J Cell Biol, 1998 Sep 7, 142(5), 1209 - 22 Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product; Powers J et al.; COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides . One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product . Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles . Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface . Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck . In erv14Delta strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates . We propose that Erv14p is required for the export of specific secretory cargo from the ER . The polarity defect of erv14Delta yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis . These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila. Genes Dev, 1998 Sep 1, 12(17), 2711 - 23 okra and spindle-B encode components of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila oogenesis; Ghabrial A et al.; okra (okr), spindle-B (spnB), and spindle-D (spnD) are three members of a group of female sterile loci that produce defects in oocyte and egg morphology, including variable dorsal-ventral defects in the eggshell and embryo, anterior-posterior defects in the follicle cell epithelium and in the oocyte, and abnormalities in oocyte nuclear morphology . Many of these phenotypes reflect defects in grk-Egfr signaling processes, and can be accounted for by a failure to accumulate wild-type levels of Gurken and Fs(1)K10 . We have cloned okr and spnB, and show that okr encodes the Drosophila homolog of the yeast DNA-repair protein Rad54, and spnB encodes a Rad51-like protein related to the meiosis-specific DMC1 gene . In functional tests of their role in DNA repair, we find that okr behaves like its yeast homolog in that it is required in both mitotic and meiotic cells . In contrast, spnB and spnD appear to be required only in meiosis . The fact that genes involved in meiotic DNA metabolism have specific effects on oocyte patterning implies that the progression of the meiotic cell cycle is coordinated with the regulation of certain developmental events during oogenesis. Genes Dev, 1998 Sep 1, 12(17), 2698 - 710 CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint; Stuart D et al.; Initiation of DNA replication during the mitotic cell cycle requires the activation of a cyclin-dependent protein kinase (CDK) . The B-type cyclins Clb5 and Clb6 are the primary activators of the S phase function of the budding yeast CDK Cdc28 . However, in mitotically growing cells this role can be fulfilled by the other B-type cyclins Clb1-Clb4 . We report here that cells undergoing meiotic development also require Clb dependent CDK activity for DNA replication . Diploid clb5/clb5 clb6/clb6 mutants are unable to perform premeiotic DNA replication . Despite this defect, the mutant cells progress into the meiotic program and undergo lethal segregation of unreplicated DNA suggesting that they fail to activate a checkpoint that restrains meiotic M phase until DNA replication is complete . We have found that a DNA replication checkpoint dependent on the ATM homolog MEC1 operates in wild-type cells during meiosis and can be invoked in response to inhibition of DNA synthesis . Although cells that lack clb5 and clb6 are unable to activate the meiotic DNA replication checkpoint, they do possess an intact DNA damage checkpoint which can restrain chromosome segregation in the face of DNA damage . We conclude that CLB5 and CLB6 are essential for premeiotic DNA replication and, consequently, for activation of a meiotic DNA replication checkpoint. Nat Struct Biol, 1998 Sep, 5(9), 793 - 802 Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p; Nicholson KL et al.; The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes . Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro . Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes . Binary and ternary complex assembly are accompanied by large increases in alpha-helical structure, indicating that folding and complex formation are linked . Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1) . An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly . Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain . Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain. Biochem Biophys Res Commun, 1998 Aug 28, 249(3), 838 - 43 Dopamine formation from tyramine by CYP2D6; Hiroi T et al.; Dopamine is formed form L-tyrosine by tyrosine hydroxylase and aromatic L-amino acid decarboxylase . In addition to this pathway, however, the formation of catecholamines, including dopamine, from trace amines such as tyramine by hepatic microsomes has been demonstrated . In this study, we investigated the formation of dopamine from trace amines, using human hepatic microsomes and human cytochrome P450 (CYP) isoforms expressed in yeast . Among the 11 isoforms of human CYP expressed in yeast, CYP2D6 was the only isoform exhibiting strong ability to convert p-tyramine and m-tyramine to dopamine . In studies with human hepatic microsomes, the hydroxylation of tyramine to dopamine was inhibited by bufuralol, a typical substrate for CYP2D isoforms, and anti-CYP2D1 antiserum . This is the first report showing that CYP2D is capable of converting tyramine to dopamine . The Km values of CYP2D6, expressed in yeast, for p-tyramine and m-tyramine were 190.1 +/- 19.5 microM and 58.2 +/- 13.8 microM, respectively . Tyramine is an endogenous compound which exists in the brain as a trace amine but is also an exogenous compound which is found in foods such as cheese and wine . Our results suggest that dopamine is formed from endogenous and/or exogenous tyramine by this CYP2D isoform. Biochem Biophys Res Commun, 1998 Aug 28, 249(3), 697 - 703 Human dynamin-like protein interacts with the glycogen synthase kinase 3beta; Hong YR et al.; Members of the dynamin superfamily are implicated in vesicle trafficking . Using human glycogen synthase kinase 3 beta (Gsk-3 beta) as bait in the yeast two-hybrid system, we identified a novel human dynamin-like protein IV (HdynIV) . When the full-length cDNA of HdynIV was sequenced, it showed that HdynIV's carboxyl terminal lacks a proline-rich domain that can bind to Gsk-3 beta . By Northern blot analysis and isoform-specific PCR, we found that HdynIV is expressed ubiquitously in all human tissues examined . Two transcripts of 2.4 and 4.4 kb are shown to be more abundant in heart, brain, and skeletal muscle . Interestingly, the 2.4-kb transcript is expressed more distinctly in the fetal liver than in the adult liver, suggesting that this protein might play a role during development . In the present report, we have demonstrated that HdynIV interacts with the Gsk-3 beta through its carboxyl-terminal region, implying than HdynIV may also be involved in cell signaling. Biochem Biophys Res Commun, 1998 Aug 28, 249(3), 678 - 82 Marked enhancement in the reductive dehalogenation of hexachloroethane by a Thr319Ala mutation of cytochrome P450 1A2; Yanagita K et al.; Mutation of the conserved Thr319 residue to Ala of cytochrome P4501A2 (CYP1A2) increased the value of Vmax 9-fold for reductive dehalogenation of hexachloroethane in the reconstituted system under anaerobic conditions . The Thr319Ala mutation also increased the elimination over substitution product ratio by 5-fold . The addition of aliphatic alcohols increased by 22-fold the activity obtained with the wild type and varied the elimination over substitution product ratio . Increasing pH increased the ratio of elimination over substitution by primarily affecting the rate of elimination. Mol Pharmacol, 1998 Sep, 54(3), 591 - 8 Pharmacological analysis of sterol delta8-delta7 isomerase proteins with {3H}ifenprodil; Moebius FF et al.; Sterol Delta8-Delta7 isomerases (SIs) catalyze the shift of the double bond from C8-9 to C7-8 in the B-ring of sterols . Surprisingly, the isoenzymes in fungi (ERG2p) and vertebrates {emopamil binding protein (EBP)} are structurally completely unrelated, whereas the sigma1 receptor, a mammalian protein of unknown function, bears significant similarity with the yeast ERG2p . Here, we compare the drug binding properties of SIs and related proteins with {3H}ifenprodil as a common high affinity radioligand (Kd = 1.4-19 nM), demonstrating an intimate pharmacological relationship among ERG2p, sigma1 receptor, and EBP . This renders SIs a remarkable example for structurally diverse enzymes with similar pharmacological profiles and the propensity to bind drugs from different chemical groups with high affinity . We identified a variety of experimental drugs with nanomolar affinity for the human EBP (Ki = 0.5-14 nM) such as MDL28815, AY9944, triparanol, and U18666A . These compounds, as well as the fungicide tridemorph and the clinically used drugs tamoxifen, clomiphene, amiodarone, and opipramol, inhibit the in vitro activity of the recombinant human EBP (IC50 = 0.015-54 microM) . The high affinity of the human EBP for 3H-tamoxifen (Kd = 3 +/- 2 nM) implies that the EBP carries the previously described microsomal antiestrogen binding site . Interactions of the EBP with structurally diverse lipophilic amines suggest that novel compounds of related structure should be counterscreened for inhibition of the enzyme to avoid interference with sterol Delta8-Delta7 isomerization. Biochemistry, 1998 Sep 8, 37(36), 12496 - 506 The 32- and 14-kilodalton subunits of replication protein A are responsible for species-specific interactions with single-stranded DNA; Sibenaller ZA et al.; Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism . RPA homologues have been identified in all eukaryotes examined . All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa . While RPA homologues are evolutionarily conserved, they are not functionally equivalent . To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively) . Both yeast and human RPA bind ssDNA with high affinity and low cooperativity . However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA . Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties . In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity . However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively . We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA . These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins. J Biomol NMR, 1998 Jul, 12(1), 73 - 88 Internal and overall motions of the translation factor eIF4E: cap binding and insertion in a CHAPS detergent micelle; McGuire AM et al.; The mRNA cap-binding protein eIF4E is the limiting factor in the eIF4F translation initiation complex, which mediates the binding of the 40S ribosome to the mRNA . 15N relaxation studies have been used to characterize the backbone dynamics of deuterated eIF4E in a CHAPS micelle for the apoprotein, the m7GDP-bound form, and the dinucleotide (m7GpppA)-bound form, as well as for CHAPS-free eIF4E . Large differences in overall correlation time between the CHAPS-free form (11.8 ns) and samples containing different concentrations of CHAPS (15.9-19.4 ns) indicate that eIF4E is embedded in a large micelle in the presence of CHAPS, with a total molecular weight in the range of 40-60 kDa . CHAPS seems to restrict the mobility of the a2-b3 and a4-b5 loops which are thought to be embedded in the micelle . No significant changes in overall mobility were seen between the m7 GDP-bound form, the m7GpppA-bound form, and the apoprotein . Amide hydrogen exchange data indicate the presence of slowly exchanging amides in two surface-exposed helices (a2 and a4), as well as the a4-b5 loop, indicating protection by the CHAPS micelle . The micelle covers the convex side of the protein away from the cap-binding site. Gene, 1998 Aug 31, 216(2), 311 - 8 Characterization of a 76 kDa endosomal, multispanning membrane protein that is highly conserved throughout evolution; Schimmoller F et al.; We report here the identification and characterization of a human 76kDa membrane protein that is found predominantly in endosomes . This protein is related to the Saccharomyces cerevisiae EMP70 gene product, a precursor protein whose 24kDa cleavage product (p24a) was found in yeast endosome-enriched membrane fractions (Singer-Kruger et al., 1993 . J . Biol . Chem . 268, 14376-14386) . Northern blot analysis indicated that p76 mRNA is highly expressed in human pancreas but could be detected in all tissues examined . p76 is highly conserved throughout evolution, as related proteins have also been detected in Caenorhabditis elegans and Arabidopsis thaliana . This family of proteins has a relatively divergent, hydrophilic N-terminal domain and a well-conserved, highly hydrophobic C-terminal domain which contains nine potential membrane-spanning domains . Transiently expressed, myc-tagged human p76 appears to be localized to endosomes by virtue of its apparent colocalization with transferrin receptors and some mannose 6-phosphate receptors . Furthermore, p76 adopts a type-I topology within the membrane, with its hydrophilic N-terminus facing the lumen of cytoplasmic membranes . The structural features of p76 suggest that it may function as a channel or small molecule transporter in intracellular compartments throughout phylogeny . 1998 Elsevier Science B.V. Eur J Dermatol, 1998 Sep, 8(6), 403 - 12 An in vitro strategy to evaluate the phototoxicity of solar UV at the molecular and cellular level: application to photoprotection assessment; Marrot L et al.; Skin cancers are among the most common human cancers and have an increasing incidence . The ultraviolet radiation components of sunlight play a major role in skin tumor induction and development . Cellular DNA has been identified as a target for most of the biological effects of UV, and the induction of photodamage is considered as the initiating step of photocarcinogenesis . Thus, effective photoprotection of DNA against harmful overex-posure to solar UV is a critical issue . The efficiency of a sunscreen is usually tested with respect to its ability to prevent skin erythema, but conceivably, more data are required at the molecular and cellular level in order to ascertain protection against photocarcinogenic risk . In the present study, we define a strategy based on the use of various in vitro models and solar-simulated light to evaluate photodamage and photoprotection: -Supercoiled circular plasmid DNA for detection of structural alterations . -The yeast Saccharomyces cerevisiae to evaluate cytotoxicity and genotoxicity . -The single-cell gel electrophoresis or comet assay to determine DNA damage and DNA repair in human keratinocytes . -p53 expression as a hallmark for genotoxic stress . -Induction of pigmentation in human melanocytes . In conditions where light source, spectrum and control of radiation delivery were precisely defined, we have demonstrated that the wide spectrum UVA sunscreen Mexoryl SX protects from the cytotoxicity and genotoxicity of solar UV. Trends Cell Biol, 1998 Sep, 8(9), 348 - 53 The control of filamentous differentiation and virulence in fungi; Madhani HD et al.; Many members of the fungal kingdom have a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a cellular yeast form and a multicellular invasive filamentous form . At least three pathways are involved in regulating the transition between these two forms in the budding yeast Saccharomyces cerevisiae, and evidence is now emerging that homologous signalling modules are involved in regulating filament formation and virulence in a range of human and plant fungal pathogens . Strikingly, components used to signal sexual differentiation in the response to mating pheromones are often reutilized to regulate dimorphic development, suggesting an ancient link between these processes. Trends Cell Biol, 1998 Sep, 8(9), 339 - 42 Telomeres and double-strand breaks: trying to make ends meet; Bertuch A et al.; Eukaryotic cells encounter two types of DNA ends: telomeres, the natural ends of linear chromosomes, and double-strand breaks, resulting from DNA damage or normal chromosomal processes such as meiotic or V(D)J recombination . These two termini have long been seen as functionally distinct, based on whether they are resistant to fusion with other ends or instead are acted upon by the DNA-repair machinery . However, a series of recent papers has shown that members of a set of proteins that are crucial for the rejoining of DNA strand breaks are also required for normal telomere function, raising new questions about how these two types of termini maintain their functional distinction. Am J Physiol, 1998 Sep, 275(3 Pt 1), L574 - 82 Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1; Moore TM et al.; Activation of Ca2+ entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells . The mode of Ca2+ entry regulating cell shape is unknown . We hypothesized that activation of store-operated Ca2+ channels (SOCs) is sufficient to promote cell shape change necessary for these processes . SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+ that was dependent on a membrane current having a net inward component of 5.45 +/- 0.90 pA/pF at -80 mV . Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+ entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin) . Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential (trp) gene have been proposed to form Ca2+ entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells . Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6 . Our study indicates that 1) Ca2+ entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs. Pediatr Res, 1998 Sep, 44(3), 271 - 6 Aceruloplasminemia; Gitlin JD; Aceruloplasminemia is an autosomal recessive disorder of iron metabolism characterized by diabetes, retinal degeneration, and neurologic symptoms . Affected patients evidence marked parenchymal iron accumulation in conjunction with an absence of circulating serum ceruloplasmin and molecular genetic analysis reveals inherited mutations in the ceruloplasmin gene . Taken together with earlier studies that characterized ceruloplasmin as a ferroxidase and recent work indicating an essential role for a homologous multicopper oxidase in iron metabolism in Saccharomyces cerevisiae, these findings reveal an essential role for ceruloplasmin in human iron metabolism . The presence of neurologic symptoms in patients with aceruloplasminemia is unique among the characterized disorders of iron metabolism, and recent findings indicate that astrocyte-specific ceruloplasmin gene expression is critical for iron metabolism and neuronal survival in the retina and basal ganglia . The discovery of this disease provides new insights into the pathways of CNS iron metabolism of direct relevance to a variety of nutritional and genetic disorders of childhood. Reprod Fertil Dev, 1998, 10(1), 1 - 16 The Y-chromosome and reproductive disorders; McDonough PG; Over the past decade the tools of modern molecular biology have provided unique insights into our fundamental understanding of developmental systems . These insights have been gleaned from the study of a wide variety of model organisms including yeast (Saccharomyces cerevisiae), fly (Drosophila), worm (Caenorhabditis elegans), and mouse . In man, the first analysis of developmental systems started with sexual differentiation and focused on the role of Y-linked genes . The presence of living developmental mutants in man affecting sexual development and the early technology of deletion mapping facilitated the isolation and identification of small segments of putative DNA suspected to contain sex-determining genes . The isolation of genes such as SRY (Sex Related gene on Y) has provided the first insights into the molecular biology of human sexual differentiation . The focus on the Y chromosome has brought further insights into chromosomal pairing, statural determinants in man, oncogenesis, spermatogenesis, haploid genomes, and the lineage of man himself . This paper provides the circumstantial and direct evidence to illustrate the importance of the Y chromosome in reproductive disorders, and in the analysis of haploid genomes. Cell, 1998 Aug 21, 94(4), 427 - 38 Structure of the histone acetyltransferase Hat1: a paradigm for the GCN5-related N-acetyltransferase superfamily; Dutnall RN et al.; We have solved the crystal structure of the yeast histone acetyltransferase Hat1-acetyl coenzyme A (AcCoA) complex at 2.3 A resolution . Hat1 has an elongated, curved structure, and the AcCoA molecule is bound in a cleft on the concave surface of the protein, marking the active site of the enzyme . A channel of variable width and depth that runs across the protein is probably the binding site for the histone substrate . A model for histone H4 binding by Hat1 is discussed in terms of possible sources of specific lysine recognition by the enzyme . The structure of Hat1 provides a model for the structures of the catalytic domains of a protein superfamily that includes other histone acetyltransferases such as Gcn5 and CBP. J Biol Chem, 1998 Sep 11, 273(37), 24131 - 8 A novel component involved in ubiquitination is required for development of Dictyostelium discoideum; Pukatzki S et al.; A novel component of the ubiquitination system, called NOSA, is essential for cellular differentiation in Dictyostelium discoideum . Disruption of nosA does not affect the growth rate but causes an arrest in development after the cells have aggregated . nosA contains seven exons and codes for a developmentally regulated 3.5-kb mRNA . The 125-kDa NOSA protein is present in the cytosol at constant levels during growth and development . The C-terminal region of NOSA has homology with ubiquitin fusion degradation protein-2 (UFD2) of Saccharomyces cerevisiae and putative homologs in Caenorhabditis elegans and humans . UFD2 is involved in the ubiquitin-mediated degradation of model substrates in which ubiquitin forms part of the translation product, but ufd2 mutants have no detected phenotype . In accord with the homology to UFD2, we found differences in the ubiquitination patterns between nosA mutants and their parental cell line . While general in vivo and in vitro ubiquitination is minimally affected, ubiquitination of individual proteins is altered throughout growth and development in nosA mutants . These findings suggest that events involving ubiquitination are critical for progression through the aggregate stage of the Dictyostelium life cycle. J Biol Chem, 1998 Sep 11, 273(37), 24088 - 94 Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure; Akhmedov AT et al.; Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation . How individual SMC proteins and their functional domains bind DNA has not been described . We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner . Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types . These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers . Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains . Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression. J Biol Chem, 1998 Sep 11, 273(37), 23781 - 5 A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID; Martinez E et al.; In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP) . In particular, yeast SPT3 has been shown to interact directly with TBP . Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3 . Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18 . Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivo either with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex . However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity . We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex. J Biol Chem, 1998 Sep 11, 273(37), 23722 - 8 Molecular cloning and functional characterization of murine sphingosine kinase; Kohama T et al.; Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function . Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1 . Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b) . Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved . Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences . One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site . From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver . Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity . The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine . The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme . Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels . Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases . Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger. J Biol Chem, 1998 Sep 11, 273(37), 23637 - 40 Loops and bulge/loops in iron-responsive element isoforms influence iron regulatory protein binding . Fine-tuning of mRNA regulation? Ke Y, Wu J, Leibold EA, Walden WE, Theil EC. A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding a family of mRNA-specific proteins, iron regulatory proteins (IRPs) . IREs are hairpins with a constant terminal loop and base-paired stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase, and transferrin receptor mRNAs) . IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding occurs with the C-G or engineered U-A . Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing natural and engineered IRE variants . Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13% . Moreover, IRP2 binding to natural IREs with the C-bulge was similar to the DeltaU6 ferritin-IRE: >90% lower than the ferritin-IRE . The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured cells . Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses to the same (iron) signal. J Biol Chem, 1998 Sep 11, 273(37), 23633 - 6 A novel regulator of p21-activated kinases; Bagrodia S et al.; Proteins of the p21-activated kinase (Pak) family have been implicated in the regulation of gene expression, cytoskeletal architecture, and apoptosis . Although the ability of Cdc42 and Rac GTPases to activate Pak is well established, relatively little else is known about Pak regulation or the identity of Pak cellular targets . Here we report the identification of two closely related Pak3-binding proteins, possibly arising from alternative splicing, designated p50 and p85(Cool-1) (cloned out of library) . Both isoforms of Cool contain a Src homology 3 domain that directly mediates interaction with Pak3 and tandem Dbl homology and pleckstrin homology domains . Despite the presence of the Dbl homology-pleckstrin homology motif, a characteristic of Rho family activators, activation of Cdc42 or Rac by Cool is not detectable . Instead binding of p50(Cool-1), but not p85(Cool-1), to Pak3 represses its activation by upstream activators such as the Dbl oncoprotein, indicating a novel mechanism of regulation of Pak signaling. Biol Cell, 1998 Jun, 90(3), 239 - 45 Leishmania major: cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family; Zemzoumi K et al.; In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms . In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (e.g . logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes) . The reactive antigen was also detected in Trypanosoma cruzi extracts . Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used . No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts . Furthermore, immunoprecipitation of {35S}methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA) . Moreover, immunofluorescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface . The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues. Mol Biol Cell, 1998 Sep, 9(9), 2577 - 93 Tim23p contains separate and distinct signals for targeting to mitochondria and insertion into the inner membrane; Davis AJ et al.; The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway . Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein . Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix . To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria . We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM . Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments . Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM . We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps. Mol Biol Cell, 1998 Sep, 9(9), 2527 - 43 Activation of androgen receptor function by a novel nuclear protein kinase; Moilanen AM et al.; Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids . In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro . The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome . However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins . ANPK is a nuclear protein that is widely expressed in mammalian tissues . Its overexpression enhances AR-dependent transcription in various cell lines . In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK . The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo . In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation. Mol Biol Cell, 1998 Sep, 9(9), 2491 - 507 Organization of highly acetylated chromatin around sites of heterogeneous nuclear RNA accumulation; Hendzel MJ et al.; Histones found within transcriptionally competent and active regions of the genome are highly acetylated . Moreover, these highly acetylated histones have very short half-lives . Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids . Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities . We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs) . The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into "chromonema" fibers . Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm . Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles . The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules . Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb . We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin . An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs. Mol Biol Cell, 1998 Sep, 9(9), 2393 - 405 Phosphorylation of sic1, a cyclin-dependent kinase (Cdk) inhibitor, by Cdk including Pho85 kinase is required for its prompt degradation; Nishizawa M et al.; In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1 . The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent . We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Delta cells . Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor . Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo . Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1. Mol Biol Cell, 1998 Sep, 9(9), 2349 - 60 An alpha-tubulin mutant destabilizes the heterodimer: phenotypic consequences and interactions with tubulin-binding proteins; Vega LR et al.; Many effectors of microtubule assembly in vitro enhance the polymerization of subunits . However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly . Here we use a mutant alpha-tubulin to probe cellular regulation of microtubule assembly . tub1-724 mutant cells arrest at low temperature with no assembled microtubules . The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and beta-tubulin is less stable than wild-type heterodimer . The unstable heterodimer explains several conditional phenotypes conferred by the mutation . These include the lethality of tub1-724 haploid cells when the beta-tubulin-binding protein Rbl2p is either overexpressed or absent . It also explains why the TUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality . Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed . These effects are explained by the ability of Pac2p to bind alpha-tubulin, a complex we demonstrate directly . The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components. Trends Genet, 1998 Aug, 14(8), 312 - 6 Splitting the ATM: distinct repair and checkpoint defects in ataxia-telangiectasia; Jeggo PA et al.; Ataxia-telangiectasia (A-T) is an autosomal recessive human disorder that, because of its multisystem nature, is of interest to scientists and clinicians from many disciplines . A-T patients have defects in the neurological and immune systems, telangiectasia in the eyes and face, and are, in addition, cancer-prone and radiation-sensitive . A-T cell lines have a range of diverse phenotypes including sensitivity to ionizing radiation and defects in cell-cycle checkpoint control . The ATM protein is a member of the PI 3-kinase-like superfamily, and it has been widely accepted that A-T cells represent mammalian cell-cycle checkpoint mutants and that the radiation sensitivity is a consequence of this defect . However, several lines of evidence suggest that A-T cells have distinct repair and checkpoint defects . A-T cells therefore appear to harbour dual checkpoint/repair defects . Here, we review the evidence supporting this contention and consider its implications for an analysis of the A-T phenotype. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10860 - 5 ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex; Wang J et al.; The t(8;21) translocation between two genes known as AML1 and ETO is seen in approximately 12-15% of all acute myeloid leukemia (AML) and is the second-most-frequently observed nonrandom genetic alteration associated with AML . AML1 up-regulates a number of target genes critical to normal hematopoiesis, whereas the AML1/ETO fusion interferes with this trans-activation . We discovered that the fusion partner ETO binds to the human homolog of the murine nuclear receptor corepressor (N-CoR) . The interaction is mediated by two unusual zinc finger motifs present at the carboxyl terminus of ETO . Human N-CoR (HuN-CoR), which we cloned and sequenced in its entirety, encodes a 2,440-amino acid polypeptide and has a central domain that binds ETO . N-CoR, mammalian Sin3 (mSin3A and B), and histone deacetylase 1 (HDAC1) form a complex that alters chromatin structure and mediates transcriptional repression by nuclear receptors and by a number of oncoregulatory proteins . We found that ETO, through its interaction with the N-CoR/mSin3/HDAC1 complex, is also a potent repressor of transcription . This observation provides a mechanism for how the AML1/ETO fusion may inhibit expression of AML1-responsive target genes and disturb normal hematopoiesis. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10553 - 8 The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides; Arnould S et al.; Protoporphyrinogen oxidase (EC 1-3-3-4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides . It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form . Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000 . The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions . N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the betaalphabeta ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor . The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme . However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein . Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments . Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling . We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10390 - 5 Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals; Ghoshal K et al.; Metallothioneins (MT) are involved in the scavenging of the toxic heavy metals and protection of cells from reactive oxygen intermediates . To investigate the potential role of the protein Ku in the expression of MT, we measured the level of MT-I mRNA in the parental rat fibroblast cell line (Rat 1) and the cell lines that stably and constitutively overexpress the small subunit, the large subunit, and the heterodimer of Ku . Treatment with CdS04 or ZnS04 elevated the MT-I mRNA level 20- to 30-fold in the parental cells and the cells (Ku-70) that overproduce the small subunit or those (Ku-7080) overexpressing the heterodimer . By contrast, the cells (Ku-80) overexpressing the large subunit of Ku failed to induce MT-I . In vitro transcription assay showed that the MT-I promoter activity was suppressed selectively in the nuclear extracts from Ku-80 cells . The specificity of the repressor function was shown by the induction of hsp 70, another Cd-inducible gene, in Ku-80 cells . Addition of the nuclear extract from Ku-80 cells at the start of the transcription reaction abolished the MT-l promoter activity in the Rat 1 cell extract . The transcript once formed in Rat 1 nuclear extract was not degraded by further incubation with the extract from Ku-80 cells . The repressor was sensitive to heat . The DNA-binding activities of at least four transcription factors that control the MT-I promoter activity were not affected in Ku-80 cells . These observations have set the stage for further exploration of the mechanisms by which the Ku subunit mediates suppression of MT induction. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10384 - 9 Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system; Bevan A et al.; The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay . A truncated prepro-alpha-factor gene encoding an alpha-factor precursor with a single alpha-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR downward arrowEAEA-) . All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made . The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-alpha-factor . Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude . All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >10(6)-fold for Trp . Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor . Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method . The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo . P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp. EMBO J, 1998 Sep 1, 17(17), 5182 - 91 Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases; Tanaka T et al.; Eukaryotic cells use multiple replication origins to replicate their large genomes . Some origins fire early during S phase whereas others fire late . In Saccharomyces cerevisiae, initiator sequences (ARSs) are bound by the origin recognition complex (ORC) . Cdc6p synthesized at the end of mitosis joins ORC and facilitates recruitment of Mcm proteins, which renders origins competent to fire . However, origins fire only upon the subsequent activation of S phase cyclin-dependent kinases (S-CDKs) and Dbf4/Cdc7 at the G1/S boundary . We have used a chromatin immunoprecipitation assay to measure the association with ARS sequences of DNA primase and the single-stranded DNA binding replication protein A (RPA) when fork movement is inhibited by hydroxyurea (HU) . RPA's association with origins requires S-CDKs, Dbf4/Cdc7 kinase and an Mcm protein . The recruitment of DNA primase depends on RPA . Furthermore, early- and late-firing origins differ not in the timing of their recruitment of an Mcm protein, but in the timing of RPA's recruitment . RPA is recruited to early but not to late origins in HU . We also show that Rad53 kinase is required to prevent RPA association with a late origin in HU . Our data suggest that the origin unwinding accompanied by RPA association is a key step, regulated by S-CDKs, Dbf4/Cdc7 and Rad53p . Thus, in the presence of active S-CDKs and Dbf4/Cdc7, Mcms may open origins and thereby facilitate the loading of RPA. EMBO J, 1998 Sep 1, 17(17), 4954 - 63 Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p; Yang X et al.; The regulation of vesicular transport in eukaryotic cells involves Ras-like GTPases of the Ypt/Rab family . Studies in yeast and mammalian cells indicate that individual family members act in vesicle docking/fusion to specific target membranes . Using the two-hybrid system, we have now identified a 248 amino acid, integral membrane protein, termed Yip1, that specifically binds to the transport GTPases Ypt1p and Ypt31p . Evidence for physical interaction of these GTPases with Yip1p was also demonstrated by affinity chromatography and/or co-immunoprecipitation . Like the two GTPases, Yip1p is essential for yeast cell viability and, according to subcellular fractionation and indirect immunofluorescence, is located to Golgi membranes at steady state . Mutant cells depleted of Yip1p and conditionally lethal yip1 mutants at the non-permissive temperature massively accumulate endoplasmic reticulum membranes and display aberrations in protein secretion and glycosylation of secreted invertase . The results suggests for a role for Yip1p in recruiting the two GTPases to Golgi target membranes in preparation for fusion. EMBO J, 1998 Sep 1, 17(17), 4930 - 42 Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities; Wurmser AE et al.; The Golgi/endosome-associated Vps34 phosphatidylinositol 3-kinase is essential for the sorting of hydrolases from the Golgi to the vacuole/lysosome . Upon inactivation of a temperature-conditional Vps34 kinase, cellular levels of PtdIns(3)P rapidly decrease and it has been proposed that this decrease is due to the continued turnover of PtdIns(3)P by cytoplasmic phosphatases . Here we show that mutations in VAM3 (vacuolar t-SNARE) and YPT7 (rab GTPase), which are required to direct protein and membrane delivery from prevacuolar endosomal compartments to the vacuole, dramatically increase/stabilize PtdIns(3)P levels in vivo by disrupting its turnover . We find that the majority of the total pool of PtdIns(3)P which has been synthesized, but not PtdIns(4)P, requires transport to the vacuole in order to be turned over . Unexpectedly, strains with impaired vacuolar hydrolase activity accumulate 4- to 5-fold higher PtdIns(3)P levels than wild-type cells, suggesting that lumenal vacuolar lipase and/or phosphatase activities degrade PtdIns(3)P . Because vacuolar hydrolases act in the lumen, PtdIns(3)P is likely to be transferred from the cytoplasmic membrane leaflet where it is synthesized, to the lumen of the vacuole . Interestingly, mutants that stabilize PtdIns(3)P accumulate small uniformly-sized vesicles (40-50 nm) within prevacuolar endosomes (multivesicular bodies) or the vacuole lumen . Based on these and other observations, we propose that PtdIns(3)P is degraded by an unexpected mechanism which involves the sorting of PtdIns(3)P into vesicles generated by invagination of the limiting membrane of the endosome or vacuole, ultimately delivering the phosphoinositide into the lumen of the compartment where it can be degraded by the resident hydrolases. EMBO J, 1998 Sep 1, 17(17), 4920 - 9 DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate; Maeda Y et al.; Biosynthesis of glycosylphosphatidylinositol and N-glycan precursor is dependent upon a mannosyl donor, dolichol phosphate-mannose (DPM) . The Thy-1negative class E mutant of mouse lymphoma and Lec15 mutant Chinese hamster ovary (CHO) cells are incapable of DPM synthesis . The class E mutant is defective in the DPM1 gene which encodes a mammalian homologue of Saccharomyces cerevisiae Dpm1p that is a DPM synthase, whereas Lec15 is a different mutant, indicating that mammalian DPM1 is not sufficient for DPM synthesis . Here we report expression cloning of a new gene, DPM2, which is defective in Lec15 cells . DPM2, an 84 amino acid membrane protein expressed in the endoplasmic reticulum (ER), makes a complex with DPM1 that is essential for the ER localization and stable expression of DPM1 . Moreover, DPM2 enhances binding of dolichol phosphate, a substrate of DPM synthase . Mammalian DPM1 is catalytic because a fusion protein of DPM1 that was stably expressed in the ER synthesized DPM without DPM2 . Therefore, biosynthesis of DPM in mammalian cells is regulated by DPM2. Curr Genet, 1998 Aug, 34(2), 112 - 9 Characterization of a galactose-1-phosphate uridylyltransferase gene from the marine red alga Gracilaria gracilis; Lluisma AO et al.; The metabolism of D-galactose is a major feature of red-algal physiology . We have cloned and sequenced a gene from the red alga Gracilaria gracilis that encodes a key enzyme of D-galactose metabolism, galactose-1-phosphate uridylyltransferase (GALT) . This gene, designated GgGALT1, is apparently devoid of introns . A potential TATA box, four potential CAAT boxes, and a repeated sequence occur in the 5'-flanking region . The predicted 369-aa peptide shares significant sequence similarity with GALTs from other organisms (human, 47%; Saccharomyces cerevisiae, 49%; Solanum tuberosum, 49%) . Southern-hybridization analysis reveals two related, but apparently not identical, GALT genes in the nuclear genome of G . gracilis . Sequence analysis indicates that the GgGALT1 enzyme lacks a rubredoxin "knuckle" motif, which in bacterial and fungal GALTs is involved in binding zinc . An open reading frame encoding a potential peptidyl tRNA hydrolase occurs 179 bp downstream from the GgGALT1 gene. Genome Res, 1998 Aug, 8(8), 834 - 41 Direct selection of conserved cDNAs from the DiGeorge critical region: isolation of a novel CDC45-like gene; McKie JM et al.; We have used a modified direct selection technique to detect transcripts that are both evolutionary conserved and developmentally expressed . The enrichment for homologous mouse cDNAs by use of human genomic DNA as template is shown to be an efficient and rapid approach for generating transcript maps . Deletions of human 22q11 are associated with several clinical syndromes, with overlapping phenotypes, for example, velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS) . A large number of transcriptional units exist within the defined critical region, many of which have been identified previously by direct selection . However, no single obvious candidate gene for the VCFS/DGS phenotype has yet been found . Our technique has been applied to the DiGeorge critical region and has resulted in the isolation of a novel candidate gene, Cdc45l2, similar to yeast Cdc45p . {The sequence data described in this paper have been submitted to the EMBL data library under accession nos . AJ0223728 and AF0223729.} Mol Cell Endocrinol, 1998 Jun 25, 141(1-2), 153 - 62 Subtype- and response element-dependent differences in transactivation by peroxisome proliferator-activated receptors alpha and gamma; Kassam A et al.; Peroxisome proliferator-activated receptors (PPAR) modulate transcription by binding to specific peroxisome proliferator-response elements (PPRE) through heterodimerization with the 9-cis retinoic acid receptor (RXR) . To investigate potential subtype- and response element-dependent differences in transcriptional activation by PPARs, we expressed PPARalpha or PPARgamma2, along with RXRalpha, in the yeast Saccharoromyces cerevisiae and compared their ability to activate transcription of reporter genes containing a PPRE from either the rat acyl-CoA oxidase (AOx) or hydratase-dehydrogenase (HD) gene . PPARgamma2 and RXRalpha, when coexpressed from low copy vectors, potently and synergistically activated transcription of the AOx-PPRE reporter gene, but only weakly stimulated transcription of the HD-PPRE reporter gene . This response element preference, which was also observed in mammalian cells, could not be attributed to differences in binding affinity of PPARgamma2/RXRalpha heterodimers to these elements in vitro . Interestingly, PPARgamma2 expressed from a high copy vector was able to strongly activate transcription of the HD-PPRE reporter gene, even in the absence of coexpressed RXRalpha . In comparison to the findings with PPARgamma2, the HD-PPRE served as a significantly more robust response element for PPARalpha as compared to the AOx-PPRE . PPRE-dependent transcriptional activation by PPARalpha correlated with binding efficiencies of PPARalpha/RXRalpha to the response element . Our findings demonstrate that the transactivation potential of PPAR subtypes can be differentially modulated by distinct PPREs. Genomics, 1998 Jul 15, 51(2), 262 - 9 Identification and mapping of human histone acetylation modifier gene homologues; Randhawa GS et al.; The products of histone acetyltransferase and deacetyltransferase genes regulate histone acetylation in eukaryotes, thereby regulating access of transcription factors to chromatin and modulating gene expression . Histone acetylation modifiers have been found to participate as cofactors in diverse mammalian transcriptional complexes involved in regulation of cellular proliferation and differentiation . A role for histone acetylase has been implicated in leukemias and developmental disorders . To gain insight into a role of additional potential histone acetylation modifier genes in human disease, we identified six histone acetyl-transferase or deacetyltransferase homologues using the dbEST database, and we mapped, using high-resolution FISH, a total of five family members to 1p34.3, 6q21-q22, 5q31, 3p24, and 17q21 . We then identified human genetic disorders for which candidate genes are not yet known and that have been mapped to the same chromosomal regions as the histone acetylation modifiers . This analysis may help identify new candidate genes for human diseases that involve disturbances of histone acetylation. J Biochem (Tokyo), 1998 Sep, 124(3), 519 - 27 High mobility group proteins 1 and 2 can function as DNA-binding regulatory components for DNA-dependent protein kinase in vitro; Yumoto Y et al.; The DNA-dependent protein kinase (DNA-PK) holoenzyme consists of a 470-kDa catalytic subunit (DNA-PKcs), a DNA-binding regulatory component known as Ku protein, and double-stranded DNA (dsDNA) with ends . We previously reported that the activity of DNA-PK in vitro is stimulated by non-histone chromosomal high mobility group proteins (HMG) 1 and 2 comprising two similar repeats, termed domains A and B, and an acidic C-terminal . Here we demonstrate that in vitro HMG1 and 2 can completely replace Ku protein as the DNA-binding regulatory component of DNA-PK . DNA-PKcs and Ku protein were separately purified from Raji nuclear extracts, and reconstituted into the DNA-PK holoenzyme in the presence of dsDNA . DNA-PKcs alone catalyzed DNA-dependent phosphorylation at a very low but significant level, and HMG1 and 2 markedly stimulated the phosphorylation of alpha-casein and a specific peptide substrate in a DNA-dependent manner . The HMG2-domains (A+B) polypeptide devoid of the C-terminal acidic region was more effective for DNA-PKcs stimulation than the full-length HMG2, and HMG2-domain A and -domain B polypeptides . Anti(Ku protein) antibodies inhibited the DNA-dependent phosphorylation activity of the DNA-PKcs:Ku protein complex, but not that of DNA-PKcs alone or when it was complexed with HMG1 or 2 . These results demonstrate that HMG1 and 2 can function as the DNA-binding regulatory component for DNA-PKcs in vitro, and imply that a conformational change of dsDNA, which is elicited by regulatory components, is important for the stimulation of DNA-PK activity of DNA-PKcs. Nucleic Acids Res, 1998 Sep 15, 26(18), 4304 - 5 A capillary electrophoresis mobility shift assay for protein-DNA binding affinities free in solution; Foulds GJ et al.; Quantitative determination of dissociation constants for DNA-protein complexes will help clarify the molecular mechanisms of transcription, replication and DNA repair . A practical capillary electrophoresis mobility shift assay (CEMSA) for protein-DNA affinities free in solution is presented . The method is fast and simple, precise and general . The speed (<2 min separations) and simplicity derive from the use of an uncoated capillary with no gel matrix . The dissociation constant for GCNK58, a DNA-binding-region construct of the yeast transcription factor GCN4, binding to the AP1 DNA site was measured ( K d = 35 +/- 4 nM) to demonstrate the utility of the method. Nucleic Acids Res, 1998 Sep 15, 26(18), 4146 - 52 Mapping of interaction domains between human repair proteins ERCC1 and XPF; de Laat WL et al.; ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes . Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions . In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion . No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype . Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency . All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins . To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits . The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1 . Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart . The ERCC1-binding domain in XPF maps to C-terminal residues 814-905 . ERCC1-XPF complex formation is established by a direct interaction between these two binding domains . A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1. J Cell Biol, 1998 Aug 24, 142(4), 913 - 22 Acidic di-leucine motif essential for AP-3-dependent sorting and restriction of the functional specificity of the Vam3p vacuolar t-SNARE; Darsow T et al.; The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p . Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway . Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways . Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Delta cells . Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein-protein interaction domains) that allow it to efficiently substitute for Pep12p . Using this screen, we identified mutations in a 7-amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome . This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor-dependent transport in both yeast and mammalian systems . Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal . Together, these data provide the first identification of an adaptor protein-specific sorting signal in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function . Regulated transport of cargoes such as Vam3p through the AP-3-dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole. J Biol Chem, 1998 Sep 4, 273(36), 23495 - 503 The malonyl-CoA-sensitive form of carnitine palmitoyltransferase is not localized exclusively in the outer membrane of rat liver mitochondria; Hoppel CL et al.; The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology . We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling . Our results indicate that the mitoplasts contain just 15-19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes . These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes . Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact . Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction . The outer membrane fraction, which consisted of membrane "ghosts," contained most (50-60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27-29% CPT-I activity . Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes. J Biol Chem, 1998 Sep 4, 273(36), 23485 - 94 Nip1p associates with 40 S ribosomes and the Prt1p subunit of eukaryotic initiation factor 3 and is required for efficient translation initiation; Greenberg JR et al.; Nip1p is an essential Saccharomyces cerevisiae protein that was identified in a screen for temperature conditional (ts) mutants exhibiting defects in nuclear transport . New results indicate that Nip1p has a primary role in translation initiation . Polysome profiles indicate that cells depleted of Nip1p and nip1-1 cells are defective in translation initiation, a conclusion that is supported by a reduced rate of protein synthesis in Nip1p-depleted cells . Nip1p cosediments with free 40 S ribosomal subunits and polysomal preinitiation complexes, but not with free or elongating 80 S ribosomes or 60 S subunits . Nip1p can be isolated in an about 670-kDa complex containing polyhistidine-tagged Prt1p, a subunit of translation initiation factor 3, by binding to Ni2+-NTA-agarose beads in a manner completely dependent on the tagged form of Prt1p . The nip1-1 ts growth defect was suppressed by the deletion of the ribosomal protein, RPL46 . Also, nip1-1 mutant cells are hypersensitive to paromomycin . These results suggest that Nip1p is a subunit of eukaryotic initiation factor 3 required for efficient translation initiation. J Biol Chem, 1998 Sep 4, 273(36), 23327 - 34 Molecular cloning of Aralar, a new member of the mitochondrial carrier superfamily that binds calcium and is present in human muscle and brain; del Arco A et al.; We have identified a new calcium-dependent subfamily of mitochondrial carrier proteins with members in Saccharomyces cerevisiae, Caenorhabditis elegans, and various mammalian species . The members of this subfamily have a bipartite structure: a carboxyl-terminal half with the characteristic features of the mitochondrial solute carrier superfamily and an amino-terminal extension harboring various EF-hand domains . A member of this subfamily (that we have termed Aralar) was cloned from a human heart cDNA library . The corresponding cDNA comprises an open reading frame of 2037 base pairs encoding a polypeptide of 678 amino acids . The carboxyl-terminal half of Aralar (amino acids 321-678) has high similarity with the oxoglutarate, citrate, and adenine nucleotide carriers (28-29% identity), whereas the amino-terminal half (amino acids 1-320) contains three canonical EF-hands . Aralar amino-terminal half was shown to bind calcium by 45Ca2+ overlay and calcium-dependent mobility shift assays . The subcellular localization of the protein in COS cells transfected with Aralar was exclusively mitochondrial . Antibodies against Aralar amino-terminal fusion protein recognized a 70-kDa protein in brain mitochondrial fractions . Northern blot analysis showed that the protein was expressed in heart, brain, and skeletal muscle . The domain structure, mitochondrial localization, and presence in excitable tissues suggests a possible function of Aralar as calcium-dependent mitochondrial solute carrier. J Biol Chem, 1998 Sep 4, 273(36), 23304 - 12 Filamin binds to the cytoplasmic domain of the beta1-integrin . Identification of amino acids responsible for this interaction; Loo DT et al.; Integrins play an important role in regulating cell adhesion, motility, and activation . In an effort to identify intracellular proteins expressed by activated T cells that interact with the cytoplasmic domain of beta1-integrin (CD29), we used the beta1-integrin cytoplasmic domain as bait in the yeast two-hybrid system . Here we report that the cytoplasmic domain of beta1-integrin specifically interacts with the cytoskeletal protein filamin . This interaction required all but the most carboxyl-terminal three residues of the cytoplasmic domain of beta1, and the carboxyl-terminal 477 residues of filamin containing the terminal 4 . 5 approximately 96-residue tandem repeats of filamin . To verify this interaction in vivo, we showed that filamin specifically coprecipitated with beta1 in mammalian cells . We also showed that recombinant filamin chimeric proteins were able to bind to the beta1 cytoplasmic domain in vitro . We observed that a subset of single point mutations in the cytoplasmic domain of beta1, which had been previously reported to impair its function, disrupt the interaction between beta1 and filamin . Taken together, these findings suggest that the interaction between beta1 and filamin, which in turn can bind actin, provides a mechanism for the interaction of this cell surface receptor with cytoskeletal proteins and that this interaction plays a role in normal receptor function. J Biol Chem, 1998 Sep 4, 273(36), 23026 - 32 Transcriptional activation capacity of the novel PLAG family of zinc finger proteins; Kas K et al.; We have isolated and characterized two novel cDNAs encoding C2H2 zinc finger proteins showing high sequence homology to PLAG1, a protein ectopically activated by promoter swapping or promoter substitution in pleomorphic adenomas with chromosomal abnormalities at chromosome 8q12 . PLAG1 and the two new PLAG1 family members (PLAGL1 and PLAGL2) constitute a novel subfamily of zinc finger proteins that recognize DNA and/or RNA . To examine the potential of the three human proteins to modulate transcription, we constructed several PLAG/GAL4 DNA binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct in different mammalian cell lines and in yeast . Although the carboxyl-terminal part of PLAGL1 shows strong overall transcriptional activity in mesenchymal (COS-1) and epithelial cells (293), both PLAG1 and PLAGL2 transactivate in mesenchymal cells only if depleted from a repressing region . This effect is less profound in epithelial cells . These data suggest that the activation in pleomorphic adenomas of PLAG1 most likely results in uncontrolled activation of downstream target genes. FEBS Lett, 1998 Aug 7, 432(3), 182 - 6 Molecular characterization and functional expression of dihydroxypterocarpan 6a-hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.); Schopfer CR et al.; Four cytochrome P450-dependent enzymes, among them dihydroxypterocarpan 6a-hydroxylase (D6aH), are specifically involved in the elicitor-inducible biosynthesis of glyceollins, the phytoalexins of soybean . Here we report that CYP93A1 cDNA, which we isolated previously from elicitor-induced soybean cells, codes for a protein with D6aH activity . Analysis of the catalytic properties of recombinant CYP93A1 expressed in yeast, its NADPH dependency, stereoselectivity and high substrate affinity confirmed that D6aH is the physiological function of CYP93A1 . It thus represents the first isoflavonoid-specific CYP to be characterized at the molecular level . In elicitor-treated soybean cells producing phytoalexins, increases in D6aH activity were correlated with elevated transcript levels which indicates that expression of the enzyme is regulated at the level of transcription . Therefore, CYP93A1 cDNA can be used as a specific molecular marker for the inducible defense response against pathogen attack. Genetica, 1998, 102-103(1-6), 29 - 39 Deleterious mutation accumulation in organelle genomes; Lynch M et al.; It is well established on theoretical grounds that the accumulation of mildly deleterious mutations in nonrecombining genomes is a major extinction risk in obligately asexual populations . Sexual populations can also incur mutational deterioration in genomic regions that experience little or no recombination, i.e., autosomal regions near centromeres, Y chromosomes, and organelle genomes . Our results suggest, for a wide array of genes (transfer RNAs, ribosomal RNAs, and proteins) in a diverse collection of species (animals, plants, and fungi), an almost universal increase in the fixation probabilities of mildly deleterious mutations arising in mitochondrial and chloroplast genomes relative to those arising in the recombining nuclear genome . This enhanced width of the selective sieve in organelle genomes does not appear to be a consequence of relaxed selection, but can be explained by the decline in the efficiency of selection that results from the reduction of effective population size induced by uniparental inheritance . Because of the very low mutation rates of organelle genomes (on the order of 10(-4) per genome per year), the reduction in fitness resulting from mutation accumulation in such genomes is a very long-term process, not likely to imperil many species on time scales of less than a million years, but perhaps playing some role in phylogenetic lineage sorting on time scales of 10 to 100 million years. Appl Microbiol Biotechnol, 1998 Jul, 50(1), 85 - 92 Utilization of the TEF1-alpha gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae; Kitamoto N et al.; For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1 alpha, was cloned from the same strain and used for expression of polygalacturonase genes . The TEF1 gene comprised 1647 bp with three introns . The TEF1-alpha protein consisted of 460 amino acids possessing high identify to other fungal TEF proteins . Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A, oryzae TEF1 gene has a strong promoter activity . Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter . A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases . PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity . PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 degrees C . PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C. Curr Opin Cell Biol, 1998 Aug, 10(4), 523 - 9 Delivery of proteins and organelles to the vacuole from the cytoplasm; Scott SV et al.; The vacuole/lysosome is a primary catabolic site in the eukaryotic cell . One implication of its cellular role is that delivery systems must exist to target both hydrolytic enzymes and substrates destined for degradation to this organelle . A number of nonclassical vacuolar targeting pathways that deliver degradative substrates and at least one resident enzyme from the cytosol to the vacuole have recently been described . The pathways identified so far include cytoplasm to vacuole targeting, macroautophagy, pexophagy and vacuolar import and degradation . Cytological, genetic and molecular genetic approaches have begun to provide insight into the molecular basis of these processes. Trends Cell Biol, 1998 Jul, 8(7), 276 - 82 Phosphatidylinositol transfer proteins: the long and winding road to physiological function; Kearns BG et al.; Phosphatidylinositol transfer proteins (PITPs) have historically been thought to help execute lipid-sorting events by transporting phospholipid monomers between membrane bilayers . Recent data, however, indicate unanticipated roles for PITPs in the coordination and/or coupling of phospholipid metabolism with vesicle trafficking and the downregulation of signal-transduction reactions . We are only now beginning to appreciate both the identities of PITP-dependent cellular reactions and the intriguing mechanisms by which PITPs execute their functions in eukaryotic cells. Nature, 1998 Aug 6, 394(6693), 592 - 5 Perinuclear localization of chromatin facilitates transcriptional silencing; Andrulis ED et al.; Transcriptional silencing in Saccharomyces cerevisiae at the HM mating-type loci and telomeres occurs through the formation of a heterochromatin-like structure . HM silencing is regulated by cis-acting elements, termed silencers, and by trans-acting factors that bind to the silencers . These factors attract the four SIR (silent information regulator) proteins, three of which (SIR2-4) spread from the silencers to alter chromatin, hence silencing nearby genes . We show here that an HMR locus with a defective silencer can be silenced by anchoring the locus to the nuclear periphery . This was accomplished by fusing integral membrane proteins to the GAL4 DNA-binding domain and overproducing the hybrid proteins, causing them to accumulate in the endoplasmic reticulum and the nuclear membrane . We expressed the hybrid proteins in a strain carrying an HMR silencer with GAL4-binding sites (UAS(G)) replacing silencer elements, causing the silencer to become anchored to the nuclear periphery and leading to silencing of a nearby reporter gene . This silencing required the hybrids of the GAL4 DNA-binding domain with membrane proteins, the UAS(G) sites and the SIR proteins . Our results indicate that perinuclear localization helps to establish transcriptionally silent chromatin. J Cell Biochem, 1998 Sep 1, 70(3), 366 - 75 Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation; Grayson J et al.; Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers . Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites . Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae . The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein . Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins . These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation. Biochemistry, 1998 Aug 25, 37(34), 11932 - 9 Interaction of Rad51 with ATP and Mg2+ induces a conformational change in Rad51; Namsaraev EA et al.; The presumptive first step in the Rad51-promoted formation of joint molecules is binding of the protein to ssDNA in the presence of ATP and Mg2+ . In this paper, we report that Rad51's ability to bind DNA is rapidly inactivated when incubated at 30-37 degrees C but is stabilized by the presence of ATP and Mg2+ . Although unable to promote binding to DNA, ATP-gamma-S also prevents inactivation of Rad51 at 37 degrees C . AMP-P-N-P lacks this property, while ADP protects partially but only at 5-10 times higher concentrations than ATP . These observations correlate with the dissociation constant of those nucleotides for Rad51 determined by equilibrium dialysis . Rad51 binds ATP and ATP-gamma-S with a 1:1 stoichiometry and Kds of 21 and 19 microM, respectively . The presence of DNA significantly increases the affinity of Rad51 for ATP, while DNA has a smaller effect on the affinity of ATP-gamma-S . Competition binding studies show that ADP and AMP-P-N-P bind with a 5- and 55-fold lower affinity, respectively, than ATP . The CD spectrum of Rad51 with negative double minima at around 210 and 222 nm is characteristic of an alpha-helical protein . Upon binding ATP and Mg2+, the CD spectrum is altered in the regions 194-208 and 208-235 nm, changes that are indicative of a more structured state; this change does not occur with Rad51 that has been inactivated at 37 degrees C . We surmise that the active conformation is more resistant to inactivation at elevated temperature . Our data suggest that one of the roles of ATP and Mg2+ in Rad51-mediated strand exchange is to induce the proper protein structure for binding the two DNA substrates. Mol Endocrinol, 1998 Aug, 12(8), 1172 - 83 Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor2); Berrevoets CA et al.; Previous studies in yeast and mammalian cells showed a functional interaction between the amino-terminal domain and the carboxy-terminal, ligand-binding domain (LBD) of the human androgen receptor (AR) . In the present study, the AR subdomains involved in this in vivo interaction were determined in more detail . Cotransfection experiments in Chinese hamster ovary (CHO) cells and two-hybrid experiments in yeast revealed that two regions in the NH2-terminal domain are involved in the functional interaction with the LBD: an interacting domain at the very NH2 terminus, located between amino acid residues 3 and 36, and a second domain, essential for transactivation, located between residues 370 and 494 . Substitution of glutamic acid by glutamine at position 888 (E888Q) in the AF-2 activation domain (AD) core region in the LBD, markedly decreased the interaction with the NH2-terminal domain . This mutation neither influenced hormone binding nor LBD homodimerization, suggesting a role of the AF-2 AD core region in the functional interaction between the NH2-terminal domain and the LBD . The AF-2 AD core region was also involved in the interaction with the coactivator TIF2 (transcriptional intermediary factor 2), as the E888Q mutation decreased the stimulatory effect of TIF2 on AR AF-2 activity . Cotransfection of TIF2 and the AR NH2-terminal domain expression vectors did not result in synergy between both factors in the induction of AR AF-2 activity . TIF2 highly induced AR AF-2 activity on a complex promoter {mouse mammary tumor virus (MMTV)}, but it was hardly active on a minimal promoter (GRE-TATA) . In contrast, the AR NH2-terminal domain induced AR AF-2 activity on both promoter constructs . These data indicate that both the AR NH2-terminal domain and the coactivator TIF2 functionally interact, either directly or indirectly, with the AF-2 AD core region in the AR-LBD, but the level of transcriptional response induced by TIF2 depends on the promoter context. Mycoses, 1998, 41 Suppl 1, 32 - 8 Cytochromes P450 in fungi; Vanden Bossche H et al.; The article gives an overview on the history of the discovery of P450 cytochromes and on their occurrence in nature, especially on their interactions with metabolic pathways in fungi . The significance of the P450 cytochromes in the ergosterol synthesis as well as in the inhibitory mechanisms caused by imidazole and triazole antimycotics is described in detail. Genes Dev, 1998 Aug 15, 12(16), 2587 - 97 Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1; Kaiser P et al.; Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins . Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit . A screen for genetic interactions with a cdc34 mutation yielded MET30, which encodes an F-box protein . MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components . Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30 complex . We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1 . Swe1 is stabilized in met30 mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo . Furthermore, extracts prepared from cdc34 or met30 mutants are defective in polyubiquitination of Swe1 . Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis . Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity. Genes Dev, 1998 Aug 15, 12(16), 2463 - 8 Nucleolar localization of early tRNA processing; Bertrand E et al.; There is little information as to the location of early tRNA biosynthesis . Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli . We also probed the RNA subunit of RNase P . The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm . These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly . The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis. Eur J Biochem, 1998 Jul 15, 255(2), 482 - 91 The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the ubiquitin-binding first component, a ubiquitin-activating enzyme; Majetschak M et al.; Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo . Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation) . The ubiquitylation of calmodulin appears to fall into this category . Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease . Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes . We therefore decided to attempt this for the calmodulin ligase . The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin . A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2) . The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited . uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity . The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1) . The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration . The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD) . N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence . No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity . The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa. Cell Growth Differ, 1998 Aug, 9(8), 595 - 610 Regulation of cyclin-dependent kinase 4 during adipogenesis involves switching of cyclin D subunits and concurrent binding of p18INK4c and p27Kip1; Phelps DE et al.; Terminal differentiation of many cell lineages involves an exit from the mitotic cycle and entry into, and maintenance of, a permanent state of G1 arrest . We found that during terminal differentiation of mouse 3T3-L1 preadipocytes, the level of cyclin-dependent kinase 4 (CDK4) remained constant, but the subunit composition of the CDK4 complex underwent a dynamic rearrangement . As 3T3-L1 cells differentiated, the levels of cyclin D1 and cyclin D1-CDK4 complexes declined to negligible levels . Meanwhile, cyclins D2 and D3 levels and their associations with CDK4 increased transiently and persistently, respectively, with cyclin D3 becoming the predominant cyclin partner of CDK4 in mature adipocytes . At least five CDK inhibitors are expressed during the differentiation program of 3T3-L1 cells . Both p15INK4b and p16INK4a continuously declined to undetectable levels immediately after differentiation induction . p21 was transiently expressed during the exit of 3T3-L1 cells from mitotic clonal expansion and then decreased to undetectable levels in mature adipocytes . The level of p27KiP1 and p27-CDK4 complexes remain high during differentiation and in mature adipocytes . Distinctly, there is a remarkable induction of p18INK4c mRNA and protein that was not seen in the closely related nondifferentiating 3T3-C2 cell line, suggesting that p18 induction in 3T3-L1 cells is related to cell differentiation, not cell cycle arrest . The pRb kinase activity of cyclin D3 and CDK4 was not detected in quiescent 3T3-L1 cells and was then induced as the cells entered the mitotic clonal expansion phase . Unexpectedly, cyclin D3 and CDK4 pRb kinase activity remained high after 3T3-L1 cells completed their mitotic division and was still readily detectable in mature adipocytes . Our study reveals an active regulation, rather than passive inhibition, of CDK4 activity during adipocyte differentiation . Two central features of this complex regulation are switching of activating cyclin D subunits and concurrent binding by the p18 and p27 CDK inhibitors. Biochim Biophys Acta, 1998 Aug 10, 1366(1-2), 127 - 37 Bcl-2 family proteins and mitochondria; Reed JC et al.; The Bcl-2 family of proteins plays a pivotal role in regulating cell life and death . Many of these proteins reside in the outer mitochondrial membrane, oriented towards the cytosol . Cytoprotective Bcl-2 family proteins such as Bcl-2 and Bcl-XL prevent mitochondrial permeability transition pore opening and release of apoptogenic proteins from mitochondria under many circumstances that would otherwise result in either apoptosis or necrosis . In contrast, some pro-apoptotic members of this family such as Bax can induce these destructive changes in mitochondria in both mammalian cells and when expressed exogenously in yeast . The mechanisms by which Bcl-2 family proteins control cell life and death remain elusive, but may include both the ability to form ion channels or pores in membranes and physical interactions with a variety of proteins implicated in apoptosis regulation. Gene, 1998 Aug 17, 216(1), 113 - 22 cDNA cloning and functional analysis of p28 (Nas6p) and p40.5 (Nas7p), two novel regulatory subunits of the 26S proteasome; Hori T et al.; We employed cDNA cloning to deduce the complete primary structures of p28 and p40.5, two novel subunits of PA700 (also called 19S complex), a 700 kDa multisubunit regulatory complex of the human 26S proteasome . These polypeptides consisted of 226 and 376 amino acids with calculated molecular masses of 24428 Da and 42945 Da, and isoelectric points of 5 . 68 and 5.46, respectively . Intriguingly, p28 contained five conserved motifs known as 'ankyrin repeats', implying that this subunit may contribute to interaction of the 26S proteasome with other protein(s) . Computer-assisted homology analysis revealed high sequence similarities of p28 and p40.5 with yeast proteins, termed Nas6p and Nas7p (non-ATPase subunits 6 and 7), respectively, whose functions are as yet unknown . Disruption of these yeast genes, NAS6 and NAS7, had no effect on cell viability, indicating that neither of the two subunits is essential for proliferation of yeast cells . However, the NAS7, but not NAS6, disruptant cells caused high sensitivity to heat stress, being unable to proliferate at 37 degreesC. Biochim Biophys Acta, 1998 Aug 14, 1404(1-2), 101 - 12 Rab proteins; Martinez O et al.; Rab proteins form the largest branch of the Ras superfamily of GTPases . They are localized to the cytoplasmic face of organelles and vesicles involved in the biosynthetic/secretory and endocytic pathways in eukaryotic cells . It is now well established that Rab proteins play an essential role in the processes that underlie the targeting and fusion of transport vesicles with their appropriate acceptor membranes . However, the recent discovery of several putative Rab effectors, which are not related to each other and which fulfil diverse functions, suggests a more complex role for Rab proteins . At least two Rab proteins act at the level of the Golgi apparatus . Rab1 and its yeast counterpart Ypt1 control transport events through early Golgi compartments . Work from our laboratory points out a role for Rab6 in intra-Golgi transport, likely in a retrograde direction. FEBS Lett, 1998 Jul 24, 431(3), 468 - 72 Clofibrate-inducible, 28-kDa peroxisomal integral membrane protein is encoded by PEX11; Abe I et al.; We cloned a human PEX11 cDNA by expressed sequence tag homology search using yeast Candida boidinii PEX11, followed by screening of human liver cDNA library . PEX11 encoded a peroxisomal protein Pex11p comprising 247 amino acids, with two transmembrane segments and a dilysine motif at the C-terminus . Pex11p comigrated in SDS-PAGE with a 28-kDa peroxisomal integral membrane protein (PMP28) isolated from the liver of clofibrate-treated rats and was crossreactive to anti-PMP28 antibody, thereby indicating PEX11 to encode PMP28 . Pex11p exposes both N- and C-terminal parts to the cytosol . PEX11 was not responsible for ten complementation groups of human peroxisome deficiency disorders. FEBS Lett, 1998 Jul 24, 431(3), 362 - 6 Glucose stimulates the activation domain potential of the PDX-1 homeodomain transcription factor; Petersen HV et al.; Glucose-stimulated expression of the insulin gene in beta cells is mediated by the PDX-1 transcription factor . In this report, we show that stimulation results from effects on activation and DNA-binding potential . Thus, glucose specifically stimulated expression in MIN6 beta cells from chimeras of PDX-1 and the GAL4 DNA-binding domain which spanned the N-terminal PDX-1 activation domain located between amino acids 1 to 79 . GAL4:PDX activity was induced over physiological glucose concentrations and was also regulated by effectors of this response . The level of endogenous PDX-1 binding and phosphorylation were also induced under these conditions . We discuss how changes in PDX-1 phosphorylation may influence activity in glucose-treated beta cells. Biochem Pharmacol, 1998 Jun 1, 55(11), 1769 - 75 Molecular specificity of a medium chain acyl-CoA synthetase for substrates and inhibitors: conformational analysis; Kasuya F et al.; Amino acid conjugation is an important route of detoxification of xenobiotic and endogenous carboxylic acids . The specificity of the purified medium chain acyl-CoA synthetase catalyzing the first reaction of amino acid conjugation was investigated further for substrates and inhibitors . Molecular modeling techniques were applied to derive the molecular characteristics of substrates and inhibitors for the medium chain acyl-CoA synthetase . The purified enzyme accepted not only straight medium chain fatty acids but also aromatic acids . Of the arylacetic acids, activity was obtained with naphthylacetic acids, whereas introduction of a methyl group at the alpha-position caused loss of activity . High activity was also observed with cyclohexanoic acid . Diflunisal, 2-hydroxydodecanoic acid, and nalidixic acid inhibited the medium chain acyl-CoA synthetase activity for hexanoic acid, with Ki values of 0.8, 4.4, and 12.3 microM, respectively . The inhibitory carboxylic acids were competitive with respect to hexanoic acid . The hydroxyl or ketone (oxo) groups at the beta-position of carboxylic acids were an important determinant for inhibitory activity . All substrates and inhibitors contained a flat hydrophobic region coplanar to the carboxylate group . In addition, the substrates had negative values for charge on the carbon in the beta-position of carboxylic acids. Biochem Biophys Res Commun, 1998 Aug 19, 249(2), 486 - 91 Cloning, chromosome mapping and expression analysis of the HIRA gene from Drosophila melanogaster; Llevadot R et al.; The human HIRA gene was identified as a putative transcriptional regulator mapping within the DiGeorge syndrome critical region at 22q11 . HIRA-related proteins have been described in a number of species, but functional information concerning family members is only available in Saccharomyces cerevisiae, where the Hir1p and Hir2p proteins are known to be transcriptional corepressors . In order to analyse conservation of HIRA-related genes and to provide resources for functional studies in another model organism we have isolated the HIRA gene from Drosophila melanogaster (dhira) . The 3374 nucleotide cDNA encodes a protein of 1047 aa, showing 42% identity with the human protein . Alignment with the predicted HIRA proteins from human, mouse, chick and pufferfish reveals strong conservation within the N-terminal region which contains seven WD domains, with less conservation of C-terminal sequences . In situ hybridisation to salivary gland chromosomes indicates that the gene resides in region 7B2-3 of the X chromosome . Dhira is expressed through embryonic development and at lower levels during larval and pupal development . The expression of dhira is dramatically increased in early embryos and in females, suggesting that the dhira mRNA could be maternally deposited in the embryos . Biochem Biophys Res Commun, 1998 Aug 19, 249(2), 361 - 5 A1 is a constitutive and inducible Bcl-2 homologue in mature human neutrophils; Chuang PI et al.; During the process of terminal differentiation toward mature neutrophils, the anti-apoptotic proteins Bcl-2 and Bcl-x become down-regulated and eventually cease to be expressed, whereas the death-promoting Bcl-2 homologue, Bax, persists . Thus, the disappearance of anti-apoptotic homologues was thought to account for the early demise of mature neutrophils . However, although the survival of mature human neutrophils can be prolonged by a variety of factors, no anti-apoptotic Bcl-2 homologues have previously been identified . Human A1 is a Bcl-2 homologue previously shown to be present in endothelial cells and to convey anti-apoptotic function in vitro . We describe here that human A1 mRNA is constitutively expressed in mature neutrophils and is up-regulated by G-CSF and LPS, agonists that promote neutrophil survival . In addition, we show progressive A1 mRNA accumulation in HL-60 cells during all-trans retinoic acid-driven neutrophilic differentiation . Our findings suggest that A1 may have an important role in neutrophilic development and in modulating mature neutrophil survival . Science, 1998 Aug 21, 281(5380), 1202 - 6 Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules; Kaiser BN et al.; Nitrogen-fixing bacteroids in legume root nodules are surrounded by the plant-derived peribacteroid membrane, which controls nutrient transfer between the symbionts . A nodule complementary DNA (GmSAT1) encoding an ammonium transporter has been isolated from soybean . GmSAT1 is preferentially transcribed in nodules and immunoblotting indicates that GmSAT1 is located on the peribacteroid membrane . {14C}methylammonium uptake and patch-clamp analysis of yeast expressing GmSAT1 demonstrated that it shares properties with a soybean peribacteroid membrane NH4<SUP ARRANGE="STAGGER">+ channel described elsewhere . GmSAT1 is likely to be involved in the transfer of fixed nitrogen from the bacteroid to the host. J Immunol, 1998 Aug 15, 161(4), 1728 - 37 Interaction of p59fyn kinase with the dynein light chain, Tctex-1, and colocalization during cytokinesis; Campbell KS et al.; The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells . We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases . Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex . The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction . Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors . Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region . Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis . Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis. Mol Cell Biol, 1998 Sep, 18(9), 5587 - 99 Functional and structural organization of Brf, the TFIIB-related component of the RNA polymerase III transcription initiation complex; Kassavetis GA et al.; Brf is the TFIIB-related component of Saccharomyces cerevisiae RNA polymerase III transcription initiation factor IIIB (TFIIIB) . An extensive set of Brf fragments has been examined for the abilities to assemble the TFIIIB-DNA complex and recruit RNA polymerase III to accurately initiate transcription . The principal TFIIIB-assembly function of Brf was found to be contributed by a C-proximal segment spanning amino acids 435 to 545, while the principal transcription-directing function was contributed by a segment of its N-proximal, TFIIB-homologous half . The diverse activities of Brf were also reconstituted from combined fragments . The fragments spanning amino acids 1 to 282 and 284 to 596 were found to assemble into TFIIIB-DNA and TFIIIC-TFIIIB-DNA complexes that were very stable, transcriptionally highly active, and indistinguishable (by in vitro footprinting) from complexes formed with intact Brf . The proximities of the individual halves of split Brf to DNA were extensively mapped by photochemical cross-linking of the TFIIIB-DNA complex . We also identified sites of interaction of Brf fragments with TATA-binding protein (TBP), taking advantage of a recently completed mutational analysis of the TBP surface . The constraints established by these analyses specify a global model of the functional segments of Brf and how they fit into the structure of the TFIIIB-DNA complex. Mol Cell Biol, 1998 Sep, 18(9), 5546 - 56 Core histones and HIRIP3, a novel histone-binding protein, directly interact with WD repeat protein HIRA; Lorain S et al.; The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae . HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q . Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA . Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex . HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts . In vitro, HIRA also interacted with core histone H4 . H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second alpha helix of H4 . HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap . In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism . Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development. Mol Cell Biol, 1998 Sep, 18(9), 5500 - 10 Transcriptional repression by the SMRT-mSin3 corepressor: multiple interactions, multiple mechanisms, and a potential role for TFIIB; Wong CW et al.; A variety of eukaryotic transcription factors, including the nuclear hormone receptors, Max-Mad, BCL-6, and PLZF, appear to mediate transcriptional repression through the ability to recruit a multiprotein corepressor complex to the target promoter . This corepressor complex includes the SMRT/N-CoR polypeptides, mSin3A or -B, and histone deacetylase 1 or 2 . The presence of a histone-modifying activity in the corepressor complex has led to the suggestion that gene silencing is mediated by modification of the chromatin template, perhaps rendering it less accessible to the transcriptional machinery . We report here, however, that the corepressor complex actually appears to exhibit multiple mechanisms of transcriptional repression, only one of which corresponds with detectable recruitment of the histone deacetylase . We provide evidence instead of an alternative pathway of repression that may be mediated by direct physical interactions between components of the corepressor complex and the general transcription factor TFIIB. Mol Cell Biol, 1998 Sep, 18(9), 5465 - 77 Structure of the chromosome VII centromere region in Neurospora crassa: degenerate transposons and simple repeats; Cambareri EB et al.; DNA from the centromere region of linkage group (LG) VII of Neurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical of Neurospora DNA in both composition (AT rich) and complexity (repetitive) . We have determined the DNA sequence of a small portion (approximately 16.1 kb) of this region and have identified a cluster of three new retrotransposon-like elements as well as degenerate fragments from the 3' end of Tad, a previously identified LINE-like retrotransposon . This region contains a novel full-length but nonmobile copia-like element, designated Tcen, that is only associated with centromere regions . Adjacent DNA contains portions of a gypsy-like element designated Tgl1 . A third new element, Tgl2, shows similarity to the Ty3 transposon of Saccharomyces cerevisiae . All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of