutations causing a wide spectrum of disturbances in male meiosis . Comparison of the proposed method with procedures of screening for yeast cell-cycle mutations showed that we succeeded in attaining the efficiency of screening in the Drosophila model close to that in the yeast model. Mol Cell Biol, 2002 Feb, 22(3), 750 - 61 A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding; Al-Khouri AM et al.; In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB) . TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription . TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction . In A . castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot . An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter . We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively . In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates . Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. Dev Biol, 2002 Jan 1, 241(1), 157 - 71 Analysis of Drosophila cyclin EI and II function during development: identification of an inhibitory zone within the morphogenetic furrow of the eye imaginal disc that blocks the function of cyclin EI but not cyclin EII; Crack D et al.; The Drosophila cyclin E (DmcycE) gene gives rise to two transcripts encoding proteins that differ at their N termini, DmcycEII and DmcycEI . This study presents the first in vivo dissection of Cyclin E function . Ectopic expression studies using N- and C-terminal deletions of DmcycEI revealed that a region of 322 residues surrounding the cyclin box is sufficient to induce entry of G1-arrested larval eye imaginal disc cells into S phase . Ectopic expression of DmcycEI in the eye disc has been previously shown to drive anterior, but not posterior, G1-phase cells within the morphogenetic furrow (MF) into S phase . Significantly, ectopic expression of DmcycEII and N-terminal deletions of DmcycEI were able to drive all G1 cells within the morphogenetic furrow into S phase, while a C-terminal deletion of DmcycEI could not . The p21 homolog Dacapo was shown by yeast two-hybrid, coimmunolocalization, and in vivo functional studies not to be the mediator of the DmcycEI inhibition in posterior part of the MF . Taken together, these results reveal a novel zone within the posterior region of the MF where DmcycEI but not DmcycEII function is inhibited, and suggest that DmcycEII is a more potent inducer of S phase. Anal Sci, 2001 Dec, 17(12), 1375 - 7 Determination of nucleic acids using calcein-neodymium complex as a fluorescence probe; Zhu Z; A novel fluorometric method has been developed for rapid determination of DNA and RNA with calcein-neodymium complex as a fluorescence probe . The method is based on the fluorescence enhancement of calcein-Nd(III) complex in the presence of DNA or RNA, with maximum excitation and emission wavelength at 489 nm and 514 nm, respectively . Under optimal conditions, the calibration graphs are linear over the range 0.5 - 3.0 microg/ml for both DNA and yeast RNA, 0.4 - 2.0 microg/ml for fish sperm DNA (FS DNA) and 0 - 3.0 microg/ml for calf thymus DNA (CT DNA) . The corresponding detection limits are 15.1 ng/ml for DNA, 21.2 ng/ml for yeast RNA, 10.5 ng/ml for FS DNA and 8.9 ng/ml for CT DNA . The interaction mechanism for the binding of calcein-Nd(III) complex to DNA is also studied . The results of absorption spectra, fluorescence polarization measurements and thermal denaturation experiments, suggested that the interaction between calcein-Nd(III) complex and DNA is an electrostatic interaction. Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 655 - 60 Epub 2002 Jan 08. tRNA elements mediate the assembly of an icosahedral RNA virus; Choi YG et al.; tRNAs, the adapter molecules in protein synthesis, also serve as metabolic cofactors and as primers for viral RNA-directed DNA synthesis . The genomic and subgenomic RNAs of some plant viruses have a 3'-terminal tRNA-like structure (TLS) that can accept a specific amino acid and serve as a site for initiation of replication and as a simple telomere . We report a previously undescribed role for the TLS of brome mosaic virus (BMV), and potentially for cellular tRNA, in mediating the assembly of its icosahedral virions . BMV genomic RNAs and subgenomic RNA lacking the TLS failed to assemble into virions when incubated with purified BMV coat protein . Assembly was restored by addition of a 201-nt RNA containing the BMV TLS . TLSs from two other plant viruses as well as tRNAs from wheat germ and yeast were similarly active in the BMV virion assembly reaction, but ribosomal RNA and polyadenylate did not facilitate assembly . Surprisingly, virions assembled from TLS-less BMV RNA in the presence of tRNAs or TLS-containing short RNA did not incorporate the latter molecules . Consistent with a critical role for the BMV TLS in virion assembly, mutations in the BMV genomic RNAs that were designed to disrupt the folding of the TLS also abolished virion assembly . We discuss the likely roles of the TLS in early stages of virion assembly. J Biol Chem, 2002 Mar 22, 277(12), 10201 - 8 Epub 2002 Jan 08. The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase itch; Traweger A et al.; Tight junctions create a highly selective diffusion barrier between epithelial and endothelial cells by preventing the free passage of molecules and ions across the paracellular pathway . Although the regulation of this barrier is still enigmatic, there is evidence that junctional transmembrane proteins are critically involved . Recent evidence confirms the notion that occludin, a four-pass integral plasma-membrane protein, is a functional component of the paracellular barrier . The overall hydrophilicity of occludin predicts two extracellular loops bounded by NH(2)- and COOH-terminal cytoplasmic domains . To date, the binding of the COOH terminus of occludin to intracellular proteins is well documented, but information concerning the function of the cytoplasmic NH(2) terminus is still lacking . Using yeast two-hybrid screening we have identified a novel interaction between occludin and the E3 ubiquitin-protein ligase Itch, a member of the HECT domain-containing ubiquitin-protein ligases . We have found that the NH(2)-terminal portion of occludin binds specifically to a multidomain of Itch, consisting of four WW motifs . This interaction has been confirmed by our results from in vivo and in vitro co-immunoprecipitation experiments . In addition, we provide evidence that Itch is specifically involved in the ubiquitination of occludin in vivo, and that the degradation of occludin is sensitive to proteasome inhibition. J Biol Chem, 2002 Apr 19, 277(16), 13479 - 87 Epub 2002 Jan 08. Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids; Li X et al.; As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta) . These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal . Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS . Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran . The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters . Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52 . The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS . One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl . A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer. J Biol Chem, 2002 Mar 15, 277(11), 9580 - 9 Epub 2002 Jan 08. Members of the Zyxin family of LIM proteins interact with members of the p130Cas family of signal transducers; Yi J et al.; Integrin binding to extracellular matrix proteins induces formation of signaling complexes at focal adhesions . Zyxin co-localizes with integrins at sites of cell-substratum adhesion and is postulated to serve as a docking site for the assembly of multimeric protein complexes involved in regulating cell motility . Recently, we identified a new member of the zyxin family called TRIP6 . TRIP6 is localized at focal adhesions and overexpression of TRIP6 slows cell migration . In an effort to define the molecular mechanism by which TRIP6 affects cell migration, the yeast two-hybrid assay was employed to identify proteins that directly bind to TRIP6 . This assay revealed that both TRIP6 and zyxin interact with CasL/HEF1, a member of the Cas family . This association is mediated by the LIM region of the zyxin family members and the SH2 domain-binding region of CasL/HEF1 . Furthermore, the association between p130(Cas) and the two zyxin family members was demonstrated to occur in vivo by co-immunoprecipitation . Zyxin and Cas family members may cooperate to regulate cell motility. Genes Dev, 2002 Jan 1, 16(1), 58 - 71 A novel zinc finger protein is associated with U7 snRNP and interacts with the stem-loop binding protein in the histone pre-mRNP to stimulate 3'-end processing; Dominski Z et al.; The stem-loop binding protein (SLBP) is the posttranscriptional regulator of histone mRNA in metazoan cells . SLBP binds histone pre-mRNAs and facilitates 3'-end processing by promoting stable association of U7 snRNP with the pre-mRNA . To identify other factors involved in histone pre-mRNA processing, we used a modified yeast two-hybrid assay in which SLBP and its RNA target were coexpressed as bait . A novel zinc finger protein, hZFP100, which interacts with the SLBP/RNA complex but not with free SLBP, was cloned . The interaction requires regions of SLBP that are important for histone pre-mRNA processing . Antibodies to hZFP100 precipitate U7 snRNA, and expression of hZFP100 in Xenopus oocytes stimulates processing of histone pre-mRNA, showing that hZFP100 is a component of the processing machinery. Oncogene, 2001 Dec 13, 20(57), 8276 - 80 The Bloom syndrome protein interacts and cooperates with p53 in regulation of transcription and cell growth control; Garkavtsev IV et al.; Bloom syndrome is an autosomal recessive disorder associated with mutations in BLM gene encoding protein that belongs to the family of DNA helicases . It is characterized by predisposition to cancer, immunodeficiency, high sensitivity to UV and genomic instability of somatic cells . Here we show physical and functional cooperation between BLM and p53 proteins . Ectopic expression of BLM causes anti-proliferative effect in p53 wild type, but not in p53-deficient cells; p53-mediated transactivation is attenuated in primary fibroblasts from Bloom syndrome patients . BLM and p53 proteins physically interact in the cells as demonstrated in yeast and mammalian two-hybrid systems; interaction sites are mapped to 237-272 aa residues of BML and 285-340 aa of p53 . Ectopic expression of the fragment of wild type BML containing p53-interactive domain suppresses p53-mediated transcription and interferes with p53-mediated growth inhibition . These observations indicate that BLM might be an important component of p53 function and suggest that Bloom Syndrome phenotype may in part be the result of the deregulation of the p53 tumor suppressor pathway. Oncogene, 2001 Dec 6, 20(56), 8045 - 56 Double-stranded RNA-dependent protein kinase, PKR, down-regulates CDC2/cyclin B1 and induces apoptosis in non-transformed but not in v-mos transformed cells; Dagon Y et al.; The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells . Paradoxically, while it can function as a tumor suppressor and inducer of apoptosis, it is overexpressed in a variety of human cancers . To resolve this enigma, we established cell-lines that overexpress PKR in non-transformed and in v-mos transformed CHO cells . Overexpression of PKR suppressed the proliferation of CHO cells by inducing a transient G0/G1 arrest, followed by a delayed G2/M arrest, which attenuated cell cycle progression . These effects were accompanied by early induction of p21/WAF-1 and delayed downregulation of CDC2 and cyclin B1 . Induction of proapoptotic activity of the ectopic PKR paralleled the onset of G2/M arrest in CHO cells . However, while transiently inducing p21/WAF-1, PKR did not impose G2/M arrest or apoptosis in v-mos-transformed cells, nor was CDC2 or cyclin B1 down-regulated in those cells . These findings link the proapoptotic activity of PKR to the arrest of cell cycle at the G2/M phase . Consequently, the apoptotic activity of PKR could be counter-acted by an oncogene-like v-mos that overrides the G2/M arrest induced by PKR. Nat Cell Biol, 2001 Dec, 3(12), 1114 - 9 Design and application of a cytokine-receptor-based interaction trap; Eyckerman S et al.; Ligand-induced clustering of type I cytokine receptor subunits leads to trans-phosphorylation and activation of associated cytosolic janus kinases (JAKs) . In turn, JAKs phosphorylate tyrosine residues in the receptor tails, leading to recruitment and activation of signalling molecules . Among these, signal transducers and activators of transcription (STATs) are important in the direct transmission of signals to the nucleus . Here, we show that incorporation of an interaction trap in a signalling-deficient receptor allows the identification of protein-protein interactions, using a STAT-dependent complementation assay . Mammalian protein-protein interaction trap (MAPPIT) adds to existing yeast two-hybrid procedures, as originally explored by Fields and Song, and permits the detection of both modification-independent and of phosphorylation-dependent interactions in intact human cells . We also demonstrate that MAPPIT can be used to screen complex complementary DNA libraries, and using this approach, we identify cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signalling-2 (SOCS-2) as interaction partners of the phosphotyrosine 402 (Tyr 402)-binding motif in the erythropoietin receptor (EpoR) . Importantly, this approach places protein-protein interactions in their normal physiological context, and is especially applicable to the in situ analysis of signal transduction pathways. Clin Microbiol Rev, 2002 Jan, 15(1), 21 - 57 Immunology of diseases associated with Malassezia species; Ashbee HR et al.; Malassezia species are members of the human cutaneous commensal flora, in addition to causing a wide range of cutaneous and systemic diseases in suitably predisposed individuals . Studies examining cellular and humoral immune responses specific to Malassezia species in patients with Malassezia-associated diseases and healthy controls have generally been unable to define significant differences in their immune response . The use of varied antigenic preparations and strains from different Malassezia classifications may partly be responsible for this, although these problems can now be overcome by using techniques based on recent work defining some important antigens and also a new taxonomy for the genus . The finding that the genus Malassezia is immunomodulatory is important in understanding its ability to cause disease . Stimulation of the reticuloendothelial system and activation of the complement cascade contrasts with its ability to suppress cytokine release and downregulate phagocytic uptake and killing . The lipid-rich layer around the yeast appears to be pivotal in this alteration of phenotype . Defining the nonspecific immune response to Malassezia species and the way in which the organisms modulate it may well be the key to understanding how Malassezia species can exist as both commensals and pathogens. Biochemistry, 2002 Jan 15, 41(2), 561 - 9 Regulation of both PDK1 and the phosphorylation of PKC-zeta and -delta by a C-terminal PRK2 fragment; Hodgkinson CP et al.; The mechanism by which PDK1 regulates AGC kinases remains unclear . To further understand this process, we performed a yeast two-hybrid screen using PDK1 as bait . PKC-zeta, PKC-delta, and PRK2 were identified as interactors of PDK1 . A combination of yeast two-hybrid binding assays and coprecipitation from mammalian cells was used to characterize the nature of the PDK1-PKC interaction . The presence of the PH domain of PDK1 inhibited the interaction of PDK1 with the PKCs . A contact region of PDK1 was mapped between residues 314 and 408 . The interaction of PDK1 with the PKCs required the full-length PKC-zeta and -delta proteins apart from their C-terminal tails . PDK1 was able to phosphorylate full-length PKC-zeta and -delta but not PKC-zeta and -delta constructs containing the PDK1 phosphorylation site but lacking the C-terminal tails . A C-terminal PRK2 fragment, normally produced by caspase-3 cleavage during apoptosis, inhibited PDK1 autophosphorylation by >90% . The ability of PDK1 to phosphorylate PKC-zeta and -delta in vitro was also markedly inhibited by the PRK2 fragment . Additionally, generation of the PRK2 fragment in vivo inhibited by >90% the phosphorylation of endogenous PKC-zeta by PDK1 . In conclusion, these results show that the C-terminal tail of PKC is a critical determinant for PKC-zeta and -delta phosphorylation by PDK1 . Moreover, the C-terminal PRK2 fragment acts as a potent negative regulator of PDK1 autophosphorylation and PDK1 kinase activity against PKC-zeta and -delta . As the C-terminal PRK2 fragment is naturally generated during apoptosis, this may provide a mechanism of restraining prosurvival signals during apoptosis. RNA, 2001 Dec, 7(12), 1833 - 44 The snoRNA domain of vertebrate telomerase RNA functions to localize the RNA within the nucleus; Lukowiak AA et al.; Telomerase RNA is an essential component of the ribonucleoprotein enzyme involved in telomere length maintenance, a process implicated in cellular senescence and cancer . Vertebrate telomerase RNAs contain a box H/ACA snoRNA motif that is not required for telomerase activity in vitro but is essential in vivo . Using the Xenopus oocyte system, we have found that the box H/ACA motif functions in the subcellular localization of telomerase RNA . We have characterized the transport and biogenesis of telomerase RNA by injecting labeled wild-type and variant RNAs into Xenopus oocytes and assaying nucleocytoplasmic distribution, intranuclear localization, modification, and protein binding . Although yeast telomerase RNA shares characteristics of spliceosomal snRNAs, we show that human telomerase RNA is not associated with Sm proteins or efficiently imported into the nucleus . In contrast, the transport properties of vertebrate telomerase RNA resemble those of snoRNAs; telomerase RNA is retained in the nucleus and targeted to nucleoli . Furthermore, both nuclear retention and nucleolar localization depend on the box H/ACA motif . Our findings suggest that the H/ACA motif confers functional localization of vertebrate telomerase RNAs to the nucleus, the compartment where telomeres are synthesized . We have also found that telomerase RNA localizes to Cajal bodies, intranuclear structures where it is thought that assembly of various cellular RNPs takes place . Our results identify the Cajal body as a potential site of telomerase RNP biogenesis. RNA, 2001 Dec, 7(12), 1817 - 32 Multiple snoRNA gene clusters from Arabidopsis; Brown JW et al.; Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modification (2'-O-ribose methylation and pseudouridylation) . In all eukaryotes, certain snoRNAs (e.g., U3) are transcribed from classical promoters . In vertebrates, the majority are encoded in introns of protein-coding genes, and are released by exonucleolytic cleavage of linearized intron lariats . In contrast, in maize and yeast, nonintronic snoRNA gene clusters are transcribed as polycistronic pre-snoRNA transcripts from which individual snoRNAs are processed . In this article, 43 clusters of snoRNA genes, an intronic snoRNA, and 10 single genes have been identified by cloning and by computer searches, giving a total of 136 snoRNA gene copies of 71 different snoRNA genes . Of these, 31 represent snoRNA genes novel to plants . A cluster of four U14 snoRNA genes and two clusters containing five different snoRNA genes (U31, snoR4, U33, U51, and snoR5) from Arabidopsis have been isolated and characterized . Of these genes, snoR4 is a novel box C/D snoRNA that has the potential to base pair with the 3' end of 5.8S rRNA and snoR5 is a box H/ACA snoRNA gene . In addition, 42 putative sites of 2'-O-ribose methylation in plant 5.8S, 18S, and 25S rRNAs have been mapped by primer extension analysis, including eight sites novel to plant rRNAs . The results clearly show that, in plants, the most common gene organization is polycistronic and that over a third of predicted and mapped methylation sites are novel to plant rRNAs . The variation in this organization among gene clusters highlights mechanisms of snoRNA evolution. RNA, 2001 Dec, 7(12), 1781 - 92 Small bristles, the Drosophila ortholog of NXF-1, is essential for mRNA export throughout development; Wilkie GS et al.; We identified a temperature-sensitive allele of small bristles (sbr), the Drosophila ortholog of human TAP/NXF-1 and yeast Mex67, in a screen for mutants defective in mRNA export . We show that sbr is essential for the nuclear export of all mRNAs tested in a wide range of tissues and times in development . High resolution and sensitive in situ hybridization detect the rapid accumulation of individual mRNA species in sbr mutant nuclei in particles that are distinct from nascent transcript foci and resemble wild-type export intermediates . The particles become more numerous and intense with increasing time at the restrictive temperature and are exported very rapidly after shifting back to the permissive temperature . The mRNA export block is not due indirectly to a defect in splicing, nuclear protein import, or aberrant nuclear ultrastructure, suggesting that in sbr mutants, mRNA is competent for export but fails to dock or translocate through NPCs . We conclude that NXF-1 is an essential ubiquitous export factor for all mRNAs throughout development in higher eukaryotes. RNA, 2001 Dec, 7(12), 1768 - 80 NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila; Herold A et al.; The conserved family of NXF proteins has been implicated in the export of messenger RNAs from the nucleus . In metazoans, NXFs heterodimerize with p15 . The yeast genome encodes a single NXF protein (Mex67p), but there are multiple nxf genes in metazoans . Whether metazoan NXFs are functionally redundant, or their multiplication reflects an adaptation to a greater substrate complexity or to tissue-specific requirements has not been established . The Drosophila genome encodes one p15 homolog and four putative NXF proteins (NXF1 to NXF4) . Here we show that depletion of the endogenous pools of NXF1 or p15 from Drosophila cells inhibits growth and results in a rapid and robust accumulation of polyadenylated RNAs within the nucleus . Fluorescence in situ hybridizations show that export of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs is inhibited . Depleting endogenous NXF2 or NXF3 has no apparent phenotype . Moreover, NXF4 is not expressed at detectable levels in cultured Drosophila cells . We conclude that Dm NXF1/p15 heterodimers only (but not NXF2-NXF4) mediate the export of the majority of mRNAs in Drosophila cells and that the other members of the NXF family play more specialized or different roles. Chin Med J (Engl), 2001 Jul, 114(7), 743 - 6 Epidemiological investigation of Histoplasma capsulatum infection in China; Zhao B et al.; OBJECTIVE: To provide reliable information concerning the presence or the absence of Histoplasma capsulatum (H . capsulatum) infection in China, and data concerning this respect . METHODS: Three hundred normal people and 435 hospitalized patients, who lived in Hunan and Jiangsu provinces, and the Xinjiang Autonomous Region, were tested with yeast-phase histoplasmin (ALK/Berkerley Biologicals Laboratories, USA) and human pure protein derivative of tuberculin (PPD) on the volar surface of the forearm . Any reaction to the antigens over 5.0 mm in diameter of induration at 48-72 hours was considered positive . RESULTS: A total of 138 subjects (18.8%) in 735 patients reacted to histoplasmin with 5.0-45.0 (9.1 +/- 4.3) mm indurations . Significant differences of positive skin reaction rates in normal subjects were found in Hunan, Jiangsu and Xinjiang (8.9% vs 15.1% vs 2.1%) . The overall positive rate of patients was 25.5% . Patients with tuberculosis {31.7% (78/246)} had a significantly higher positive skin reaction rate in comparison with those suffering from pneumonia {17.7% (11/62)}, lung cancer {20.9% (9/43)}, chronic obstructive pulmonary disease {17.3% (9/52)} and other diseases {12.5% (4/32)} (P < 0.01) . Of 562 cases, 292 cases (52.0%) reacted to PPD with indurations of 5-50 (13.7 +/- 4.9) mm in diameter, 63 cases (11.2%) reacted to both histoplasmin and PPD, while 38 cases (6.9%) reacted to histoplasmin but not to PPD . CONCLUSIONS: The data suggest that there is H . capsulatum herd infection in China . The infection rate in Southeast China is higher than that in the Northwest, and the infection rate of patients with pulmonary tuberculosis is higher than that of normal persons and other pneumonopathy patients. Nature, 2002 Jan 3, 415(6867), 92 - 6 IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA; Calfon M et al.; The unfolded protein response (UPR), caused by stress, matches the folding capacity of endoplasmic reticulum (ER) to the load of client proteins in the organelle . In yeast, processing of HAC1 mRNA by activated Ire1 leads to synthesis of the transcription factor Hac1 and activation of the UPR . The responses to activated IRE1 in metazoans are less well understood . Here we demonstrate that mutations in either ire-1 or the transcription-factor-encoding xbp-1 gene abolished the UPR in Caenorhabditis elegans . Mammalian XBP-1 is essential for immunoglobulin secretion and development of plasma cells, and high levels of XBP-1 messenger RNA are found in specialized secretory cells . Activation of the UPR causes IRE1-dependent splicing of a small intron from the XBP-1 mRNA both in C . elegans and mice . The protein encoded by the processed murine XBP-1 mRNA accumulated during the UPR, whereas the protein encoded by unprocessed mRNA did not . Purified mouse IRE1 accurately cleaved XBP-1 mRNA in vitro, indicating that XBP-1 mRNA is a direct target of IRE1 endonucleolytic activity . Our findings suggest that physiological ER load regulates a developmental decision in higher eukaryotes. Nature, 2002 Jan 3, 415(6867), 88 - 92 Identification of a host protein essential for assembly of immature HIV-1 capsids; Zimmerman C et al.; To form an immature HIV-1 capsid, 1,500 HIV-1 Gag (p55) polypeptides must assemble properly along the host cell plasma membrane . Insect cells and many higher eukaryotic cell types support efficient capsid assembly, but yeast and murine cells do not, indicating that host machinery is required for immature HIV-1 capsid formation . Additionally, in a cell-free system that reconstitutes HIV-1 capsid formation, post-translational assembly events require ATP and a subcellular fraction, suggesting a requirement for a cellular ATP-binding protein . Here we identify such a protein (HP68), described previously as an RNase L inhibitor, and demonstrate that it associates post-translationally with HIV-1 Gag in a cell-free system and human T cells infected with HIV-1 . Using a dominant negative mutant of HP68 in mammalian cells and depletion-reconstitution experiments in the cell-free system, we demonstrate that HP68 is essential for post-translational events in immature HIV-1 capsid assembly . Furthermore, in cells the HP68-Gag complex is associated with HIV-1 Vif, which is involved in virion morphogenesis and infectivity . These findings support a critical role for HP68 in post-translational events of HIV-1 assembly and reveal a previously unappreciated dimension of host-viral interaction. J Biol Chem, 2002 Mar 22, 277(12), 10226 - 35 Epub 2002 Jan 04. The FXXLF motif mediates androgen receptor-specific interactions with coregulators; He B et al.; The androgen receptor (AR) activation function 2 region of the ligand binding domain binds the LXXLL motifs of p160 coactivators weakly, engaging instead in an androgen-dependent, interdomain interaction with an FXXLF motif in the AR NH(2) terminus . Here we show that FXXLF motifs are present in previously reported AR coactivators ARA70/RFG, ARA55/Hic-5, and ARA54, which account for their selection in yeast two-hybrid screens . Mammalian two-hybrid assays, ligand dissociation rate studies, and glutathione S-transferase adsorption assays indicate androgen-dependent selective interactions of these FXXLF motifs with the AR ligand binding domain . Mutagenesis of residues within activation function 2 indicates distinct but overlapping binding sites where specificity depends on sequences within and flanking the FXXLF motif . Mutagenesis of the FXXLF motifs eliminated interaction with the ligand binding domain but only modestly reduced AR coactivation in transcription assays . The studies indicate that the FXXLF binding motif is specific for the AR and mediates interactions both within the AR and with coregulatory proteins. J Biol Chem, 2002 Mar 15, 277(11), 9326 - 34 Epub 2002 Jan 04. A novel zinc finger transcription factor with two isoforms that are differentially repressed by estrogen receptor-alpha; Conroy AT et al.; Estrogen receptor-alpha (ERalpha) can induce the expression of genes in response to estrogen by binding to estrogen response elements in the promoters of target genes . There is growing evidence that ERalpha can alter patterns of gene expression in response to ligand by regulating the activity of other factors through a direct protein-protein interaction . To identify other factors that are regulated by ERalpha, a yeast two-hybrid screen was performed that identified a novel Cys(2)His(2) zinc finger protein named ZER6 . The ZER6 protein contains a Kruppel-associated box domain and six Cys(2)His(2) zinc fingers . Transcripts from the ZER6 gene can have alternate 5' exons and encode either a p71 or p52 isoform . The p52-ZER6 protein interacts strongly with ERalpha in the presence of 17beta-estradiol, whereas the p71-ZER6 isoform has a HUB-1 amino-terminal domain that inhibits the interaction with ERalpha . A consensus ZER6 binding element was defined using PCR-assisted binding site selection . In COS-1 cells, both the p52 and p71 isoforms can activate transcription through the ZER6 binding element; however, in the presence of ERalpha, transactivation by the p52 isoform is specifically repressed . Overexpression of the p52 isoform was able to abrogate activation by p71-ZER6 . Expression of ZER6 was largely restricted to the mammary gland with a lower level of expression in the kidney . We conclude that ZER6 is a novel zinc finger transcription factor in which regulation of transcription in hormone-responsive cells can be controlled by the relative level of expression of two distinct isoforms. Genome Res, 2002 Jan, 12(1), 132 - 44 Analysis of the 5S RNA pool in Arabidopsis thaliana: RNAs are heterogeneous and only two of the genomic 5S loci produce mature 5S RNA; Cloix C et al.; One major 5S RNA, 120 bases long, was revealed by an analysis of mature 5S RNA from tissues, developmental stages, and polysomes in Arabidopsis thaliana . Minor 5S RNA were also found, varying from the major one by one or two base substitutions; 5S rDNA units from each 5S array of the Arabidopsis genome were isolated by PCR using CIC yeast artificial chromosomes (YACs) mapped on the different loci . By using a comparison of the 5S DNA and RNA sequences, we could show that both major and minor 5S transcripts come from only two of the genomic 5S loci: chromosome 4 and chromosome 5 major block . Other 5S loci are either not transcribed or produce rapidly degraded 5S transcripts . Analysis of the 5'- and 3'-DNA flanking sequence has permitted the definition of specific signatures for each 5S rDNA array. Genome Res, 2002 Jan, 12(1), 37 - 46 Relating whole-genome expression data with protein-protein interactions; Jansen R et al.; We investigate the relationship of protein-protein interactions with mRNA expression levels, by integrating a variety of data sources for yeast . We focus on known protein complexes that have clearly defined interactions between their subunits . We find that subunits of the same protein complex show significant coexpression, both in terms of similarities of absolute mRNA levels and expression profiles, e.g., we can often see subunits of a complex having correlated patterns of expression over a time course . We classify the yeast protein complexes as either permanent or transient, with permanent ones being maintained through most cellular conditions . We find that, generally, permanent complexes, such as the ribosome and proteasome, have a particularly strong relationship with expression, while transient ones do not . However, we note that several transient complexes, such as the RNA polymerase II holoenzyme and the replication complex, can