Virology, 1998 Feb 1, 241(1), 49 - 60 The intracellular expression pattern of the human papillomavirus type 11 E1--E4 protein correlates with its ability to self associate; Bryan JT et al.; The function of the human papillomavirus type 11 (HPV 11) E1--E4 spliced protein is not known . E1--E4 protein in HPV-infected tissue is detected in the cytoplasm of differentiated epithelial cells and as immunoreactive bands corresponding to potential monomers, dimers and trimers in immunoblots . The yeast two-hybrid system was employed to test for self association of the HPV 11 E1--E4 protein . To confirm the results of the yeast two-hybrid experiments, coimmunofluorescence studies of a green fluorescent fusion protein (GFP-E1--E4) and a T7 epitope-tagged E1--E4 protein were performed in C33a keratinocytes . E1--E4 protein was shown to self associate in the yeast two-hybrid system, and this result was confirmed by colocalization of GFP-E1--E4 and T7-E1(wedge)E4 proteins in keratinocytes . Analysis of E1--E4 mutants established that the C-terminus was required for self association and that sequences in the N-terminus influenced the intracellular localization of E1--E4 protein . The intracellular expression patterns of GFP-E1--E4 and GFP-E1--E4 mutants were correlated with E1--E4 binding in the yeast two-hybrid system . Those E1--E4 mutants that did not self associate in the yeast two-hybrid system were detected as diffuse cellular fluorescence when expressed as GFP fusions . In contrast, GFP-E1--E4 was detected as a perinuclear aggregate . All E1--E4 mutants capable of associating with E1--E4 in the yeast two-hybrid system were detected as aggregates when expressed as GFP fusion proteins in keratinocytes . Science, 1998 Feb 6, 279(5352), 876 - 8 Meiotic synapsis in the absence of recombination; McKim KS et al.; Although in Saccharomyces cerevisiae the initiation of meiotic recombination, as indicated by double-strand break formation, appears to be functionally linked to the initiation of synapsis, meiotic chromosome synapsis in Drosophila females occurs in the absence of meiotic exchange . Electron microscopy of oocytes from females homozygous for either of two meiotic mutants (mei-W68 and mei-P22), which eliminate both meiotic crossing over and gene conversion, revealed normal synaptonemal complex formation . Thus, synapsis in Drosophila is independent of meiotic recombination, consistent with a model in which synapsis is required for the initiation of meiotic recombination . Furthermore, the basic processes of early meiosis may have different functional or temporal relations, or both, in yeast and Drosophila. Science, 1998 Feb 6, 279(5352), 857 - 60 A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles; Raghunathan PL et al.; The spliceosome removes introns from pre-messenger RNAs by a mechanism that entails extensive remodeling of RNA structure . The most conspicuous rearrangement involves disruption of 24 base pairs between U4 and U6 small nuclear RNAs (snRNAs) . Here, the yeast RNA binding protein Prp24 is shown to reanneal these snRNAs . When Prp24 is absent, unpaired U4 and U6 small nuclear ribonucleoprotein particles (snRNPs) accumulate; with time, splicing becomes inhibited . Addition of purified Prp24 protein regenerates duplex U4/U6 snRNPs for new rounds of splicing . The reannealing reaction catalyzed by Prp24 proceeds more efficiently with snRNPs than with deproteinized snRNAs. Genes Dev, 1998 Feb 1, 12(3), 370 - 81 Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity; Larochelle S et al.; Cdk7 has been shown previously to be able to phosphorylate and activate many different Cdks in vitro . However, conclusive evidence that Cdk7 acts as a Cdk-activating kinase (CAK) in vivo has remained elusive . Adding to the controversy is the fact that in the budding yeast Saccharomyces cerevisiae, CAK activity is provided by the CAK1/Civ1 protein, which is unrelated to Cdk7 . Furthermore Kin28, the budding yeast Cdk7 homolog, functions not as a CAK but as the catalytic subunit of TFIIH . Vertebrate Cdk7 is also known to be part of TFIIH . Therefore, in the absence of better genetic evidence, it was proposed that the CAK activity of Cdk7 may be an in vitro artifact . In an attempt to resolve this issue, we cloned the Drosophila cdk7 homolog and created null and temperature-sensitive mutations . Here we demonstrate that cdk7 is necessary for CAK activity in vivo in a multicellular organism . We show that cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division . These results suggest that there may be a fundamental difference in the way metazoans and budding yeast effect a key modification of Cdks. Genes Dev, 1998 Feb 1, 12(3), 343 - 56 DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs; Wada T et al.; We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) . This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8 . DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits . Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5 . Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5 . In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates . A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG . The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo. J Cell Biol, 1998 Jan 26, 140(2), 271 - 81 Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2; Hua XH et al.; Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes . Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway . If this displacement is inhibited, DNA replication fails to initiate . We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication . Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity . Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity. J Cell Biol, 1998 Jan 26, 140(2), 259 - 70 Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association; Mahajan R et al.; The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1 . Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1 . SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101- amino acid protein are proteolytically removed before its attachment to RanGAP1 . Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo . In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1 . Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import . Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1 . RanGAP1-SUMO-1 remains stably associated with the NE during many cycles of in vitro import . This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role. EMBO J, 1998 Jan 15, 17(2), 609 - 14 Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks; Ramsden DA et al.; Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase . Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks . We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell . This effect of Ku is specific to eukaryotic DNA ligases . Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase. EMBO J, 1998 Jan 15, 17(2), 575 - 89 Identifying the right stop: determining how the surveillance complex recognizes and degrades an aberrant mRNA; Ruiz-Echevarria MJ et al.; The nonsense-mediated mRNA decay (NMD) pathway functions by checking whether translation termination has occurred prematurely and subsequently degrading the aberrant mRNAs . In Saccharomyces cerevisiae, it has been proposed that a surveillance complex scans 3' of the premature termination codon and searches for the downstream element (DSE), whose recognition by the complex identifies the transcript as aberrant and promotes its rapid decay . The results presented here suggest that translation termination is important for assembly of the surveillance complex . Neither the activity of the initiation ternary complex after premature translation termination has occurred nor the elongation phase of translation are essential for the activity of the NMD pathway . Once assembled, the surveillance complex is active for searching and recognizing a DSE for approximately 200 nt 3' of the stop codon . We have also identified a stabilizer sequence (STE) in the GCN4 leader region that inactivates the NMD pathway . Inactivation of the NMD pathway, as a consequence of either the DSE being too far from a stop codon or the presence of the STE, can be circumvented by inserting sequences containing a new translation initiation/termination cycle immediately 5' of the DSE . Further, the results indicate that the STE functions in the context of the GCN4 transcript to inactivate the NMD pathway. EMBO J, 1998 Jan 15, 17(2), 507 - 19 The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways; Voegel JJ et al.; The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2 . The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL . Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs . The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity . Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation . TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain . In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP . TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells . Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs. EMBO J, 1998 Jan 15, 17(2), 368 - 83 Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway; Lisztwan J et al.; In normal and transformed cells, the F-box protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2 . Here we identify human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo . CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiprotein complex . In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34 . The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 ubiquitin protein ligase . We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase . Finally, we show that multiprotein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2 . The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway. EMBO J, 1998 Jan 2, 17(1), 288 - 96 The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins; O'Neill RE et al.; Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes . Influenza virus replicates its RNA genome in the nucleus of infected cells . The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell . A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein . Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process . Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP . We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex. EMBO J, 1998 Jan 2, 17(1), 204 - 14 Integrity of a Zn finger-like domain in SamB is crucial for morphogenesis in ascomycetous fungi; Kruger M et al.; Genetic features determine the site of polarized growth in filamentous fungi and lead to hyphal tip extension or subapical branching . We have isolated the samB gene (suppressor of anucleate metulae) of Aspergillus nidulans which encodes a 66 kDa protein carrying an atypical Cys4 and an additional Cys2/His/Cys Zn finger motif at the carboxy-terminus . Such novel Zn finger-like domains have recently been found in several other developmental regulators in organisms ranging from yeast to man . Deletion of this domain at the carboxy-terminus of SamB led to premature hyphal ramification, mislocalization of septa and suppression of the asporogenous phenotype of the developmental mutant aps (anucleate primary sterigmata) . A DeltasamB deletion strain displayed an identical phenotype . A homologous gene in Saccharomyces cerevisiae was also characterized whose deletion resulted in a multi-budding phenotype; thus it was named MUB1 . An underlying common mechanism for both genes in determination of the onset of polarized growth and its links to other cellular developmental processes is discussed. EMBO J, 1998 Jan 2, 17(1), 149 - 58 The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3; Nagata K et al.; The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family . In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif . The C-terminal regions of the proteins are essentially unrelated . Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac . Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38 . When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules . In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library . MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function. Biochem J, 1998 Jan 15, 329 ( Pt 2), 395 - 403 Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains; Tell G et al.; The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung . TTF-1 contains two transcriptional activation domains (N and C domain) . The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains . In aqueous solution the N domain exists in a random-coil conformation . The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure . Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced . Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains . Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1 . In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast . Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain . Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains . TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development. Biochem J, 1998 Jan 15, 329 ( Pt 2), 321 - 8 Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past; Price NT et al.; Measurement of monocarboxylate transport kinetics in a range of cell types has provided strong circumstantial evidence for a family of monocarboxylate transporters (MCTs) . Two mammalian MCT isoforms (MCT1 and MCT2) and a chicken isoform (REMP or MCT3) have already been cloned, sequenced and expressed, and another MCT-like sequence (XPCT) has been identified . Here we report the identification of new human MCT homologues in the database of expression sequence tags and the cloning and sequencing of four new full-length MCT-like sequences from human cDNA libraries, which we have denoted MCT3, MCT4, MCT5 and MCT6 . Northern blotting revealed a unique tissue distribution for the expression of mRNA for each of the seven putative MCT isoforms (MCT1-MCT6 and XPCT) . All sequences were predicted to have 12 transmembrane (TM) helical domains with a large intracellular loop between TM6 and TM7 . Multiple sequence alignments showed identities ranging from 20% to 55%, with the greatest conservation in the predicted TM regions and more variation in the C-terminal than the N-terminal region . Searching of additional sequence databases identified candidate MCT homologues from the yeast Saccharomyces cerevisiae, the nematode worm Caenorhabditis elegans and the archaebacterium Sulfolobus solfataricus . Together these sequences constitute a new family of transporters with some strongly conserved sequence motifs, the possible functions of which are discussed. Nucleic Acids Res, 1998 Jan 15, 26(2), 623 - 30 Green fluorescent protein as a scaffold for intracellular presentation of peptides; Abedi MR et al.; Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules . Such aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory activities . Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical . To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display of conformationally constrained peptides . The GFP-peptide libraries permit analysis of library diversity and expression levels in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of correctly folded protein, by selection for high fluorescence. Nucleic Acids Res, 1997 Dec 15, 25(24), 5085 - 94 Use of an engineered ribozyme to produce a circular human exon; Mikheeva S et al.; We report the use of an engineered ribozyme to produce a circular human exon in vitro . Specifically, we have designed a derivative of a yeast self-splicing group II intron that is able to catalyze the formation of a circular exon encoding the first kringle domain (K1) of the human tissue plasminogen activator protein . We show that the circular K1 exon is formed with high fidelity in vitro . Furthermore, the system is designed such that the circular exon that is produced consists entirely of human exon sequence . Thus, our results demonstrate that all yeast exon sequences are dispensable for group II intron catalyzed inverse splicing . This is the first demonstration that an engineered ribozyme can be used to create a circular exon containing only human sequences, linked together at a precise desired ligation point . We expect these results to be generalizable, so that similar ribozymes can be designed to precisely create circular derivatives of any nucleotide sequence. Nucleic Acids Res, 1997 Dec 15, 25(24), 5033 - 40 Genetic interactions of conserved regions in the DEAD-box protein Prp28p; Chang TH et al.; The yeast PRP28 g ene has been implicated in nuclear precursor messenger RNA (pre-mRNA) splicing, a two-step reaction involved in a multitude of RNA structural alterations . Prp28p, the gene product of PRP28 , is a member of the evolutionarily conserved DEAD-box proteins (DBPs) . Members of DBPs are involved in a variety of RNA-related biochemical processes, presumably by their putative RNA helicase activities . Prp28p has been speculated to play a role in melting the duplex between U4 and U6 small nuclear RNAs (snRNAs), leading to the formation of an active spliceosome . To study the function of Prp28p and its interactions with other components of the splicing machinery, we have isolated and characterized a large number of prp28 conditional mutants . Strikingly, many of these prp28 mutations are localized in the highly conserved motifs found in all the DBPs . Intragenic reversion analysis suggests that regions of motifs II, III and V, as well as of motifs I and IV, in Prp28p are likely to be in close proximity to each other . Our results thus provide the first hint of the local structural arrangement for Prp28p, and perhaps for other DBPs as well. J Exp Biol, 1998 Jan, 201(Pt 1), 115 - 20 Metal ions suppress the abnormal taste behavior of the Drosophila mutant malvolio; Orgad S et al.; A mutation in the malvolio (mvl) gene affects taste behavior in Drosophila melanogaster . The malvolio gene encodes a protein (MVL) that exhibits homology to the mammalian natural resistance-associated macrophage proteins . It is also homologous to the Smf1 protein from Saccharomyces cerevisiae, which we have recently demonstrated to function as a Mn2+/Zn2+ transporter . We proposed that the Drosophila and mammalian proteins, like the yeast SMF1 gene product, are metal-ion transporters . To test this hypothesis, malvolio mutant flies were allowed to develop, from egg to adulthood, on a medium containing elevated concentrations of metal ions . Mutant flies that were reared in the presence of 10 mmol l-1 MnCl2 or FeCl2 developed into adults with recovered taste behavior . CaCl2 or MgCl2 had no effect on the mutant's taste perception . ZnCl2 inhibited the effect of MnCl2 when both ions were supplied together . Similar suppression of the abnormal taste behavior was observed when mvl mutants were fed MnCl2 or FeCl2 only at the adult stage . Furthermore, exposure of adult mutant flies to these ions in the testing plate for only 2 h was sufficient to restore normal taste behavior . The suppression of the defective taste behavior suggests that MVL functions as a Mn2+/Fe2+ transporter and that Mn2+ and/or Fe2+ are involved in the signal transduction of taste perception in Drosophila adults. Curr Biol, 1997 Nov 1, 7(11), 850 - 9 Translational attenuation mediated by an mRNA intron; Chapman RE et al.; BACKGROUND: The unfolded protein response (UPR) is an intracellular signaling pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) . The UPR results in an increase in transcription of ER-resident proteins that facilitate protein folding in the ER . A key regulatory step in UPR activation is the regulated splicing of HAC1 mRNA, which encodes Hac1p, a transcription factor dedicated to this pathway . Hac1p can be detected only when the spliced form of HAC1 mRNA (termed HAC1i mRNA, for induced) is produced; this was surprising because the unspliced HAC1u mRNA (HAC1u for uninduced) is equally stable in cells . RESULTS: We show that in contrast to most other unspliced pre-mRNAs, the HAC1u mRNA is transported from the nucleus into the cytosol . Although HAC1u mRNA is associated with polyribosomes, no detectable Hac1pu is produced unless the intron is removed, indicating that the presence of the intron prevents mRNA translation . When it is produced, Hac1pu has a stability similar to that of Hac1pi, the form of the Hac1p that is produced from the spliced mRNA and that differs from Hac1pu by a short carboxy-terminal tail sequence . Hac1pu, however, is differently modified and less active in activating transcription . Interestingly, when transplanted into the 3' untranslated region of a completely unrelated mRNA, the HAC1 intron is sufficient to attenuate translation of the preceding open reading frame . CONCLUSIONS: We have shown that the HAC1 mRNA intron is both necessary and sufficient to prevent complete translation of polyribosome-associated mRNAs . To our knowledge, this identifies a new way by which translation of a mRNA can be attenuated. Curr Biol, 1997 Nov 1, 7(11), 905 - 8 Association of homologous chromosomes during floral development; Aragon-Alcaide L et al.; Reduction in chromosome number and genetic recombination during meiosis require the prior association of homologous chromosomes, and this has been assumed to be a central event in meiosis . Various studies have suggested, however, that while the reduction division of meiosis is a universally conserved process, the pre-meiotic association of homologues differs among organisms . In the fruit fly Drosophila melanogaster, some somatic tissues also show association of homologues {1,2} . In the budding yeast Saccharomyces cerevisiae, there is some evidence for homologue association during the interphase before meiotic division {3,4}, and it has been argued that such associations lead directly to meiotic homologue pairing during prophase I {5} . The available evidence for mammals suggests that homologous chromosomes do not associate in germ cells prior to meiotic prophase {6} . To study the occurrence of homologue pairing in wheat, we have used vibratome tissue sections of wheat florets to determine the location of homologous chromosomes, centromeres and telomeres in different cell types of developing anthers . Fluorescence in situ hybridization followed by confocal microscopy demonstrated that homologous chromosomes associate pre-meiotically in meiocytes (germ-line cells) . Surprisingly, association of homologues was observed simultaneously in all the surrounding somatic tapetum cells . Homologues failed to associate at equivalent stages in a homologue recognition mutant . These results demonstrate that the factors responsible for the recognition and association of homologues in wheat act before the onset of meiotic prophase . The observation of homologue association in somatic tapetum cells demonstrates that this process and meiotic division are separable. J Mol Recognit, 1997 Jul-Aug, 10(4), 182 - 7 Studies of metabolic control using NMR and molecular genetics; Brindle KM et al.; The techniques of NMR spectroscopy and molecular genetics have provided new and powerful approaches to studying the control and organisation of cellular metabolism in vivo . We review here our recent applications of these methodologies to the study of energy metabolism in yeast and mammalian cells. Comput Appl Biosci, 1997 Dec, 13(6), 609 - 15 SAMBA: hardware accelerator for biological sequence comparison; Guerdoux-Jamet P et al.; MOTIVATION: SAMBA (Systolic Accelerator for Molecular Biological Applications) is a 128 processor hardware accelerator for speeding up the sequence comparison process . The short-term objective is to provide a low-cost board to boost PC or workstation performance on this class of applications . This paper places SAMBA amongst other existing systems and highlights the original features . RESULTS: Real performance obtained from the prototype is demonstrated . For example, a sequence of 300 amino acids is scanned against SWISS-PROT-34 (21 210 389 residues) in 30 s using the Smith and Waterman algorithm . More time-consuming applications, like the bank-to-bank comparison, are computed in a few hours instead of days on standard workstations . Technology allows the prototype to fit onto a single PCI board for plugging into any PC or workstation . AVAILABILITY: SAMBA can be tested on the WEB server at URL http://www.irisa.fr/SAMBA/. J Neurovirol, 1997 Dec, 3(6), 455 - 9 C2H2-546: a zinc finger protein differentially expressed in HTLV-1 infected T cells; Drew PD et al.; We report the cloning and characterization of a novel cDNA termed C2H2-546 which encodes a C2H2-type zinc finger protein . C2H2-546 RNA is expressed in the HTLV-1 infected T cells examined which were derived from HAM-TSP patients, but not in T cells derived from ATL patients . The C2H2-546 gene is conserved in humans and primates and maps to chromosome 10q11.2, a site associated with a variety of cancers . Thus, C2H2-546 is a candidate regulatory molecule important in the formation of these tumors, and may serve as an important marker to distinguish HTLV-1 infected ATL versus HAM-TSP T cell lineages. Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 73 - 8 Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal reorganization; Miki H et al.; Verprolin is a yeast protein whose inactivation leads to a cytoskeletal defect characterized by the abnormal organization of actin filaments . Recently, two mammalian proteins previously shown to regulate the actin cytoskeleton, Wiskott-Aldrich Syndrome Protein (WASP) and its homolog expressed in neurons (N-WASP), were found to possess short peptide motifs homologous to one part of verprolin . However, the physiological function of the homologous regions (verprolin-homology domain, VPH domain) remains unknown . Here we report the importance of the VPH domain as the direct actin binding region . In the case of N-WASP, the VPH domain co-acts with the cofilinhomologous region to sever actin filaments in vitro . Furthermore, the VPH domain is indispensable for the reorganization of the actin cytoskeleton by N-WASP downstream of tyrosine kinases in living cells . All data demonstrate that the VPH domain plays critical roles in the regulation of the actin cytoskeleton. Science, 1998 Jan 30, 279(5351), 703 - 7 Transcription factor-specific requirements for coactivators and their acetyltransferase functions; Korzus E et al.; Different classes of mammalian transcription factors-nuclear receptors, cyclic adenosine 3',5'-monophosphate-regulated enhancer binding protein (CREB), and signal transducer and activator of transcription-1 (STAT-1)-functionally require distinct components of the coactivator complex, including CREB-binding protein (CBP/p300), nuclear receptor coactivators (NCoAs), and p300/CBP-associated factor (p/CAF), based on their platform or assembly properties . Retinoic acid receptor, CREB, and STAT-1 also require different histone acetyltransferase (HAT) activities to activate transcription . Thus, transcription factor-specific differences in configuration and content of the coactivator complex dictate requirements for specific acetyltransferase activities, providing an explanation, at least in part, for the presence of multiple HAT components of the complex. Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 382 - 7 Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase; Gu T et al.; Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing) . This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified . Using the yeast two-hybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases . In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent . Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response. Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 150 - 5 Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response; Mandala SM et al.; The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells . This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that influences growth and survival of yeast . Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G1 arrest and cell death . We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide . LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis . LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity . In vitro characterization of Lbp1p shows that this phosphatase is Mg2+-independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine . The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels . Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock . Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells. Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 132 - 7 Drosophila NURF-55, a WD repeat protein involved in histone metabolism; Martinez-Balbas MA et al.; The Drosophila nucleosome remodeling factor (NURF) is a protein complex of four distinct subunits that assists transcription factor-mediated chromatin remodeling . One NURF subunit, ISWI, is related to the transcriptional regulators Drosophila brahma and yeast SWI2/SNF2 . We have determined peptide sequences and isolated cDNA clones for a second NURF component (the 55-kDa subunit) . Immunological studies show that p55 is an integral subunit of NURF and is generally associated with polytene chromosomes . The predicted sequence of p55 reveals a WD repeat protein that is identical with the 55-kDa subunit of the Drosophila chromatin assembly factor (CAF-1) . Given that WD repeat proteins related to p55 are associated with histone deacetylase and histone acetyltransferase, our findings suggest that p55 and its homologs may function as a common platform for the assembly of protein complexes involved in chromatin metabolism. EMBO J, 1997 Dec 15, 16(24), 7317 - 25 Autocatalytic processing of the ATP-dependent PIM1 protease: crucial function of a pro-region for sorting to mitochondria; Wagner I et al.; The biogenesis of the ATP-dependent PIM1 protease of mitochondria was studied by mutational analysis . The ATPase and proteolytic activities of PIM1 were shown to be essential for mitochondrial function . A proteolytically inactive mutant form of PIM1 protease accumulated as a pro-form in mitochondria, revealing a two-step processing of PIM1: the matrix targeting signal is removed by the mitochondrial processing peptidase and then a pro-region of 61 amino acids is cleaved off in an autocatalytic reaction . This latter process depended on the ATP-dependent assembly of PIM1 protease subunits and can occur by an intermolecular and, most probably, also an intramolecular pathway . The respiratory competence of cells harboring mutant PIM1 protease lacking the pro-region was strongly impaired . Subcellular fractionation revealed a cytosolic localization of mutant PIM1 protease . This demonstrates the requirement for the propeptide for efficient sorting of PIM1 protease to mitochondria. Curr Biol, 1997 Dec 1, 7(12), 930 - 9 Interphase chromosomes undergo constrained diffusional motion in living cells; Marshall WF et al.; BACKGROUND: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies . Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus . RESULTS: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll . Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions . We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus . Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility . Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture . CONCLUSIONS: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus . This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place . These results lead to a model for the regulation of chromosome interactions by nuclear architecture. J Cell Sci, 1997 Dec, 110 ( Pt 24), 3019 - 30 Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts; Nicolas FJ et al.; Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation . By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1) . Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-beta/karyopherin-beta and importin-alpha/karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins . These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport . Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran . Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin . They fail to actively import proteins and do not undergo DNA replication . By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted . However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei . These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1. Curr Opin Genet Dev, 1997 Dec, 7(6), 822 - 8 Chromosome ends: all the same under their caps; Pryde FE et al.; The recent characterisation of subtelomeric regions from a variety of organisms from yeast to man has led to the realisation that all chromosome ends are similar in structure although maintenance of the terminus varies . The mosaic of repeats and proteins associated with telomeres has an architectural role which divides the genome into two domains, allowing for the adaptive use of the region as well as the evolution of non-telomerase-mediated telomere maintenance. FEBS Lett, 1998 Jan 16, 421(3), 185 - 90 ABI2, a second protein phosphatase 2C involved in abscisic acid signal transduction in Arabidopsis; Rodriguez PL et al.; The abi2-1 (abscisic acid insensitive) mutant of Arabidopsis thaliana shows abscisic acid (ABA) insensitivity with respect to seed germination and vegetative ABA responses . We identified the ABI2 gene by a combination of positional mapping and homology to ABI1 . The ABI2 protein shows 80% amino acid sequence identity to ABI1, a protein phosphatase 2C (PP2C) involved in ABA signaling . The mutation that confers the abi2-1 phenotype is equivalent to the mutation previously identified in abi1-1 and the resulting Gly168Asp abi2 protein shows a reduced PP2C activity . Thus, a pair of highly homologous PP2Cs regulate ABA signaling. FEBS Lett, 1998 Jan 9, 421(2), 109 - 14 Substrate and sequential site specificity of cytoplasmic histone acetyltransferases of maize and rat liver; Kolle D et al.; The cytoplasmic B-type histone acetyltransferase was purified to apparent homogeneity from maize embryos . We established a novel protocol for easy large-scale preparation of acetylated core histone species, using preparative acetic acid-urea-Triton PAGE . The pure maize histone acetyltransferase B was highly specific for histone H4 under various assay conditions, modifying H4 up to the di-acetylated isoform . Only non-acetylated H4 isoform was accepted as substrate, whereas mono-acetylated H4 could not be further acetylated . The enzyme selectively acetylated lysines 12 and 5 in a sequential manner . The same results were obtained with a partially purified cytoplasmic histone acetyltransferase of rat liver . Protein sequencing results were supported by immunological characterization of acetylated H4 subspecies with site-specific H4-acetyllysine antibodies. Oncogene, 1998 Jan 8, 16(1), 43 - 52 Association of Bovine Papillomavirus Type 1 E6 oncoprotein with the focal adhesion protein paxillin through a conserved protein interaction motif; Vande Pol SB et al.; We have found that the E6 oncoprotein of Bovine Papillomavirus Type 1 (BE6) as well as the E6 protein of the cancer associated HPV-16 (16E6) interact with the focal adhesion protein paxillin . Mutational analysis of paxillin revealed that BE6 binds paxillin through small protein interaction motifs called LD motifs that have been previously identified as important in regulating association of paxillin with vinculin and focal adhesion kinase (FAK), and that BE6 can interact with at least two separate binding sites on paxillin . The LD motifs of paxillin that bind BE6 share homology with the E6 binding site of E6-AP, a ubiquitin ligase that together with 16E6 targets the degradation of the p53 tumor suppressor . Paxillin binding to BE6 excludes simultaneous binding to E6-AP . Mutational analysis of BE6 can distinguish the interaction of BE6 with E6-AP compared to paxillin and revealed that the interaction of BE6 with paxillin may be necessary for the induction of anchorage independent growth of cells by BE6. Biochemistry (Mosc), 1997 Nov, 62(11), 1202 - 5 Telomerase is a true reverse transcriptase . A review; Cech TR et al.; Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme . The RNA subunit, which contains the template for DNA synthesis, has been identified in many organisms . Recently, the protein subunit that catalyzes telomeric DNA extension has also been identified in Euplotes aediculatus and Saccharomyces cerevisiae . It has sequence and functional characteristics of a reverse transcriptase related to retrotransposon and retroviral reverse transcriptases, so this new family of telomerase subunits has been named TRT (Telomerase Reverse Transcriptase) . We find it remarkable that the same type of protein structure required for retroviral replication is now seen to be essential for normal chromosome telomere replication in diverse eukaryotes. Biochemistry, 1998 Jan 27, 37(4), 965 - 70 Structure-based design of a dimeric zinc finger protein; Pomerantz JL et al.; Designing DNA-binding proteins with novel sequence specificities may provide valuable tools for biological research and gene therapy . Computer modeling was used to design a dimeric zinc finger protein, ZFGD1, containing zinc fingers 1 and 2 from Zif268 and a portion of the dimerization domain of GAL4 . ZFGD1 binds with high affinity and specificity to the predicted binding site, which contains two 6 base-pair symmetry-related zinc finger subsites separated by a 13 base-pair spacer . The DNA-binding specificity of this fusion protein is determined primarily by the zinc fingers and can be systematically altered through the substitution of the zinc fingers with variants selected by phage display . This zinc finger-GAL4 fusion may serve as a prototype for designed DNA-binding proteins that could exploit advantages of homo- and heterodimer formation, and the adaptability of the Cys2His2 zinc finger motif, to target virtually any site in the genome. J Mol Biol, 1998 Jan 16, 275(2), 379 - 88 Native tertiary structure in an A-state; Marmorino JL et al.; The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway protein folding intermediate with native secondary structure and non-native, fluctuating tertiary structure . We used yeast iso-1-ferricytochrome c to test for an evolutionary-invariant tertiary interaction in its A-state . Thermal denaturation monitored by circular dichroism (CD)spectropolarimetry was used to determine A-state and native-state stabilities, delta GA reversible D and delta GN reversible D . We examined the wild-type protein, seven variants with substitutions at the interface between the N and C-terminal helices, and four control variants . The controls have the same amino acid changes as the interface variants, but the changes are close to, not at, the interface . We also examined the pH and sulfate concentration dependencies and found that while these factors affect the far-UV CD spectra of the least stable variants, they do not alter the difference in stability between the wild-type protein and the variants . A delta GA reversible D versus-delta GN reversible D plot for the interface variants has a slope near unity and the control variants have near-wild-type stability . These results show that the helix-helix interaction stabilizes the A-state and the native state to the same degree, confirming our preliminary report . We determined that the heat capacity change for A-state denaturation is approximately 60% of the value for native-state denaturation, indicating that the A-state interior is native-like . We discuss our results in relation to ferricytochrome c folding kinetics. Int J Cancer, 1998 Feb 9, 75(4), 568 - 77 Tumor-suppressive activity of the growth arrest-specific gene GAS1 in human tumor cell lines; Evdokiou A et al.; The GAS1 gene product induces growth arrest through a p53-dependent mechanism . To investigate whether GAS1 is a tumor suppressor gene, we transfected GAS1-negative human tumor cells with GAS1 plasmids and analyzed growth characteristics of stable transfectants . When a constitutively expressing GAS1 plasmid was transfected into A549 cells, no stable colonies expressing GAS1 were isolated . When A549 cells were transfected with a dexamethasone-inducible GAS1 plasmid, expression of GAS1 inhibited growth in vitro, and fewer slow-growing tumors arose in nude mice . GAS1 also inhibited proliferation of an HT1080 subline with wild-type (wt) p53 and normal MDM2 . However, when the HT1080 subline HTD114 was transfected with the constitutive GAS1 plasmid, there was no reduction in colony number . GAS1-transfectant clones had unaltered growth in vitro, were morphologically unchanged and showed no difference in their ability to form tumors in nude mice . Although HTD114 cells contain wt p53, levels of MDM2 were elevated by 10-15 fold . The HT1080-6TGc5 subline with mutant p53 and normal MDM2 was also refractory to GAS1 . Our results show that GAS1 suppresses the growth and tumorigenicity of human tumor cells and overexpression of MDM2 or p53 mutation inhibits the GAS1-mediated growth-suppressing pathway. Int Immunol, 1997 Dec, 9(12), 1885 - 95 Regulation of the human IgE receptor (Fc epsilonRII/CD23) by Epstein-Barr virus (EBV): Ku autoantigen binds specifically to an EBV-responsive enhancer of CD23; Shieh B et al.; An early and critical event in immortalization of human B cells by Epstein-Barr virus (EBV) is induction of CD23 expression . CD23 is constitutively expressed in all EBV-immortalized B cells and its expression is tightly linked with immortalization . We have previously shown that activation of CD23 by EBV occurs at the transcriptional level and is mediated, in part, by EBV-responsive enhancer elements in the region of the type a promoter . We have localized one EBV-responsive enhancer (designated EBVRE) to a 37 bp sequence in intron 1 of type a CD23 that contains a GC-rich sequence that binds nuclear protein(s) from EBV-positive but not EBV-negative cells with sequence specificity . This EBVRE-binding activity was enhanced by protein phosphorylation and did not react with antibodies to the ubiquitous GC box transcription factor, Sp1 . We have now shown by protein purification with peptide sequencing and immunological reactivity that p70/p80 Ku autoantigen {the DNA-binding component of DNA-dependent protein kinase (DNA-PK)} binds to this EBVRE with high affinity and sequence specificity . Although Ku autoantigen is ubiquitously expressed, an EBV-specific DNA-protein complex that contains Ku was elicited from EBV-positive but not EBV-negative nuclear extracts . Furthermore, the formation of this EBV-specific DNA-Ku complex was dramatically enhanced by protein phosphorylation . Thus, we have identified EBVRE-binding activity that contains the Ku autoantigen, is DNA sequence specific and is present in EBV-positive but not EBV-negative nuclear extracts . The possible functional significance of the Ku autoantigen-EBVRE interaction is discussed in light of the role of DNA-PK in phosphorylation and activation of several transcription factors . We suggest that phosphorylation of the EBV-specific EBVRE-binding activity by DNA-PK may modulate its activity as a transcription factor. Nat Genet, 1998 Feb, 18(2), 147 - 9 Mutations in PROP1 cause familial combined pituitary hormone deficiency; Wu W et al.; Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones . Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) is preserved . The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism . We have recently positionally cloned the putative Ames dwarf gene Prop1, which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression . In this report, we have identified four CPHD families with homozygosity or compound heterozygosity for inactivating mutations of PROP1 . These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation . In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously . Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes. J Math Biol, 1997 Dec, 36(2), 119 - 32 Local and global bifurcations at infinity in models of glycolytic oscillations; Sturis J et al.; We investigate two models of glycolytic oscillations . Each model consists of two coupled nonlinear ordinary differential equations . Both models are found to have a saddle point at infinity and to exhibit a saddle-node bifurcation at infinity, giving rise to a second saddle and a stable node at infinity . Depending on model parameters, a stable limit cycle may blow up to infinite period and amplitude and disappear in the bifurcation, and after the bifurcation, the stable node at infinity then attracts all trajectories . Alternatively, the stable node at infinity may coexist with either a stable sink (not at infinity) or a stable limit cycle . This limit cycle may then disappear in a heteroclinic bifurcation at infinity in which the unstable manifold from one saddle at infinity joins the stable manifold of the other saddle at infinity . These results explain prior reports for one of the models concerning parameter values for which the system does not admit any physical (bounded) behavior . Analytic results on the scaling of amplitude and period close to the bifurcations are obtained and confirmed by numerical computations . Finally, we consider more realistic modified models where all solutions are bounded and show that some of the features stemming from the bifurcations at infinity are still present. Nat Genet, 1998 Feb, 18(2), 143 - 6 The itchy locus encodes a novel ubiquitin protein ligase that is disrupted in a18H mice; Perry WL et al.; Non-agouti-lethal 18H (a18H) mice are dark agouti with black pinna hairs . What makes these mice unique is that they develop a spectrum of immunological diseases not seen in other agouti mutant mice . On the JU/Ct background, a18H mice develop an inflammatory disease of the large intestine . On the C57BL/6J background, they develop a fatal disease characterized by pulmonary chronic interstitial inflammation and alveolar proteinosis, inflammation of the glandular stomach and skin resulting in scarring due to constant itching, and hyperplasia of lymphoid cells, haematopoietic cells and the forestomach epithelium . Previous studies suggested that the a18H mutation results from a paracentric inversion that affects two loci: agouti and another, as yet unidentified locus designated itchy (the provisional gene symbol is Itch), that is responsible for the immunological phenotype of a18H mice . Here we confirm that a18H results from an inversion and show that Itch encodes a novel E3 ubiquitin protein ligase, a protein involved in ubiquitin-mediated protein degradation . Our results indicate that ubiquitin-dependent proteolysis is an important mediator of the immune response in vivo and provide evidence for Itch's role in inflammation and the regulation of epithelial and haematopoietic cell growth. Hepatology, 1998 Feb, 27(2), 557 - 62 Enhanced expression of hepatic acyl-coenzyme A synthetase and microsomal triglyceride transfer protein messenger RNAs in the obese and hypertriglyceridemic rat with visceral fat accumulation; Kuriyama H et al.; The liver plays a central role in lipoprotein metabolism . In particular, very-low density lipoprotein (VLDL) is assembled in the hepatocytes and secreted into the blood circulation . The VLDL is then catabolized to low-density lipoprotein by lipoprotein lipase and hepatic triglyceride lipase . Obese subjects, especially those with visceral fat accumulation, are frequently associated with hyperlipidemia, non-insulin-dependent diabetes mellitus (NIDDM), and hypertension . The mechanism of hyperlipidemia in visceral fat obesity has not yet been elucidated . Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of NIDDM, characterized by obesity with visceral fat accumulation, hyperlipidemia, and late-onset insulin resistance . To elucidate the mechanism of hyperlipidemia observed in OLETF rats, we focused on the production of VLDL by the liver and investigated hepatic messenger RNA (mRNA) levels of microsomal triglyceride transfer protein (MTP), acyl-coenzyme A synthetase (ACS), and apolipoprotein B (apo B), which play important roles in VLDL synthesis and secretion . In 6-week-old OLETF rats, in which insulin resistance had not been manifested, visceral fat weight was already higher and portal free fatty acid (FFA) and VLDL-triglyceride levels were elevated compared with the control rats . Hepatic ACS activity and mRNA levels, and MTP mRNA levels were also increased in OLETF rats, whereas apo B mRNA levels were similar; these results suggest that the enhanced expression of both ACS and MTP genes associated with visceral fat accumulation before developing insulin resistance may be involved in the pathogenesis of hyperlipidemia in obese animal models with NIDDM. Gene, 1998 Jan 5, 206(1), 137 - 43 AtGDI2, a novel Arabidopsis gene encoding a Rab GDP dissociation inhibitor; Ueda T et al.; The GTPase cycle of Rab/Ypt proteins is strictly controlled by several classes of regulators to ensure their proper roles in membrane traffic . GDP dissociation inhibitor (GDI) is known to play essential roles in regulating nucleotide states and subcellular localizations of Rab/Ypt proteins . To obtain further knowledge on this regulator molecule in plants, we isolated and characterized two genes of Arabidopsis thaliana that encode different GDIs . AtGDI1 has been identified by a novel functional cloning in yeast {Ueda et al . (1996) Plant Cell, 8, 2079-2091} and AtGDI2 was isolated by cross-hybridization in this study . AtGDI2, as well as AtGDI1, complements the yeast sec19/gdi1 mutant, indicating that they can replace the function of yeast GDI . Evidence is shown that both AtGDI1 and AtGDI2 can interact with Ara4, an Arabidopsis Rab protein, in the yeast ypt1 mutant cells . AtGDI2 is ubiquitously expressed in Arabidopsis tissues with some difference from AtGDI1 in expression level . Genomic DNA hybridization using specific probes reveals the presence of one more GDI gene in Arabidopsis . This may imply differentiated roles of GDI in higher plants. Biol Chem, 1997 Dec, 378(12), 1433 - 43 Resolution of RNA polymerase I into dimers and monomers and their function in transcription; Milkereit P et al.; We have further analyzed the requirements of yeast RNA polymerase I (pol I) to initiate transcription at the ribosomal gene promoter . Resolution of yeast whole cell extracts through several chromatographic steps yielded three protein fractions required for accurate initiation . One fraction is composed of TBP associated within a 240 kDa protein complex . The fraction contributing the RNA polymerase I (pol I) activity consists of dimeric and monomeric pol I under conditions optimal for in vitro transcription . The capability to utilize the ribosomal gene promoter correlates with monomeric pol I complexes which are possibly associated with further transcription factors . These initiation competent pol I complexes appeared to be resistant to high salt concentrations . Pol I dimers which represent the majority of the isolated pol I, can be reversibly dissociated into monomers and are only active in non-specific RNA synthesis, if single stranded DNA serves as a template . We suggest a model in which dimeric inactive pol I is converted into an active monomeric form that might be associated with other transcription factors to maintain a stable initiation competent complex. Nat Struct Biol, 1998 Feb, 5(2), 110 - 4 Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family; Krapp S et al.; RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases . We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae . It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop . The strands are connected in classic Greek-key fashion . The overall topology is unusual and contains a striking C2 rotational symmetry . Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class. J Biomol NMR, 1997 Dec, 10(4), 397 - 401 Refined solution structure of the DNA-binding domain of GAL4 and use of 3J(113Cd,1H) in structure determination; Baleja JD et al.; We have refined the solution structure of cadmium-bound GAL4 and present its 15N and 1H NMR assignments . The root-mean-square (rms) deviation to the average structure was 0.4 +/- 0.05 A for backbone atoms, and 0.9 +/- 0.1 A for all heavy atoms . The three-bond heteronuclear 3J(113Cd,1H) coupling constants were found to disobey a Karplus-type relationship, which was attributable to the unusual constraints imposed by the bimetal-thiolate cluster in GAL4 . We conclude that the structural parameters that correlate to 3J(113Cd,1H) are complex. J Cell Biol, 1998 Jan 12, 140(1), 81 - 90 Relocalization of phospholipase D activity mediates membrane formation during meiosis; Rudge SA et al.; Phospholipase D (PLD) enzymes catalyze the hydrolysis of phosphatidylcholine and are involved in membrane trafficking and cytoskeletal reorganization . The Saccharomyces cerevisiae SPO14 gene encodes a PLD that is essential for meiosis . We have analyzed the role of PLD in meiosis by examining two mutant proteins, one with a point mutation in a conserved residue (Spo14pK--> H) and one with an amino-terminal deletion (Spo14pDeltaN), neither of which can restore meiosis in a spo14 deletion strain . Spo14pK--> H is enzymatically inactive, indicating that PLD activity is required, whereas Spo14pDeltaN retains PLD catalytic activity in vitro, indicating that PLD activity is not sufficient for meiosis . To explore other aspects of Spo14 function, we followed the localization of the enzyme during meiosis . Spo14p is initially distributed throughout the cell, becomes concentrated at the spindle pole bodies after the meiosis I division, and at meiosis II localizes to the new spore membrane as it surrounds the nuclei and then expands to encapsulate the associated cytoplasm during the formation of spores . The catalytically inactive protein also undergoes relocalization during meiosis; however, in the absence of PLD activity, no membrane is formed . In contrast, Spo14pDeltaN does not relocalize properly, indicating that the failure of this protein to complement a spo14 mutant is due to its inability to localize its PLD activity . Furthermore, we find that Spo14p movement is correlated with phosphorylation of the protein . These experiments indicate that PLD participates in regulated membrane formation during meiosis, and that both its catalytic activity and subcellular redistribution are essential for this function. J Cell Biol, 1998 Jan 12, 140(1), 61 - 9 A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion; Ungermann C et al.; Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide-sensitive fusion protein {NSF}), Sec17p (soluble NSF attachment protein {alpha-SNAP}), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs) . We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v-t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion . In the priming reaction, Sec18p and ATP dissociate this v-t-SNARE complex, accompanied by the release of Sec17p . SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs . Sec17p physically and functionally interacts largely with the t-SNARE . (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release . (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE . (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not . Sec18p thus acts, upon ATP hydrolysis, to disassemble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion . These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, alpha-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes. J Biol Chem, 1998 Jan 9, 273(2), 1099 - 106 A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain; Harper JF et al.; To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2) . ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain . In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes . ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80) . Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1 . Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM) . In contrast, the truncated pump was fully active and insensitive to calmodulin . A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins . Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization. J Biol Chem, 1998 Jan 9, 273(2), 1038 - 43 Kanadaptin is a protein that interacts with the kidney but not the erythroid form of band 3; Chen J et al.; Although epithelial membrane proteins are separately targeted to apical or basolateral domains, some are apically located in one cell type but are basolateral in others . More dramatically, the anion exchanger of a clonal cell line of intercalated cells derived from the kidney can be retargeted from the apical to basolateral domain . This Cl:HCO3 exchanger, kAE1, is an alternately spliced form of the erythroid anion exchanger (AE1, band 3), but unlike band 3 it does not bind ankyrin . Here we identify a new protein (kanadaptin) that binds to the cytoplasmic domain of kAE1 in vitro and in vivo but not to the erythroid AE1 or to ankyrin . No significant homologous proteins have been reported so far . Kanadaptin is widely expressed in epithelial (kidney, lung, and liver) and non-epithelial cells (brain and skeletal and cardiac muscle) . In kidney, we found by immunocytochemistry that kanadaptin was only expressed in the collecting tubule . In the intercalated cells of this segment, it colocalized with kAE1 in cytoplasmic vesicles but not when the exchanger was in the basolateral membrane . These results raised the possibility that this protein is involved in the targeting of kAE1 vesicles to their final destination. J Biol Chem, 1998 Jan 9, 273(2), 842 - 8 Identification of two domains of the p70 Ku protein mediating dimerization with p80 and DNA binding; Wang J et al.; The Ku autoantigen is a heterodimer of 70 (p70) and approximately 80 kDa (p80) subunits that is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) complex involved in DNA repair and V(D)J recombination . Binding to DNA ends is critical to the function of DNA-PK, but how Ku interacts with DNA is not completely understood . To define the role of p70 and p80 and their dimerization in DNA binding, heterodimers were assembled by co-expressing the subunits using recombinant baculoviruses . Two p70 dimerization sites, amino acids 1-115 and 430-482, respectively, were identified . Binding of p70 to linear double-stranded DNA could be demonstrated by an immunoprecipitation assay, and required the C-terminal portion (amino acids 430-609), but not interaction with p80 . The p70 mutants 1-600, 1-542, 1-115, and 430-600 did not bind DNA efficiently . However, DNA binding of 1-600, 1-542, and 1-115, but not 430-600, was restored by dimerization with p80, indicating that p70 has two DNA binding sites, each partially overlapping one of the dimerization sites . The C-terminal domain can bind DNA by itself, but the N-terminal domain requires dimerization with p80 . These observations could be relevant to the multiple functional activities of Ku and explain controversies regarding the role of dimerization in DNA binding. Mol Endocrinol, 1997 Dec, 11(13), 2025 - 37 Characterization of receptor interaction and transcriptional repression by the corepressor SMRT; Li H et al.; SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription . To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR . Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation . Coordinately, two SMRT domains are found to interact independently with the receptors . Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription . Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay . Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A . Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters . Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression. Mol Endocrinol, 1997 Dec, 11(13), 1911 - 23 Interaction of wild type and dominant-negative p55PIK regulatory subunit of phosphatidylinositol 3-kinase with insulin-like growth factor-1 signaling proteins; Mothe I et al.; In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55PIK, and several of its potential signaling partners . The region between the Src homology 2 (SH2) domains of p55PIK bound to the NH2 terminus region of p110alpha, as previously shown for p85alpha . Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55PIK; the interaction occurred at the receptor tyrosine 1316 and involved both p55PIK SH2 domains . Interaction between p55PIK and IGF-IR was seen not only in the yeast two-hybrid system, but also using in vitro binding and coimmunoprecipitation of lysates from IGF-1 stimulated 293 cells overexpressing p55PIK . Further, IGF-I stimulation of these cells led to tyrosine phosphorylation of p55PIK . In 293 cells association of p55PIK with insulin receptor substrate-1 and with IGF-IR was dependent on PI 3-kinase, since it was increased by wortmannin, an inhibitor of PI 3-kinase . Further, by deleting amino acids 203-217 of p55PIK inter-SH2 domain, we engineered a p55PIK mutant unable to bind to the p110alpha catalytic subunit of PI 3-kinase . This mutant had a dominant-negative action on insulin-stimulated glucose transport, since insulin's effect on Glut 4 myc translocation was inhibited in adipocytes expressing mutant p55PIK . Importantly, this dominant-negative mutant was more efficient than wild type p55PIK in associating to IGF-IR and insulin receptor substrate-1 in 293 cells . Taken together, our results show that p55PIK interacts with key elements in the IGF-I signaling pathway, and that these interactions are negatively modulated by PI 3-kinase itself, providing circuitry for regulatory feedback control. Science, 1997 Dec 19, 278(5346), 2123 - 6 Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex; Beckmann R et al.; An oligomer of the Sec61 trimeric complex is thought to form the protein-conducting channel for protein transport across the endoplasmic reticulum . A purified yeast Sec61 complex bound to monomeric yeast ribosomes as an oligomer in a saturable fashion . Cryo-electron microscopy of the ribosome-Sec61 complex and a three-dimensional reconstruction showed that the Sec61 oligomer is attached to the large ribosomal subunit by a single connection . Moreover, a funnel-shaped pore in the Sec61 oligomer aligned with the exit of a tunnel traversing the large ribosomal subunit, strongly suggesting that both structures function together in the translocation of proteins across the endoplasmic reticulum membrane. J Cell Biol, 1997 Dec 15, 139(6), 1383 - 96 Probing the architecture of a simple kinetochore using DNA-protein crosslinking; Espelin CW et al.; In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome . In this paper, we report the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres . We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp . A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in direct contact with DNA forms at the genetically defined center of CDEIII . This core complex spans approximately 56 bp of CEN3 . An extended complex comprising the core complex and additional DNA-bound p110(NDC10) also forms . It spans approximately 80 bp of DNA . CBF3 makes sequence-specific and -nonspecific contacts with DNA . Both contribute significantly to the energy of CBF3-DNA interaction . Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres . These findings provide a foundation for understanding the organization of the CBF3-centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores . These results also have implications for understanding centromere-binding proteins in higher cells. J Biol Chem, 1997 Dec 12, 272(50), 31801 - 8 The recycling of ERGIC-53 in the early secretory pathway . ERGIC-53 carries a cytosolic endoplasmic reticulum-exit determinant interacting with COPII; Kappeler F et al.; Further investigation of the targeting of the intracellular membrane lectin endoplasmic reticulum (ER)-Golgi intermediate compartment-53 (ERGIC-53) by site-directed mutagenesis revealed that its lumenal and transmembrane domains together confer ER retention . In addition we show that the cytoplasmic domain is required for exit from the ER indicating that ERGIC-53 carries an ER-exit determinant . Two phenylalanines at the C terminus are essential for ER-exit . Thus, ERGIC-53 contains determinants for ER retention as well as anterograde transport which, in conjunction with a dilysine ER retrieval signal, control the continuous recycling of ERGIC-53 in the early secretory pathway . In vitro binding studies revealed a specific phenylalanine-dependent interaction between an ERGIC-53 cytosolic tail peptide and the COPII coat component Sec23p . These results suggest that the ER-exit of ERGIC-53 is mediated by direct interaction of its cytosolic tail with the Sec23p.Sec24p complex of COPII and that protein sorting at the level of the ER occurs by a mechanism similar to receptor-mediated endocytosis or Golgi to ER retrograde transport. J Biol Chem, 1997 Dec 12, 272(50), 31589 - 97 MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interaction domains; Dobrosotskaya I et al.; Membrane-associated guanylate kinase (MAGUK) proteins participate in the assembly of multiprotein complexes on the inner surface of the plasma membrane at regions of cell-cell contact . MAGUKs are characterized by three types of protein-protein interaction modules: the PDZ domain, the Src homology 3 (SH3) domain, and the guanylate kinase (GuK) domain . The arrangement of these domains is conserved in all previously known MAGUKs: either one or three PDZ domains in the NH2-terminal half, followed by the SH3 domain, followed by a COOH-terminal GuK domain . In this report, we describe the cDNA cloning and subcellular distribution of MAGI-1, a MAGUK with three unique structural features: 1) the GuK domain is at the NH2 terminus, 2) the SH3 domain is replaced by two WW domains, and 3) it contains five PDZ domains . MAGI-1 mRNA was detected in several adult mouse tissues . Sequence analysis of overlapping cDNAs revealed the existence of three splice variants that are predicted to encode MAGI-1 proteins with different COOH termini . The longest variant, MAGI-1c, contains three bipartite nuclear localization signals in its unique COOH-terminal sequence and was found predominantly in the nucleus of Madin-Darby canine kidney cells . A shorter form lacking these signals was found primarily in membrane and cytoplasmic fractions . This distribution, which is reminiscent of that seen for the tight junction protein ZO-1, suggests that MAGI-1 may participate in the transmission of regulatory signals from the cell surface to the nucleus. J Biol Chem, 1997 Dec 12, 272(50), 31459 - 64 Mints, Munc18-interacting proteins in synaptic vesicle exocytosis; Okamoto M et al.; Munc18-1 is a neuronal protein that interacts with syntaxin 1 and is required for synaptic vesicle exocytosis . We have now identified two Munc18-1-interacting proteins called Mint1 and Mint2 that may mediate the function of Munc18-1 . Mint proteins are detectable only in brain and are composed of an N-terminal sequence that binds Munc18-1, a middle phosphotyrosine-binding domain, and two C-terminal PDZ domains thought to attach proteins to the plasma membrane . In brain, Mint proteins are part of a multimeric complex containing Munc18-1 and syntaxin that likely functions as an intermediate in synaptic vesicle docking/fusion . The phosphotyrosine-binding domain specifically binds to phosphatidylinositol phosphates known to be produced during vesicle exocytosis (Hay, J . C., Fisette, P . L., Jenkins, G . H., Fukami, K., Takonawa, T., Anderson, R . A., and Martin, T . F . J . (1995) Nature 374, 173-177) . Our data suggest a model whereby local production of phosphatidylinositol phosphates may trigger the binding of vesicles to the active zone via the Mint.Munc18-1 complex in conjunction with syntaxin 1. J Biol Chem, 1997 Dec 12, 272(50), 31235 - 40 Characterization of substrate phosphorylation and use of calmodulin mutants to address implications from the enzyme crystal structure of calmodulin-dependent protein kinase I; Chin D et al.; Calcium/calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to the enzyme and indirectly promotes the phosphorylation and synergistic activation of CaMKI by an exogenous kinase . We have evaluated the initial CaM-dependent activation of the unphosphorylated form of CaMKI . The kinetics of bacterially expressed human CaMKI show that the peptide syntide-2 is a relatively poor substrate, whereas the synapsin site-1 peptide is 17-fold more specific . The peptide ADR1G is 400-fold more specific than syntide-2, and its catalytic rate is among the highest reported for a kinase peptide substrate . To understand how CaM activates CaMKI, we have characterized the activation of the enzyme by CaM mutants with substitutions at hydrophobic residues . The point mutant M124Q located in the C-terminal domain of CaM produced a 57-fold increase in the CaM activation constant for CaMKI and suggests the involvement of methionine 124 in an important hydrophobic interaction with tryptophan 303 of CaMKI . Substituting two, three, and five hydrophobic residues in the N-terminal domain of CaM increased the CaM activation constant for CaMKI by 10-190-fold and lowered the maximal enzyme activity by more than 80% . Two of these N-terminal mutants of CaM do not affect the Km for peptide substrate but instead produce a 5-10-fold higher Km for ATP . This result demonstrates the critical role of the N-terminal domain of CaM in regulating the access of ATP to CaMKI. Curr Biol, 1997 Mar 1, 7(3), 202 - 10 Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells; Sells MA et al.; BACKGROUND: The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors . In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers . Recently, the serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity towards exogenous substrates . This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors . RESULTS: Here, we report that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles . The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures . This phenotype resembles that seen in motile fibroblasts . The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains . Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton . CONCLUSIONS: These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility. Curr Biol, 1997 Mar 1, 7(3), 184 - 90 The lipid transfer activity of phosphatidylinositol transfer protein is sufficient to account for enhanced phospholipase C activity in turkey erythrocyte ghosts; Currie RA et al.; BACKGROUND: The minor membrane phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP2) has been implicated in the control of a number of cellular processes . Efficient synthesis of this lipid from phosphatidylinositol has been proposed to require the presence of a phosphatidylinositol/phosphatidylcholine transfer protein (PITP), which transfers phosphatidylinositol and phosphatidylcholine between membranes, but the mechanism by which PITP exerts its effects is currently unknown . The simplest hypothesis is that PITP replenishes agonist-sensitive pools of inositol lipids by transferring phosphatidylinositol from its site of synthesis to sites of consumption . Recent cellular studies, however, led to the proposal that PITP may play a more active role as a co-factor which stimulates the activity of phosphoinositide kinases and phospholipase C (PLC) by presenting protein-bound lipid substrates to these enzymes . We have exploited turkey erythrocyte membranes as a model system in which it has proved possible to distinguish between the above hypotheses of PITP function . RESULTS: In turkey erythrocyte ghosts, agonist-stimulated PIP2 hydrolysis is initially rapid, but it declines and reaches a plateau when approximately 15% of the phosphatidylinositol has been consumed . PITP did not affect the initial rate of PIP2 hydrolysis, but greatly prolonged the linear phase of PLC activity until at least 70% of phosphatidylinositol was consumed . PITP did not enhance the initial rate of phosphatidylinositol 4-kinase activity but did increase the unstimulated steady-state levels of both phosphatidylinositol 4-phosphate and PIP2 by a catalytic mechanism, because the amount of polyphosphoinositides synthesized greatly exceeded the molar amount of PITP in the assay . Furthermore, when polyphosphoinositide synthesis was allowed to proceed in the presence of exogenous PITP, after washing ghosts to remove PITP before activation of PLC, enhanced inositol phosphate production was observed, whether or not PITP was present in the subsequent PLC assay . CONCLUSION: PITP acts by catalytically transferring phosphatidylinositol down a chemical gradient which is created as a result of the depletion of phosphatidylinositol at its site of use by the concerted actions of the phosphoinositide kinases and PLC . PITP is therefore not a co-factor for the phosphoinositide-metabolizing enzymes present in turkey erythrocyte ghosts. Curr Biol, 1997 Mar 1, 7(3), 157 - 65 DNA methylation directs a time-dependent repression of transcription initiation; Kass SU et al.; BACKGROUND: The regulation of DNA methylation is required for differential expression of imprinted genes during vertebrate development . Earlier studies that monitored the activity of the Herpes simplex virus (HSV) thymidine kinase (tk) gene after injection into rodent cells have suggested that assembly of chromatin influences the methylation-dependent repression of gene activity . Here, we examine the mechanism of methylation-dependent HSV tk gene regulation by direct determination of nucleoprotein organization during the establishment of a transcriptionally silenced state after microinjection of templates with defined methylation states into Xenopus oocyte nuclei . RESULTS: The transcriptional silencing conferred by a methylated DNA segment was not immediate, as methylated templates were initially assembled into active transcription complexes . The eventual loss of DNase I hypersenitive sites and inhibition of transcription at the HSV tk promoter only occurred after several hours . Flanking methylated vector DNA silenced the adjacent unmethylated HSV tk promoter, indicative of a dominant transmissible repression originating from a center of methylation . The resulting repressive nucleoprotein structure silenced transcription in the presence of activators that are able to overcome repression of transcription by nucleosomes . CONCLUSIONS: Silencing of transcription by DNA methylation is achieved at the level of transcription initiation and involves the removal of transcriptional machinery from active templates . This transcriptional repression can occur by indirect mechanisms involving the time-dependent assembly of repressive nucleoprotein complexes, which are able to inhibit transcription more effectively than nucleosomes alone. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13938 - 43 Chaperone-supervised conversion of prion protein to its protease-resistant form; DebBurman SK et al.; Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system . A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE) . PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet . The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection . To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state . None affected conversion in the absence of pre-existing PrPSc . In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion . Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct . In contrast, chemical chaperones inhibited conversion . Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13683 - 8 Syntenin, a PDZ protein that binds syndecan cytoplasmic domains; Grootjans JJ et al.; The syndecans are transmembrane proteoglycans that place structurally heterogeneous heparan sulfate chains at the cell surface and a highly conserved polypeptide in the cytoplasm . Their versatile heparan sulfate moieties support various processes of molecular recognition, signaling, and trafficking . Here we report the identification of a protein that binds to the cytoplasmic domains of the syndecans in yeast two-hybrid screens, surface plasmon resonance experiments, and ligand-overlay assays . This protein, syntenin, contains a tandem repeat of PDZ domains that reacts with the FYA C-terminal amino acid sequence of the syndecans . Recombinant enhanced green fluorescent protein (eGFP)-syntenin fusion proteins decorate the plasmamembrane and intracellular vesicles, where they colocalize and cosegregate with syndecans . Cells that overexpress eGFP-syntenin show numerous cell surface extensions, suggesting effects of syntenin on cytoskeleton-membrane organization . We propose that syntenin may function as an adaptor that couples syndecans to cytoskeletal proteins or cytosolic downstream signal-effectors. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13588 - 93 Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: the effects of ionizing radiation on growth, survival, and development; Nussenzweig A et al.; We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination . These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks . To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation . Ku80(-/-) embryonic stem cells are more sensitive than controls to gamma-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and gamma-ray-induced apoptosis . We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice . Ku80(-/-) mice display a hypersensitivity to gamma-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality . Exposure of newborn Ku80(-/-) mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation . However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80(-/-) mice is not accompanied by extensive thymocyte proliferation . The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13536 - 41 Functional separation of pre-rRNA processing steps revealed by truncation of the U3 small nucleolar ribonucleoprotein component, Mpp10; Lee SJ et al.; The U3 small nucleolar ribonucleoprotein (snoRNP) is required for three cleavage events that generate the mature 18S rRNA from the pre-rRNA . In Saccharomyces cerevisiae, depletion of Mpp10, a U3 snoRNP-specific protein, halts 18S rRNA production and impairs cleavage at the three U3 snoRNP-dependent sites: A0, A1, and A2 . We have identified truncation mutations of Mpp10 that affect 18S rRNA synthesis and confer cold-sensitivity and slow growth . However, distinct from yeast cells depleted of Mpp10, the mutants carrying these truncated Mpp10 proteins accumulate a novel precursor, resulting from cleavage at only A0 . The Mpp10 truncations do not alter association of Mpp10 with the U3 snoRNA, nor do they affect snoRNA or protein stability . Thus, the role in processing of the U3 snoRNP can be separated into cleavage at the A0 site, which occurs in the presence of truncated Mpp10, and cleavage at the A1/A2 sites, which occurs only with intact Mpp10 . These results strongly argue for a role for Mpp10 in processing at the A1/A2 sites. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13499 - 503 Ultraspiracle: an invertebrate nuclear receptor for juvenile hormones; Jones G et al.; Juvenile hormones (JH), a sesquiterpenoid group of ligands that regulate developmental transitions in insects, bind to the nuclear receptor ultraspiracle (USP) . In fluorescence-based binding assays, USP protein binds JH III and JH III acid with specificity, adopting for each ligand a different final conformational state . JH III treatment of Saccharomyces cerevisiae expressing a LexA-USP fusion protein stabilizes an oligomeric association containing this protein, as detected by formation of a protein-DNA complex, and induces USP-dependent transcription in a reporter assay . We propose that regulation of morphogenetic transitions in invertebrates involves binding of JH or JH-like structures to USP. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13475 - 80 Bidirectional binding of the TATA box binding protein to the TATA box; Cox JM et al.; By selective attachment of a DNA cleavage agent to specific residues in the yeast TATA box binding protein (yTBP), we demonstrate that, in solution, yTBP binds to the TATA boxes of both the adenovirus major late promoter and the yeast CYC1 promoter with only a modest preference in orientation and binds well to several overlapping binding sites . The general factors TFIIA and TFIIB each increase the rotational and translational selectivity of yTBP but are not sufficient, at least individually, to confer a unique polarity to the preinitiation complex . We conclude that TBP alone cannot define the productive orientation of general factor assembly on a promoter. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13402 - 6 A nonnatural transcriptional coactivator; Nyanguile O et al.; In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts . In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators . Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators . We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo . The present strategy provides a means of gaining external control over gene activation through intervention using small molecules. Genes Dev, 1997 Dec 1, 11(23), 3109 - 15 Human telomerase contains evolutionarily conserved catalytic and structural subunits; Harrington L et al.; We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein 2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus . Indirect immunofluorescence revealed that TP2 was localized to the nucleus . Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1 . Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity . These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits. J Cell Sci, 1997 Oct, 110 ( Pt 20), 2533 - 45 Identification of an Spc110p-related protein in vertebrates; Tassin AM et al.; Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components . In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly . Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner . Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells . This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it . Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin . This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex . To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract . We performed a partial purification of the gamma-tubulin-ring complex from egg extract . Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex . Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation. Nucleic Acids Res, 1997 Nov 15, 25(22), 4455 - 63 The ETS family member ERM contains an alpha-helical acidic activation domain that contacts TAFII60; Defossez PA et al.; Transcription factors are modular entities built up of discrete domains, some devoted to DNA binding and others permitting transcriptional modulation . The structure of DNA binding domains has been thoroughly investigated and structural classes clearly defined . In sharp contrast, the structural constraints put on transactivating regions, if any, are mostly unknown . Our investigations focus on ERM, a eukaryotic transcription factor of the ETS family . We have previously shown that ERM harbours two transactivating domains (TADs) with distinct functional features: AD1 lies in the first 72 amino acids of ERM, while AD2 sits in the last 62 . Here we show that AD1 is a bona fide acidic TAD, for it activated transcription in yeast cells, while AD2 did not . AD1 contains a 20 amino acid stretch predicted to form an alpha-helix that is found unchanged in the related PEA3 and ER81 transcription factors . Circular dichroism analysis revealed that a 32 amino acid peptide encompassing this region is unstructured in water but folds into a helix when the hydrophobic solvent trifluoroethanol is added . The isolated helix was sufficient to activate transcription and mutations predicted to disrupt it dramatically affected AD1-driven transactivation, whereas mutations decreasing its acidity had more gentle effects . A phenylalanine residue within the helix was particularly sensitive to mutations . Finally, we observed that ERM bound TAFII60 via AD1 and bound TBP and TAFII40, presumably via other activation domains. Mol Gen Genet, 1997 Sep, 256(2), 179 - 86 Molecular characterization of the glnA gene encoding glutamine synthetase from the edible mushroom Agaricus bisporus; Kersten MA et al.; The gene encoding glutamine synthetase (glnA) was isolated from an Agaricus bisporus H39 recombinant lambda phage library . The deduced A . bisporus glutamine synthetase amino acid sequence contains 354 residues . The amino acid sequence is very similar to that derived from the gene coding for glutamine synthetase of the yeast Saccharomyces cerevisiae . The open reading frame is interrupted by four introns . Northern analysis revealed that transcription of the gene is repressed upon addition of ammonium to the culture but the repression was not as strong as that of the gene encoding NADP+ -dependent glutamate dehydrogenase (gdhA) . Enzyme activities are low in the presence of ammonium, glutamine and albumin and do not correlate with the mRNA levels revealed by Northern analysis . This suggests that glutamine synthetase expression in A . bisporus is also post-transcriptionally regulated by the nitrogen source. Mol Gen Genet, 1997 Sep, 256(2), 104 - 14 Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient; Prokisch H et al.; The function of Neurospora crassa calcineurin was investigated in N . crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1) . Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct integrations . Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N . crassa . Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae . This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation . In vitro inhibition of N . crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N . crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin . The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea. J Bacteriol, 1998 Feb, 180(3), 478 - 82 Synthesis and differential turnover of the CYS3 regulatory protein of Neurospora crassa are subject to sulfur control; Tao Y et al.; The transcription factor CYS3 of Neurospora crassa is a positive regulator of the sulfur regulatory circuit which contains many structural genes involved in sulfur metabolism . Expression and degradation of the CYS3 protein are precisely regulated in a sulfur-dependent manner . cys-3 expression was found to be fully repressed by high concentrations of methionine or inorganic sulfate present in the culture medium and to be derepressed when these favored sulfur sources were limited . cys-3 transcripts could be readily detected within 2 h after derepression, whereas the CYS3 protein was not found until after 4 h . CYS3 is stable, with a half-life greater than 4 h under low-sulfur conditions when it is required for cell growth . However, it is degraded relatively quickly when methionine or inorganic sulfate becomes available . Upon sulfur repression, cys-3 transcripts disappeared within 30 min with an estimated half-life of 5 min whereas CYS3 protein almost entirely disappeared in 1 h with a half-life of approximately 10 min . These results suggest that a selective elimination of CYS3 is a highly regulated process . Site-directed mutagenesis showed that Lys-105 of CYS3 is important for its instability . The change of this single residue from lysine to glutamine resulted in a prolonged half life of CYS3 and impaired responsiveness of CYS3 degradation to sulfur level changes. Biochemistry, 1998 Jan 27, 37(4), 1060 - 6 Electrostatic effects in DNA bending by GCN4 mutants; Strauss-Soukup JK et al.; DNA architecture has been shown to be important for cellular processes such as activation of transcription, recombination, and replication . Many proteins reconfigure the shape of duplex DNA upon binding . Previous experiments have shown that some members of the eukaryotic bZIP family of DNA binding proteins appear to bend DNA, while others do not . We are exploring the role of electrostatic effects in DNA bending by bZIP proteins . The yeast bZIP transcription factor GCN4 does not induce DNA bending in vitro . Previously we substituted basic residues for three neutral amino acids in GCN4 to produce a GCN4 derivative that bends DNA by approximately 15 degrees . This result is consistent with a model of induced DNA bending wherein excess positive charge in proximity to one face of the double helix neutralizes local phosphate diester anions resulting in a laterally-asymmetric charge distribution along the DNA . Such an unbalanced charge distribution can result in collapse of the DNA toward the neutralized surface . We now present a more comprehensive analysis of electrostatic effects in DNA bending by GCN4 derivatives . It is shown that the direction and extent of DNA bending by these derivatives are a linear function of the charges of the amino acids adjacent to the basic domain of the protein . This relation holds over the charge range +6 (16 degrees bend toward the minor groove) to -6 (25 degrees bend toward the major groove). Virology, 1998 Jan 20, 240(2), 221 - 31 Identification of conserved hydrophobic C-terminal residues of the human papillomavirus type 1 E1E4 protein necessary for E4 oligomerisation in vivo; Ashmole I et al.; Previous studies have shown that human papillomavirus (HPV) E4 proteins undergo oligomerisation, although the precise sequences involved have not been identified . Using the yeast two-hybrid system we have identified HPV 1 E4 sequences that are critical to multimerisation . Fusion proteins were created by linking wild-type and mutant E4 proteins to a LexA DNA-binding domain or a B42 transactivation domain . HPV 1 E4:E4 interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae . This assay showed that (1) amino acid residues 95 to 115 at the carboxy-terminus were critical for oligomerisation and (2) hydrophobic residues (isoleucine 107, phenylalanine 114) in this domain are major determinants in the formation of oligomers . Interestingly, the carboxy-terminal domain shares homology with other E4 proteins of cutaneous HPV types and, furthermore, positions 107 and 114 are conserved residues . Substitution of the conserved aspartate amino acids (residues 110 and 112) did not abrogate E4 oligomerisation . Chemical cross-linking of wart and recombinant (baculovirus-expressed) HPV 1 E4 protein indicated that in solution this viral protein forms complexes consistent in size with either trimers or tetramers . These complexes were resistant to urea denaturation and are not dependent on the formation of disulphide linkages . A mutant protein containing a deletion of residues 110 to 115 was unable to form oligomers following cross-linking supporting a role for this region in mediating E4:E4 interactions . We conclude that oligomerisation of the HPV 1 E4 protein is likely to be mediated by carboxy-terminal residues and that conserved hydrophobic residues of this domain play a major role in E4 oligomerisation. Biochemistry, 1998 Jan 6, 37(1), 3 - 8 H+ transport by uncoupling protein (UCP-1) is dependent on a histidine pair, absent in UCP-2 and UCP-3; Bienengraeber M et al.; UCP from brown adipose tissue of hamster (now UCP-1) expressed in Saccharomyces cerevisiae was used to examine the role of a conspicuous histidine pair H145 and H147 which is conserved among UCP-1 from various animals . Single and double mutants were generated by converting H145 and H147 into neutral residues (H145Q and H147N) . As measured by fluorescence of dansyl-GTP binding, the level of expression of the mutant UCP was the same as wild-type (wt) in the isolated mitochondria . With the isolated and reconstituted UCP, transport of H+ and Cl- were measured . The fatty acid dependent H+ transport was reduced to about 10% in the single mutant H145Q and H147N and almost abolished in the double mutant, whereas Cl- transport into these vesicles was not affected as compared to wt . The possible involvement of the His pair in nucleotide binding and its pH dependence were examined by determining the KD and the kinetics for {14C}GTP and {14C}ADP binding . There were no marked changes in the affinity as well as in the binding and dissociation rates toward both these nucleotides in the mutant versus wt . Thus, the involvement in nucleotide binding can be excluded . The His pair is localized on the matrix side, probably at the entrance of the H+ translocation channel in UCP-1 . It is absent in the recently discovered UCP-2, and therefore, UCP-2 might b