J Steroid Biochem Mol Biol, 1997 Feb, 60(3-4), 181 - 7 In vitro binding of vitamin D receptor occupied by 24R,25-dihydroxyvitamin D3 to vitamin D responsive element of human osteocalcin gene; Uchida M et al.; We previously reported that 24R,25-dihydroxyvitamin D3 {24R,25(OH)2D3} activates the human osteocalcin gene (hOC) through vitamin D receptor (VDR) and vitamin D responsive element (VDRE) in the same manner as 1 alpha,25-dihydroxyvitamin D3 {1 alpha,25(OH)2 D3} {17} . In the present study, the interaction of 24R,25(OH)2D3-liganded VDR {24R,25(OH)2D3-VDR} with the hOC VDRE in vitro was investigated . The electrophoretic mobility shift assay (EMSA) revealed that the binding of 24R,25(OH)2D3-liganded VDR to the hOC VDRE was weak, even at concentrations of 24R,25(OH)2D3 10(5)-fold higher than 1 alpha,25(OH)2D3 . The effect of the nuclear accessory factor (NAF), which is required for the high affinity interaction of the VDR to the VDRE, on the binding of the 24R,25(OH)2D3-VDR to the VDRE was studied using hOC VDRE affinity column chromatographic assays . In the absence of NAF, the 24R,25(OH)2D3-VDR associated weakly with the VDRE compared to the 1 alpha,25(OH)2D3-liganded VDR {1 alpha,25(OH)2D3-VDR}, whereas the NAF enhanced the binding of the 24R,25(OH)2D3-VDR for the VDRE . In the absence of the hOC VDRE, the binding affinity of the 24R,25(OH)2D3-VDR for the NAF was weaker than that of 1 alpha,25(OH)2D3-VDR . These results suggest that the weak interaction of the 24R,25(OH)2D3-VDR with both NAF and hOC VDRE is responsible for the weak binding of the 24R,25(OH)2D3-VDR to the VDRE detected in EMSA . In terms of VDR function, 24R,25(OH)2D3 was more potent in transactivation than in vitro binding. Mol Biol Cell, 1997 Feb, 8(2), 205 - 17 Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum; Salama NR et al.; The COPII vesicle coat protein promotes the formation of endoplasmic reticulum- (ER) derived transport vesicles that carry secretory proteins to the Golgi complex in Saccharomyces cerevisiae . This coat protein consists of Sar1p, the Sec23p protein complex containing Sec23p and Sec24p, and the Sec13p protein complex containing Sec13p and a novel 150-kDa protein, p150 . Here, we report the cloning and characterization of the p150 gene . p150 is encoded by an essential gene . Depletion of this protein in vivo blocks the exit of secretory proteins from the ER and causes an elaboration of ER membranes, indicating that p150 is encoded by a SEC gene . Additionally, overproduction of the p150 gene product compromises the growth of two ER to Golgi sec mutants: sec16-2 and sec23-1 . p150 is encoded by SEC31, a gene isolated in a genetic screen for mutations that accumulate unprocessed forms of the secretory protein alpha-factor . The sec31-1 mutation was mapped by gap repair, and sequence analysis revealed an alanine to valine change at position 1239, near the carboxyl terminus . Sec31p is a phosphoprotein and treatment of the Sec31p-containing fraction with alkaline phosphatase results in a 50-75% inhibition of transport vesicle formation activity in an ER membrane budding assay. Genes Cells, 1997 Feb, 2(2), 129 - 41 The highly conserved DAD1 protein involved in apoptosis is required for N-linked glycosylation; Makishima T et al.; BACKGROUND: The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation which have been isolated from the BHK21 cell line derived from the golden hamster . It has a mutation in the DAD1 gene encoding a 12.5kDa highly conserved protein through evolution, and enters apoptosis at the restrictive temperature due to this mutation . RESULTS: DAD1 was recovered in light membrane fractions after differential centrifugation . It could not be released from the membrane, even by carbonate extraction, without a detergent . Upon digestion with proteinase K, both N and C terminal portions-but not the middle portions of DAD1- were released from the membrane . Thus, DAD1 appears to be an integral membrane protein in which both termini are located in the cytosol . DAD1 was localized in the endoplasmic reticulum . In accordance with a similarity to the yeast protein Ost2p, which is a subunit of the oligosaccharyltransferase, at the restrictive temperature, loss of DAD1 function caused a defect of N-linked glycosylation in tsBN7 cells resulting in apoptosis . However, tunicamycin, which is known to inhibit N-linked glycosylation did not induce apoptosis in either tsBN7 or BHK21 cells . CONCLUSION: tsBN7 cells have a defect in N-linked glycosylation caused by the loss of DAD1. Neuropediatrics, 1997 Feb, 28(1), 42 - 4 Farnesylation of Batten disease CLN3 protein; Pullarkat RK et al.; The carboxyl terminal of the predicted amino acid sequence of the Batten disease CLN3 gene protein is CQLS . This motif is expected to be a site for farnesylation at the cysteine residue . In order to determine whether this is indeed farnesylated we have carried out the in-vitro prenylation of tetrapeptides CVLS, CAIL and CQLS using a farnesyl transferase preparation from bovine brain . The data shows that the CQLS is a good acceptor of a farnesyl group similar to CVLS while it is a poor acceptor of a geranylgeranyl group unlike CAIL, which is a good acceptor of a geranylgeranyl group . This suggests that the CLN3 gene product may be a farnesylated protein. Neuropediatrics, 1997 Feb, 28(1), 21 - 2 Genetic linkage analysis of a variant of juvenile onset neuronal ceroid lipofuscinosis with granular osmiophilic deposits; O'Rawe A et al.; A number of variant forms of the neuronal ceroid lipofuscinoses (NCL) have been described and remain unmapped . The genes for infantile (CLN1), juvenile (CLN3) and Finnish-variant late-infantile (CLN5) have previously been mapped to chromosome regions 1p32, 16p12 and 13q21.1-32 respectively . The locus for a variant form of juvenile onset NCL characterised by cytosomal granular osmiophilic deposits (GROD) has been excluded from the CLN3 region of chromosome 16 . This study describes the outcome of genetic linkage analysis in four families with this variant at the loci for the CLN1 and CLN5 genes . Using highly informative microsatellite markers tightly linked to the CLN5 locus we have excluded the JNCL variant with GROD from this region . Marker typing across the CLN1 region suggests that JNCL with GROD may be an allelic variant of infantile NCL. Neuropediatrics, 1997 Feb, 28(1), 15 - 7 Strategy for mutation detection in CLN3: characterisation of two Finnish mutations; Munroe PB et al.; A strategy for detection of mutations in CLN3, the gene for Batten disease or juvenile onset neuronal ceroid lipofuscinosis, has been devised using a technique which detects conformation polymorphisms and direct sequencing of genomic DNA fragments . We define two mutations found uniquely in Finnish patients, one a large deletion (2.8 kb), the other a point mutation affecting the 5'splice donor site of an intron. Neuropediatrics, 1997 Feb, 28(1), 12 - 4 Structure of the CLN3 gene and predicted structure, location and function of CLN3 protein; Mitchison HM et al.; The genomic sequence of the human CLN3 gene, which is defective in juvenile onset neuronal ceroid lipofuscinosis (Batten disease) is being delineated using a variety of methods . A Saccharomyces cerevisiae gene, YHC3 (for Yeast Homologue to human CLN3), which is highly similar to the human disease gene, has been identified by computer-aided homology searching . Topology predictions indicate the CLN3 protein contains six transmembrane segments . Most similarity between the human and yeast proteins lies either in the transmembrane segments or along one face of the predicted protein structure. Mech Dev, 1997 Feb, 62(1), 51 - 60 Homeotic transformation of legs to mouthparts by proboscipedia expression in Drosophila imaginal discs; Aplin AC et al.; The Drosophila homeotic gene proboscipedia (pb) specifies labial identify and directs formation of the adult distiproboscis from the labial imaginal discs . pb null alleles result in the homeotic transformation of the distiproboscis into prothoracic (T1) legs {Kaufman (1978) Genetics 90, 579-596; Pultz et al . (1988) Genes Dev . 2, 901-920} . Homology with other transcription factors, localization to the nucleus, and restricted embryonic and imaginal expression implicate the pb protein (PB) as a transcription factor . In order to examine the possible roles that PB may play in the specification of adult mouthparts, we have expressed PB in cells of wing, leg and eye-antennal imaginal discs and assayed for effects on the development of adult structures . We report here that the ectopic expression of PB in the imaginal discs under the control of the inducible GAL4 system {Brand and Perrimon (1993) Development 118, 401-415} alters the developmental program of adult legs into maxillary or labial palps . These homeotic transformations have an equal effect on all three sets of legs, indicating an activity that is not solely dependent upon the unique combinations of other homeotic genes present in each of the leg discs . Segment polarity genes required for establishing the AP compartment boundary were found to be undisturbed by ectopic PB . Furthermore, normal patterns of apoptosis are observed in animals expressing ectopic PB, indicating that PB does not alter or affect cell death . These results suggest that molecular events occurring downstream of the establishment of the compartment boundary are affected by ectopic PB expression in imaginal discs and point to a general role in 'palp' formation in addition to the specification of labial identity. Curr Biol, 1997 Feb 1, 7(2), R82 - 4 Histone acetyltransferases in control; Wade PA et al.; Several transcriptional regulators have been found to act as enzymes that acetylate histones . The targeted post-translational modification of histones within regulatory nucleoprotein complexes provides an attractive mechanism for controlling transcription within a chromatin environment. Curr Biol, 1997 Feb 1, 7(2), R67 - 70 Splicing together the unfolded-protein response; Shamu CE; Recent work has identified a transcription factor, Hac1p, in the yeast Saccharomyces cerevisiae, as a component of a pathway that signals to the nucleus the presence of unfolded proteins in the endoplasmic reticulum and has shown that Hac1p expression is regulated by a novel RNA splicing pathway. Plant J, 1997 Feb, 11(2), 347 - 52 Tissue-specific expression of ATCYS-3A, a gene encoding the cytosolic isoform of O-acetylserine(thiol)lyase in Arabidopsis; Gotor C et al.; Atcys-3A from Arabidopsis encodes the cytosolic isoform of O-acetylserine(thiol)lyase that catalyzes the last step of cysteine biosynthesis . The Atcys-3A transcript is present in different organs of mature plants, being more abundant in roots and declining to 40-50% in rosette leaves and flowers . In situ hybridization studies have shown a high Atcys-3A signal in root tissues, mainly localized to the cortex and xylem parenchyma . In a flower before anthesis, the transcript is detected exclusively in anthers and sepals and evenly distributed throughout the receptacle of the flower . An unexpected observation from these studies is the highest expression of Atcys-3A mRNA found in trichomes of either leaf or stem . The presence of high levels of the transcript is observed very early in trichome cell development . This is the first report describing the cellular localization of any plant O-acetylserine(thiol)lyase mRNA . The high level of Atcys-3A expression in trichomes raises new aspects to the biological function of trichomes, related to sulfate metabolism. J Toxicol Sci, 1997 Feb, 22(1), 45 - 56 Molecular cloning of mouse liver flavin containing monooxygenase (FMO1) cDNA and characterization of the expression product: metabolism of the neurotoxin, 1,2,3,4-tetrahydroisoquinoline (TIQ); Itoh K et al.; A mouse liver cDNA clone, MFMO1, coding for a flavin-containing monooxygenase (FMO) was isolated . This cDNA clone encoded a protein of 532 amino acids . Based upon its predicted amino acid sequence, this clone was assumed to belong to the FMO1 subfamily . The deduced amino acid sequence showed 94, 84, 83, and 83% identity with FMO1s of rats, pigs, rabbits and humans, respectively, while it showed only 50-59% identity with human FMO3 and FMO4, rabbit FMO2, FMO3, FMO4 and FMO5, and guinea-pig FMO2 . RNA blot analysis showed that the mouse FMO1 was also expressed in the lung and kidney and to lesser extents in the heart, spleen, testis and brain . Mouse FMO1 expressed in yeast showed activities of thiobenzamide S-oxidation, and NADPH oxidation associated with the S- or N-oxidation of chlorpromazine, N,N-dimethylaniline, N,N-dimethyl-hydrazine, imipramine, nicotine, thioacetamide, thiourea and trimethylamine . Moreover, 1,2,3,4-tetrahydroisoquinoline (TIQ), a substance known to induce a parkinsonism-like syndrome in monkeys, was also metabolized by the mouse FMO1 . The K(m) values for chlorpromazine, imipramine and TIQ were determined to be 2,4, 16.0, 435 mM, respectively . This is the first report to show that an expressed FMO can metabolize a neurotoxin, TIQ. Fungal Genet Biol, 1997 Feb, 21(1), 92 - 100 The Aspergillus nidulans Septin Encoding Gene, aspB, Is Essential for Growth Momany M, Hamer JE. Septins are a highly conserved family of presumed cytoskeletal proteins involved in cytokinesis and morphogenesis in Saccharomyces cerevisiae and Drosophila melanogaster . To investigate the role of septins in filamentous fungi, we have identified presumptive septin homologues in Aspergillus nidulans . One of these septins, aspB, is expressed during vegetative growth and asexual sporulation . Based on cDNA and genomic sequences, the predicted aspB protein shares a P-loop motif and coiled coil regions with septins from other organisms . Antibodies generated against an aspB fusion protein recognized an A . nidulans protein of approximately 50 kDa, but could not localize the septin product in cells by immunofluorescence . Hybridization to a chromosome-specific ordered cosmid library placed the aspB gene on the right arm of chromosome I . Disruption of aspB showed that it is an essential gene. Fungal Genet Biol, 1997 Feb, 21(1), 50 - 63 The amdA Regulatory Gene of Aspergillus nidulans: Characterization of Gain-of-Function Mutations and Identification of Binding Sites for the Gene Product Andrianopoulos A, Brons J, Davis MA, Hynes MJ. Genetic evidence suggests that the amdA gene of Aspergillus nidulans encodes a protein which controls the expression of the amdS and aciA structural genes . The amdI66 and amdI666 mutations in the 5' regulatory region of amdS lead to higher levels of amdA-dependent amdS expression . We show here that the putative DNA binding domain of amdA is capable of binding specific regions of the amdS and aciA promoters in vitro and this region includes sequences duplicated and triplicated in the amdI66 and amdI666 mutations, respectively . Footprinting analysis has shown that AmdA binds to two sites in this region represented by the sequences 5'-GCGGGG-3' and 5'-GAGGGG-3' . A number of gain-of-function mutations in amdA were localized to a region rich in acidic and hydrophobic amino acid residues and shown to be involved in transcriptional activation by studies of fusions with the GAL4 DNA binding domain in Saccharomyces cerevisiae . Therefore, both an increased probability of AmdA binding to the amdS promoter and an increased activation potential of AmdA can result in higher levels of expression. Plant Cell, 1997 Feb, 9(2), 171 - 84 The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes; Albani D et al.; The conserved bifactorial endosperm box found in the promoter of wheat storage protein genes comprises two different cis elements that are thought to be involved in regulating endosperm-specific gene expression . Endosperm nuclear extracts contain binding activities . One is called ESBF-I, which binds to the endosperm motif (EM), and the other is called ESBF-II, which binds to the GCN4-like motif(GLM) . Here, we present a functional analysis of the endosperm box of a low-molecular-weight glutenin gene found on the 1D1 chromosome of hexaploid wheat (LMWG-1D1) in transgenic tobacco plants . Our analysis demonstrates the necessity of the EM and GLM for endosperm-specific gene expression and suggests the presence in tobacco of functional counterparts of wheat ESBF-I and ESBF-II . Furthermore, we describe the isolation and characterization of cDNA clones encoding SPA, a seed-specific basic leucine zipper protein from wheat that can activate transcription from the GLMs of the -326-bp LMWG-1D1 promoter in both maize and tobacco leaf protoplasts . This activation is also partially dependent on the presence of functional EMs, suggesting interactions between SPA with ESBF-I-like activities. J Cell Sci, 1997 Feb, 110 ( Pt 3), 295 - 300 Centrosome-microtubule nucleation; Pereira G et al.; In many cell types the formation of microtubules from tubulin subunits is initiated at defined nucleation sites at the centrosome . These sites contain the conserved gamma-tubulin which is in association with additional not very will characterised proteins, identified as components of a gamma-tubulin ring complex from Xenopus egg extracts or from suppressor screens in the yeast Saccharomyces cerevisiae . In this review we discuss two recently proposed models of how the gamma-tubulin complex assists in the assembly of tubulin to form microtubules . These models propose different roles for gamma-tubulin and the other proteins in the complex in tubulin assembly . While the structure and composition of a microtubule nucleation site is becoming clearer, it is still unknown how the cell-cycle dependent regulation of microtubule nucleation sites is achieved and whether they disassemble after microtubule formation in order to allow microtubule fluxes towards the centrosome which have been observed in mitotic cells. J Membr Biol, 1997 Feb 1, 155(3), 189 - 97 Protein translocation across the membrane of the endoplasmic reticulum; Wilkinson BM et al.; Eukaryotic cells are characterized by the existence of membrane-bound subcellular compartments which perform a variety of specialized functions . Proteins destined for these compartments begin their synthesis in the cytosol and must be subsequently targeted to their functional compartment by specific signal sequences present in the newly synthesized polypeptide chain . The translocation of preproteins across biological membranes is a fundamental process of intracellular trafficking and organelle biogenesis . Entry into the secretory pathway occurs by translocation of proteins into or across the membrane of the endoplasmic reticulum (ER) . This process involves two distinct steps which are dependent on the orchestrated action of several proteins . The initial step of targeting involves recognition of the signal sequence and delivery of the protein precursor to the ER in a translocation competent conformation . The subsequent translocation event is characterized by interaction of the preprotein with the translocation channel followed by unidirectional movement across the lipid bilayer of the ER membrane into the lumenal space . The study of the mechanism of the translocation process is one of the most intriguing and rapidly advancing areas in cell biology . Here we review recent findings in both the yeast Saccharomyces cerevisiae and mammals concerning the mechanisms of the translocation step and discuss the roles of the proteins implicated in this process. Trends Biochem Sci, 1997 Feb, 22(2), 43 - 7 Recognition of telomeric DNA; Konig P et al.; The three-dimensional structure of the yeast telomere-binding protein RAP1 in complex with DNA provides the first insight into telomeric DNA recognition . RAP1 binds to DNA via two Myb/homeodomain-like motifs, which are DNA-binding folds previously identified in transcription factors . This, together with the finding that human TRF1 and other telomere-binding factors contain Myb-like motifs, has led us to speculate that a conserved protein fold might be used for telomeric DNA recognition. Development, 1997 Feb, 124(4), 761 - 71 The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells; Ito K et al.; The mushroom body (MB) is an important centre for higher order sensory integration and learning in insects . To analyse the development and organisation of the MB neuropile in Drosophila, we performed cell lineage analysis in the adult brain with a new technique that combines the Flippase (flp)/FRT system and the GAL4/UAS system . We showed that the four mushroom body neuroblasts (MBNbs) give birth exclusively to the neurones and glial cells of the MB, and that each of the four MBNb clones contributes to the entire MB structure . The expression patterns of 19 GAL4 enhancer-trap strains that mark various subsets of MB cells revealed overlapping cell types in all four of the MBNb lineages . Partial ablation of MBNbs using hydroxyurea showed that each of the four neuroblasts autonomously generates the entire repertoire of the known MB substructures. RNA, 1997 Feb, 3(2), 175 - 85 Nuclear pre-tRNA terminal structure and RNase P recognition; Lee Y et al.; Nuclear pre-tRNA transcripts often contain an extension of the aminoacyl stem formed by base pairing between the 5'-leader and 3'-trailing sequences, but the -1 position preceding the mature 5' end is usually left unpaired . Considering recently proposed tertiary structural models for RNase P RNAs, we hypothesize that the -1 mismatch prevents a strong, coaxially extended aminoacyl stem, which might otherwise sterically interfere with substrate positioning in the RNase P active site . This hypothesis is tested by creating uninterrupted aminoacyl stem extensions in four nuclear tRNA precursors that normally have a mismatched nucleotide at position -1, and comparing their cleavage rates with those of the normal precursors . Determinations of Km and kcat values for a normal and an altered pre-tRNA(SUP53), which exhibits the most subtle structural alteration immediately upstream of the cleavage site, indicate that the mismatch at position -1 is an important structural requirement for both substrate affinity and efficient catalysis (and/or product release) by nuclear RNase P . This conclusion is further supported in vivo, where the pre-tRNA(SUP53) mutant precursor lacking the -1 mismatch is shown to accumulate. Curr Opin Neurobiol, 1997 Feb, 7(1), 81 - 6 Rho family GTP-binding proteins in growth cone signalling; Luo L et al.; Rho family GTP-binding proteins regulate various aspects of the actin cytoskeleton in a wide variety of organisms . Recent evidence suggests that they may also be important components of the signalling pathways that link the reception of extracellular cues to the regulation of the cytoskeleton in neuronal growth cones. Genes Dev, 1997 Feb 1, 11(3), 321 - 33 A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme; Yamanaka S et al.; Transgene expression of the apolipoprotein B mRNA-editing enzyme (APOBEC-1) causes dysplasia and carcinoma in mouse and rabbit livers . Using a modified differential display technique, we identified a novel mRNA (NAT1 for novel APOBEC-1 target no . 1) that is extensively edited at multiple sites in these livers . The aberrant editing alters encoded amino acids, creates stop codons, and results in markedly reduced levels of the NAT1 protein in transgenic mouse livers . NAT1 is expressed ubiquitously and is extraordinarily conserved among species . It has homology to the carboxy-terminal portion of the eukaryotic translation initiation factor (eIF) 4G that binds eIF4A and eIF4E to form eIF4F . NAT1 binds eIF4A but not eIF4E and inhibits both cap-dependent and cap-independent translation . NAT1 is likely to be a fundamental translational repressor, and its aberrant editing could contribute to the potent oncogenesis induced by overexpression of APOBEC-1. Curr Opin Genet Dev, 1997 Feb, 7(1), 93 - 8 Control of cell cycle arrest by the Mec1sc/Rad3sp DNA structure checkpoint pathway; Carr AM; The Mec1(sc)/Rad3(sp) protein family is central to the checkpoint pathways of cells . Functions upstream and downstream of Mec1(sc)/Rad3(sp) show both similarities and differences when compared between organisms . Analogy with a related protein, DNAPKcs, suggests that different subunits may activate Mec1(sc)/Rad3(sp) in response to specific DNA or DNA-protein structures. Curr Opin Genet Dev, 1997 Feb, 7(1), 99 - 104 Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double-strand breaks; Lieber MR et al.; A convergence of information from biochemistry, yeast and mammalian genetics, immunology, and radiation biology has permitted identification of some of the protein participants - Ku, DNA-PK, XRCC4 - and the reaction intermediates in DNA end joining, suggesting how broken chromosomal ends may be recognized and repaired in eukaryotic cells . Some components may be defective in inherited disorders. J Pharmacol Exp Ther, 1997 Feb, 280(2), 527 - 32 Acetaldehyde as well as ethanol is metabolized by human CYP2E1; Kunitoh S et al.; Acetaldehyde was oxidized by rat and human hepatic microsomes in the presence of NADPH . We designated this NADPH-dependent oxidation system MAOS (microsomal acetaldehyde-oxidizing system), to distinguish it from the NAD-dependent acetaldehyde oxidation system of acetaldehyde dehydrogenase in mitochondria and cytosol . This activity was increased 2.3-fold by giving rats ethanol . Judging from the Vmax/Km values, the metabolic capacity of rat hepatic microsomes for MAOS activity was increased 24-fold by ethanol . The acetaldehyde oxidation activity of eight forms of purified rat cytochrome P450 was investigated in a reconstituted system . CYP2E1 had the highest level, followed by CYP1A2 and 4A2 . Immunoinhibition studies showed that an anti-CYP2E1 antibody inhibited 90% of the MAOS activity in rats given ethanol . NADPH-dependent acetate formation was 12% or 33.6% of the NAD-dependent acetate formation in liver homogenates of control rats and those treated with ethanol, respectively . We investigated human MAOS activity further . Among the 10 forms of human cytochrome P450 expressed in yeast, CYP2E1 had especially high acetaldehyde oxidation activity . The correlation of MAOS activity with the levels of immunoreactive CYP2E1 in individual human microsomes was highly significant (r2 = 0.88, P < .01) . These results indicate that hepatic CYP2E1 mainly contributes to MAOS in rats and humans, the pathway of which may play an alternative role against acetaldehyde in the liver after alcohol consumption together with acetaldehyde dehydrogenase in the metabolism of acetaldehyde. Hepatology, 1997 Feb, 25(2), 496 - 8 The hepatitis B virus X protein: the quest for a role in viral replication and pathogenesis; Seeger C; Although the biological importance of hepatitis B virus X protein (HBX) in the life cycle of hepatitis B virus has been well established, the cellular and molecular basis of its function remains largely undefined . Despite the association of multiple activities with HBX, none of them appear to provide a unifying hypothesis regarding the true biological function of HBX . Identification and characterization of cellular targets of HBX remain an essential goal in the elucidation of the molecular mechanisms of HBX . Using the Saccharomyces cerevisiae two-hybrid system, we have identified and characterized a novel subunit of the proteasome complex (XAPC7) that interacts specifically with HBX . We also showed that HBX binds specifically to XAPC7 in vitro . Mutagenesis studies have defined the domains of interaction to be critical for the function of HBX . Furthermore, overexpression of XAPC7 appeared to activate transcription by itself and antisense expression of XAPC7 was able to block transactivation by HBX . Therefore, the proteasome complex is possibly a functional target of HBX in cells. Curr Genet, 1997 Feb, 31(2), 97 - 105 Mutations in SPK1/RAD53 that specifically abolish checkpoint but not growth-related functions; Fay DS et al.; SPK1/RAD53/SAD1/MEC2 encodes an essential protein kinase of Saccharomyces cerevisiae and is required for the execution of checkpoint arrest at multiple stages of the cell cycle . We have isolated two mutant alleles of SPK1 (spk1K227A and spk1-1A208P) that are defective for checkpoint-arrest functions but retain wild-type levels of SPK1-associated growth activity . Both mutations occur within conserved regions of the kinase domain of SPK1 resulting in a substantial reduction in the catalytic activity of Spk1 . Thus, while minimal levels of Spk1 kinase activity are capable of supporting normal rates of growth, higher levels are required for checkpoint functions . In addition, using deletional analysis we have identified a region within the N-terminus of Spk1 outside of the conserved kinase domain that is required for checkpoint functions . Interestingly, this region may be important in the regulation of Spk1 kinase activity. Curr Opin Cell Biol, 1997 Feb, 9(1), 86 - 92 Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton; Tapon N et al.; Rho, Rac and Cdc42 are three Ras-related GTP-binding proteins that control the assembly and disassembly of the actin cytoskeleton in response to extracellular signals . During the past year, numerous candidate downstream targets for these GTPases have been identified using affinity chromatography and yeast two-hybrid techniques . These techniques have revealed that Rho regulates the myosin light chain phosphatase and that Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton. Mol Cell Biol, 1997 Feb, 17(2), 934 - 45 Role of hydrophobic amino acid clusters in the transactivation activity of the human glucocorticoid receptor; Almlof T et al.; We have performed a mutagenesis analysis of the 58-amino-acid tau1-core peptide, which represents the core transactivation activity of the tau1 transactivation domain from the glucocorticoid receptor . Mutants with altered activity were identified by phenotypic screening in the yeast Saccharomyces cerevisiae . Most mutants with reduced activity had substitutions of hydrophobic amino acids . Most single-substitution mutants with reduced activity were localized near the N terminus of the tau1-core within a segment that has been shown previously to have a propensity for alpha-helix conformation, suggesting that this helical region is of predominant importance . The particular importance of hydrophobic residues within this region was confirmed by comparing the activities of alanine substitutions of the hydrophobic residues in this and two other helical regions . The hydrophobic residues were shown to be important for the transactivation activity of both the isolated tau1-core and the intact glucocorticoid receptor in mammalian cells . Rare mutations in helical regions I and II gave rise to increased transcriptional activation activity . These mutations increase the hydrophobicity of hydrophobic patches on each of these helices, suggesting a relationship between the hydrophobicity of the patches and transactivation activity . However, certain nonhydrophobic residues are also important for activity . Interestingly, helical region I partially matches a consensus motif found in the retinoic acid receptor, VP16, and several other activator proteins. Mol Cell Biol, 1997 Feb, 17(2), 751 - 9 Role of alpha2 protein in donor locus selection during mating type interconversion; Szeto L et al.; The homeodomain protein alpha2p plays a role both in transcriptional repression in the process of cell type determination and in donor selection during mating interconversion . We have explored the mechanism of alpha2p-directed donor selection by examining the effects on donor preference of mutants deficient in alpha2p-mediated transcriptional repression . As a transcriptional regulator, alpha2p interacts with Mcm1p, Tup1p, and Ssn6p to repress a-specific genes and with a1p, Tup1p, and Ssn6p to repress haploid-specific genes . We have found that mutant alleles of MATalpha2 that specifically diminish the interaction of alpha2p with Mcm1p or Tup1p behave as null alleles with regard to donor preference, while mutations of MATalpha2 that specifically diminish interaction of alpha2p with a1p behave as wild-type MATalpha2 in this capacity . Tup1p plays an essential role in alpha2p-mediated transcriptional repression, while Ssn6p has only a modest effect in repression . In a similar vein, we find that TUP1, but not SSN6, is required for proper donor selection . These results suggest that, in addition to regulating a-specific gene expression to establish the mating type of the cell, alpha2p-Mcm1p-Tup1p complex may indirectly regulate donor preference through transcriptional control of an a-specific gene . Alternatively, this complex may play a direct role in establishing donor preference via its DNA binding and chromatin organization capacity. Mol Cell Biol, 1997 Feb, 17(2), 537 - 44 A novel, transformation-relevant activation domain in Fos proteins; Funk M et al.; We have previously demonstrated that transformation by Fos is critically dependent on an intact DNA-binding domain (bZip) and a functional N-terminal transactivation motif (N-TM) . We now show that a novel motif (C-terminal transactivation motif {C-TM}) near the C terminus also plays an important role in both transformation and the activation of AP1-dependent transcription and that the hydrophobic amino acids in the C-TM are functionally essential . The C-TM is the most crucial element in the C-terminal transactivation domain in Fos, as indicated by its relative strength and context-independent function . The C-TM is clearly different from the previously identified HOB2 domain, located N terminally to the C-TM, and the C-terminally positioned TATA-binding protein-binding domain . We also show that the C-terminal transactivation domain strongly synergizes with the HOB1-like N-TM, even when both domains are present on different proteins within a dimeric complex, and that the C-TM plays a crucial role in this cooperation . These observations can be corroborated in a model in which multiple contacts with the basal machinery are established either to stabilize the transcription complex or to facilitate its sequential assembly. J Virol, 1997 Feb, 71(2), 1019 - 24 Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery; Kawaguchi Y et al.; The herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a promiscuous transactivator, and by necessity, its functions must be mediated through cellular gene products . In an attempt to identify cellular factors interacting with ICP0, we used the carboxyl-terminal domain of ICP0 as "bait" in the yeast (Saccharomyces cerevisiae) two-hybrid system . Our results were as follows: (i) All 43 cDNAs in positive yeast colonies were found to encode the same translation factor, elongation factor delta-1 (EF-1delta) . (ii) Purified chimeric protein consisting of glutathione S-transferase (GST) fused to EF-1delta specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate . (iii) Fractionation of infected HEp-2 cells and immunofluorescence studies revealed that ICP0 was localized both in the nucleus and in the cytoplasm . In primary human foreskin fibroblasts, ICP0 was localized predominantly in the cytoplasm throughout HSV-1 infection even early in infection . (iv) Addition of the chimeric protein GST-carboxyl-terminal domain of ICP0 to the rabbit reticulocyte lysate in vitro translation system resulted in a dose-dependent decrease in protein synthesis . In contrast, GST alone or GST fused to the amino-terminal domain of ICP0 had no effect on the in vitro translation system . (v) The predominant forms of EF-1delta on electrophoresis in denaturing gels have apparent Mrs of 38,000 and 40,000 . The higher-Mr form is a minor species in mock-infected cells, whereas in human fibroblasts and Vero cells infected with HSV-1, this isoform becomes dominant . These results indicate that ICP0 is present and may have a significant role in the cytoplasm of infected cells, possibly by altering the efficiency of translation of viral mRNAs. J Biol Chem, 1997 Jan 31, 272(5), 2668 - 74 Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by an inducible mitogen-activated protein Kinase/ERK kinase kinase 3 (MEKK) derivative; Ellinger-Ziegelbauer H et al.; The extracellular signal-regulated kinase (ERK) pathway, the stress-activated protein kinase (SAPK) pathway, and the p38 pathway are three major mitogen-activated protein kinase (MAPK) cascades known to participate in the regulation of cellular responses to a variety of extracellular signals . Upstream regulatory components of these kinase cascades, the MAPK/ERK kinase kinases (MEKK), have been described in several systems . We have isolated a cDNA encoding human MEKK3 . Transfected MEKK3 has the ability to activate both SAPK and ERK pathways, but does not induce p38 activity, in agreement with a previous report on murine MEKK3 (Blank, J . L., Gerwins, P., Elliott, E . M., Sather, S., and Johnson, G . L . (1996) J . Biol . Chem . 271, 5361-5368) . We now demonstrate that MEKK3 activates SEK and MEK, the known kinases targeting SAPK and ERK, respectively . Utilizing an estrogen ligand-activated MEKK3 derivative, we furthermore demonstrate that MEKK3 regulates the SAPK and the ERK pathway directly . Consistent with the fact that several SAPK-inducing agents activate the transcription factor NFkappaB, we now show that MEKK3 also enhances transcription from an NFkappaB-dependent reporter gene in cotransfection assays . The ability of MEKK3 to simultaneously activate the SAPK and ERK pathways is remarkable, given that they have divergent roles in cellular homeostasis. Biochemistry, 1997 Jan 28, 36(4), 941 - 51 Site-directed mutagenesis of the sodium pump: analysis of mutations to amino acids in the proposed nucleotide binding site by stable oxygen isotope exchange; Farley RA et al.; A model for the active site of P type ATPases has been tested by site-directed mutagenesis of amino acids in two conserved sequences of Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase . The mutants K501R, K501E, D586E, D586N, P587A, and P588A were expressed in yeast cells and compared with wild type . In addition to previously published assays of adenosine 5'-triphosphate binding and hydrolysis, measurements of 18O exchange between Pi and water have been used to identify steps in the E2 half of the reaction cycle affected by the mutations . The study supports the prediction that K501 in the KGAP sequence interacts with adenosine 5'-triphosphate . However, quantitative comparisons of the effect of mutation K501E on the activity with the effects of mutations to an enzyme of known structure that also catalyzes phosphoryl group transfer make a direct role for the positive charge on the side chain of K501 in catalysis by stabilizing the transition state unlikely . No evidence for the predicted interaction between D586 and the hydroxyl groups of ribose was found . However, the data do indicate that the spatial organization of the loop containing the DPPR sequence is critical for phosphorylation of the enzyme . A role for D586 in coordinating the Mg2+ that is required for activity is proposed. Mol Gen Genet, 1997 Jan 27, 253(4), 507 - 11 Glutamic acid-371 of the barnase homology domain in RNA polymerase II is not required for SII-activated RNA cleavage; Powell W et al.; RNA polymerase II contains a ribonuclease activity which is stimulated by the transcription elongation factor SII . This nuclease shortens the nascent RNA and facilitates relief of transcriptional arrest by allowing the enzyme to make multiple attempts to read through an obstacle to transcription . The catalytic center of this ribonuclease is unknown, although a region of the enzyme's second largest subunit shares local sequence similarly with barnase and other bacterial ribonucleases . To test the role of the barnase homology region in SII-activated cleavage, we engineered a single amino acid change in the Saccharomyces cerevisiae enzyme at a position homologous to a catalytic residue of barnase (Glu-371) and has been suggested as a participant in active site chemistry of RNA polymerase II . We purified RNA polymerase II from mutant yeast and assayed its ability to cleave and re-extend the nascent RNA following SII treatment . We find no defects in this function of the mutant enzyme, suggesting that the barnase homology region does not represent the active site of the SII-activated nuclease . These mutant yeast cells were also resistant to mycophenolic acid, which slows the growth of some yeast mutants bearing elongation defective RNA polymerase II or mutant elongation factor SII. Biochem Pharmacol, 1997 Jan 24, 53(2), 179 - 87 Mechanistic studies on metabolic chiral inversion of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of the new anti-rheumatic agent 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), in rats; Yoshida H et al.; The chiral inversion properties of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), were compared with those of ibuprofen in rats . After administration of R(-)-{2 alpha-2H}KE-748, S(+)-KE-748 was present in the rat plasma, and the deuterium atoms of the S(+)-enantiomer were almost all replaced by hydrogen atoms . After administration of S(+)-{2 alpha-2H}KE-748, the deuterium content of S(+)-KE-748 in the plasma remained intact . In the in vitro study, using a cell-free system and rat liver homogenates, the chiral inversion of ibuprofen was apparent when both CoA and ATP were present; however, KE-748 was not inverted . In the study on isolated rat hepatocytes, the unidirectional chiral inversion from R(-)-to S(+)-enantiomer was observed for both ibuprofen and KE-748 . When R(-)-ibuprofen was incubated with medium and long chain fatty acids (carbon chain length C6 to C16), using isolated hepatocytes, the chiral inversion decreased significantly . On the other hand, when R(-)-KE-748 was incubated with short and medium chain fatty acids (carbon chain length C3 to C8), chiral inversion was inhibited markedly . To induce hepatic microsomal long chain fatty acid CoA ligase, rats were treated with clofibric acid (CF rats) . In both in vitro and in vivo experiments on CF rats, chiral inversion from R(-)-to S(+)-ibuprofen was enhanced significantly compared with that in controls, whereas the enhancement was not observed in the case of R(-)-KE-748 . There was no influence of benzoic acid, a typical substrate on medium chain fatty acid CoA ligase in the mitochondrial matrix, on chiral inversion of R(-)-ibuprofen, using, isolated hepatocytes . In contrast, the chiral inversion from R(-)-to S(+)-KE-748 was strongly inhibited in the presence of benzoic acid . These results indicate that chiral inversion of R(-)-KE-748 may proceed via formation of the CoA-thioester intermediate with loss of the 2 alpha-methine proton, in a manner similar to that seem with R(-)-ibuprofen . However, the enzymes needed to form CoA-thioester of R(-)-KE-748 differ from those for R(-)-ibuprofen. J Biol Chem, 1997 Jan 24, 272(4), 2477 - 85 Characterization of p150, an adaptor protein for the human phosphatidylinositol (PtdIns) 3-kinase . Substrate presentation by phosphatidylinositol transfer protein to the p150.Ptdins 3-kinase complex; Panaretou C et al.; Genetic and biochemical studies have shown that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is required for the efficient sorting and delivery of proteins to the vacuole . A human homologue of the yeast VPS34 gene product has recently been characterized as part of a complex with a cellular protein of 150 kDa (Volinia, S., Dhand, R., Vanhaesebroeck, B., MacDougall, L . K., Stein, R., Zvelebil, M . J., Domin, J., Panaretou, C., and Waterfield, M . D . (1995) EMBO J . 14, 3339-3348) . Here, cDNA cloning is used to show that the amino acid sequence of this protein, termed p150, is 29.6% identical and 53% similar to the yeast Vps15p protein, an established regulator of Vps34p . Northern blot analysis showed a ubiquitous tissue distribution for p150 similar to that previously observed with PtdIns 3-kinase . Recombinant p150 associated with PtdIns 3-kinase in vitro in a stable manner, resulting in a 2-fold increase in lipid kinase activity . Addition of phosphatidylinositol transfer protein (PI-TP) further stimulated the lipid kinase activity of the p150.PtdIns 3-kinase complex 3-fold . A PtdIns 3-kinase activity could also be co-immunoprecipitated from human cell lysates using anti-PI-TP antisera . This observation demonstrates that an interaction between a PtdIns 3-kinase and PI-TP occurs in vivo, which further implicates lipid transfer proteins in the regulation of PtdIns 3-kinase activity . These results suggest that the Vps15p.Vps34p complex has been conserved from yeast to man and in both species is involved in protein trafficking. J Biol Chem, 1997 Jan 24, 272(4), 2056 - 9 Structural determinants for interaction with three different effectors on the G protein beta subunit; Yan K et al.; In the yeast two-hybrid system, a 100-residue fragment (beta1A) from the N terminus of the beta1 subunit interacts with domains specific to adenylyl cyclase 2 (AC2), the muscarinic atrial potassium channel (GIRK1), and phospholipase C-beta2 (PLC-beta2) . Based on the crystal structure of the G protein, beta1A is composed of an N-terminal alpha helix, a loop, and five beta strands in which the C-terminal four beta strands form a beta sheet, the first of seven sheets that make up the propeller structure of the beta subunit . A mutant of beta1A (L4P, L7P, and L14P), in which the alpha helix was potentially destroyed, interacted poorly with the G protein gamma subunit but effectively with domains of AC2, GIRK1, and PLC-beta2 . In contrast, another mutant of beta1A (S72A, D76A, and W82A), in which a network of hydrogen bonds was disrupted, interacted poorly with GIRK1 and PLC-beta2 domains, but effectively with the gamma subunit and the AC2 domain . These results suggest that the proper folding of the first five beta strands in the G protein beta subunit is a requirement for appropriately positioning residues that interact with GIRK1 and PLC-beta2 . Furthermore, since mutations that potentially disrupted the folding of these beta strands did not affect interaction with AC2, the structural determinants on the G protein beta subunit for interaction with various effectors may be different. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 762 - 7 A homolog of the mammalian GTPase Rab2 is present in Arabidopsis and is expressed predominantly in pollen grains and seedlings; Moore I et al.; Vesicle traffic between the endoplasmic reticulum and the Golgi apparatus in mammals requires the small GTP-binding protein Rab2, but Saccharomyces cerevisiae appears not to have a Rab2 homolog . Here it is shown that the higher plant, Arabidopsis thaliana, contains a gene, At-RAB2, whose predicted product shares 79% identity with human Rab2 protein . Transgenic plants containing fusions between beta-glucuronidase and sequences upstream of At-RAB2 demonstrated histochemical staining predominantly in maturing pollen and rapidly growing organs of germinating seedlings . beta-glucuronidase activity in pollen is first detectable at microspore mitosis and increases thereafter . In this respect, the promoter of At-RAB2 behaves like those of class II pollen-specific genes, whose products are often required after germination for pollen tube growth . Seedling germination and pollen tube growth are notable for their unusually high rates of cell wall and membrane biosynthesis . These results are consistent with a role for At-RAB2 in secretory activity. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 448 - 52 Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway; Pumiglia KM et al.; The mitogen-activated protein kinase (MAPK) cascade plays a crucial role in the transduction of extracellular signals into responses governing growth and differentiation . The effects of a specific inhibitor of the MAPK kinase (MEK)/MAPK pathway (PD98059) on nerve growth factor (NGF)-induced growth arrest and inhibition of cell cycle-dependent kinases (CDKs) have been examined . Treatment of NIH 3T3 cells expressing TRKA with PD98059 dramatically reversed the complete inhibition of growth of these cells caused by NGF . PD98059 also blocked the ability of NGF to inhibit the activities of CDK4 and CDK2, while partially preventing NGF induction of p21Cip1/WAF1 . To independently evaluate the involvement of the MEK/MAPK pathway in growth arrest, an inducible activated form of the Raf-1 protooncogene (delta RAF-1:ER) was expressed in these cells . Activation of delta RAF-1:ER resulted in a prolonged increase in MAPK activity and growth arrest of these cells, with concomitant induction of p21Cip1/WAF1 and inhibition of CDK2 activity . These effects of delta RAF-1:ER activation were all reversed by treatment of cells with PD98059 . These data indicate that in addition to functioning as a positive effector of growth, stimulation of the MEK/MAPK pathway can result in an inhibition of CDK activity and cell cycle arrest. Biochemistry, 1997 Jan 21, 36(3), 469 - 80 Histones in transit: cytosolic histone complexes and diacetylation of H4 during nucleosome assembly in human cells; Chang L et al.; The organization and acetylation of nascent histones prior to their stable incorporation into chromatin were examined . Through sedimentation and immunoprecipitation analyses of HeLa cytosolic extracts, two somatic non-nucleosomal histone complexes were detected: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1 . The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins . H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin . The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly . As part of our studies of the nascent H3/H4 complex, the cytoplasmic histone acetyltransferase most likely responsible for acetylating newly synthesized H4 was also investigated . HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate . Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4 (Sobel et al., 1995), and has a native molecular weight of approximately 100 kDa . Based on our findings a model is presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo. Virology, 1997 Jan 20, 227(2), 431 - 8 Inhibition of HIV-1 transcription and virus replication using soluble Tat peptide analogs; Kashanchi F et al.; The human immunodeficiency virus type 1 (HIV-1) transactivator Tat protein is essential for efficient viral gene expression and virus replication . The Tat core domain, a stretch of 12 amino acids between the cysteine-rich and the basic domain, is conserved in all HIV isolates and required for interaction with a number of cellular transcriptional regulatory proteins . Here we demonstrate that soluble peptide analogs of the Tat core domain (amino acid 36-50) are able to effectively block LTR transactivation . In transfection experiments, Tat core peptide analogs containing amino acid substitutions at position 41 and 44 inhibited Tat transactivation of an HIV-1 LTR-CAT reporter construct up to 80-fold . In contrast, inhibition of other promoters such as HTLV-I and CMV was approximately 2-fold . Tat peptide analog 36-50 (41/44) inhibited HIV virus replication by 85% in latently infected U1 cells induced with Tat . Furthermore, U1 cells treated with the Tat peptide 36-50 (41/44) analog showed markedly delayed virus transmission when cocultivated with parental U937 cells . Interestingly, while both short and long peptide analogs (amino acids 36-50 vs 36-72) inhibited Tat transactivation in transient assays, the short peptides were more effective inhibitors of virus replication in U1 cells . The Tat peptide analog did not decrease expression of cellular genes including beta-actin, GAPDH, and histone H2B. J Biol Chem, 1997 Jan 17, 272(3), 1970 - 5 The transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum; Yang M et al.; UBC6 is a C-terminal membrane-anchored (type IV) protein, native to Saccharomyces cerevisiae, where it is found in the endoplasmic reticulum . When expressed in mammalian cells, this novel ubiquitin-conjugating enzyme also localizes to the endoplasmic reticulum . UBC6 lacks a lumenal domain and contains no known endoplasmic reticulum retention signals . Analysis of chimeric proteins in which the cytosolic domain of UBC is linked to a heterologous transmembrane domain, or in which the UBC6 transmembrane domain is appended to an unrelated soluble protein, led to the determination that the transmembrane domain of UBC6 plays a dominant role in its compartmental localization . The basis for the transmembrane domain-mediated subcellular targeting of UBC6 was evaluated by lengthening the wild type UBC6 hydrophobic segment from 17 to 21 amino acids, which resulted in re-targeting to the Golgi complex . A further increase in length to 26 amino acids allowed this modified protein to traverse the secretory pathway and gain expression at the plasma membrane . These findings are consistent with models in which, in the absence of dominant cytosolic or lumenal targeting determinants, proteins may be sorted within the secretory pathway based on interactions between their transmembrane domains and the surrounding lipid bilayer. J Biol Chem, 1997 Jan 17, 272(3), 1864 - 9 Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis; Nilsson U et al.; The three-dimensional structure of the quaternary complex of Saccharomyces cerevisiae transketolase, thiamin diphosphate, Ca2+, and the acceptor substrate erythrose-4-phosphate has been determined to 2.4 A resolution by protein crystallographic methods . Erythrose-4-phosphate was generated by enzymatic cleavage of fructose-6-phosphate . The overall structure of the enzyme in the quaternary complex is very similar to the structure of the holoenzyme; no large conformational changes upon substrate binding were found . The substrate binds in a deep cleft between the two subunits . The phosphate group of the substrate interacts with the side chains of the conserved residues Arg359, Arg528, His469, and Ser386 at the entrance of this cleft . The aldehyde moiety of the sugar phosphate is located in the vicinity of the C-2 carbon atom of the thiazolium ring of the cofactor . The aldehyde oxygen forms hydrogen bonds to the side chains of the residues His30 and His263 . One of the hydroxyl groups of the sugar phosphate forms a hydrogen bond to the side chain of Asp477 . The preference of the enzyme for donor substrates with D-threo configuration at the C-3 and C-4 positions and for alpha-hydroxylated acceptor substrates can be understood from the pattern of hydrogen bonds between enzyme and substrate . Amino acid replacements by site-directed mutagenesis of residues Arg359, Arg528, and His469 at the phosphate binding site yield mutant enzymes with considerable residual catalytic activity but increased Km values for the donor and in particular acceptor substrate, consistent with a role for these residues in phosphate binding . Replacement of Asp477 by alanine results in a mutant enzyme impaired in catalytic activity and with increased Km values for donor and acceptor substrates . These findings suggest a role for this amino acid in substrate binding and catalysis. J Biol Chem, 1997 Jan 17, 272(3), 1769 - 76 Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen; Mossi R et al.; Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair . It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon . To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus . Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side) . A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hubscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433) . Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA . The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution. Science, 1997 Jan 17, 275(5298), 400 - 2 Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases; Musti AM et al.; The proto-oncogene-encoded transcription factor c-Jun activates genes in response to a number of inducers that act through mitogen-activated protein kinase (MAPK) signal transduction pathways . The activation of c-Jun after phosphorylation by MAPK is accompanied by a reduction in c-Jun ubiquitination and consequent stabilization of the protein . These results illustrate the relevance of regulated protein degradation in the signal-dependent control of gene expression. Science, 1997 Jan 17, 275(5298), 387 - 9 Functional specificity among Hsp70 molecular chaperones; James P et al.; Molecular chaperones of the 70-kilodalton heat shock protein (Hsp70) class bind to partially unfolded polypeptide substrates and participate in a wide variety of cellular processes . Differences in peptide-binding specificity among Hsp70s have led to the hypothesis that peptide binding determines specific Hsp70 functions . Protein domains were identified that were required for two separate functions of a yeast Hsp70 family . The peptide-binding domain was not required for either of these specific Hsp70 functions, which suggests that peptide-binding specificity plays little or no role in determining Hsp70 functions in vivo. Biochim Biophys Acta, 1997 Jan 16, 1318(1-2), 71 - 8 What is the driving force for protein import into mitochondria? Horst M, Azem A, Schatz G, Glick BS. Nuclear-encoded mitochondrial proteins are synthesized in the cytosol as precursors and then imported into mitochondria . Protein import into the matrix space requires the function of the mitochondrial hsp70 (mhsp70) chaperone . mhsp70 is an ATPase that acts in conjunction with two partner proteins: the Tim44 subunit of the inner membrane import complex, and the nucleotide exchange factor mGrpE . A central question concerns how mhsp70 uses the energy of ATP hydrolysis to transport precursor proteins into the matrix . Recent evidence suggests that mhsp70 is a mechanochemical enzyme that actively pulls precursors across the inner membrane. Gene, 1997 Jan 15, 184(2), 229 - 35 Conservation of a putative inhibitory domain in the GAL4 family members; Poch O; The GAL4 family members are fungal transcriptional activators composed of several functional domains: a characteristic cysteine-rich DNA-binding domain common to all members, a dimerization domain, various transactivation domains generally exhibiting a high acidic content and a highly variable central region supposed to be involved in regulation and in effector recognition . We report here that the central region of the GAL4 family members share eight conserved motifs embedded in a large functional domain of 225 up to 405 residues . This domain may also be present in four proteins belonging to another family of transcriptional activators sharing a C2H2-type zinc finger . Analysis of the biochemical data available on the well-studied GAL4 protein suggests that this domain may be involved in the regulation of the activity of the protein, particularly in an inhibitory function . This hypothesis is further supported by deletion and site-directed mutagenesis experiments on other GAL4 family members . The mean secondary structure prediction performed on the eight motifs strongly suggests that the inhibitory activity may be mediated by hydrophobic interactions linked to the presence of amphipathic alpha-helices. Eur J Biochem, 1997 Jan 15, 243(1-2), 72 - 84 Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant; Bujons J et al.; A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant . Between pH 4 and 8, this variant forms three pH-linked spectroscopic species . The electronic absorption and 1H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system . Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand . At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation . The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers . Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme . The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation {Erman, J . E., Vitello, L . B., Miller, M . A . & Kraut, J . (1992) J . Am . Chem . Soc . 114, 6592-6593; Vitello, L . B., Erman, J . E., Miller, M . A., Wang, J . & Kraut, J . (1993) Biochemistry 32, 9807-9818} . Stopped-flow studies failed to detect even transient formation of a porphyrin-centered radical following addition of hydrogen peroxide to the Fe(III)-enzyme . The consequences of this drastic electrostatic modification of the active site on the steady-state kinetics of the variant are relatively minor. Nucleic Acids Res, 1997 Jan 15, 25(2), 379 - 87 HTLV-I Tax self-association in optimal trans-activation function; Jin DY et al.; HTLV-I Tax protein is a potent transcriptional activator of viral and cellular genes . Tax does not bind DNA directly but interacts through protein-protein contact with host cell factors that recognize the viral long terminal repeat (LTR) . Domains within Tax needed for protein-protein interaction have not been fully characterized . In studying transcriptional function in yeast cells, we unexpectedly found that Tax functions optimally not as a monomer, but as a homodimer . Here we have used the one hybrid and two hybrid genetic approaches in yeast to investigate the region(s) within Tax necessary for self-association . Dimer formation was also confirmed biochemically by using electrophoretic mobility shift (EMSA) and supershift assays . Twenty two Tax point mutants were utilized to map relevant residues . Genetic results from this series of mutants revealed that a necessary region for dimerization is contained within a previously characterized zinc finger domain . Two loss-of-function Tax mutants, each poorly active when assayed individually, were found to have complementing activity when co-expressed together . This genetic complementation suggests a mechanism fortrans-activation resulting from simultaneous but non-identical contact with a responsive target by each of two Tax monomers in a dimer. Biochem Biophys Res Commun, 1997 Jan 13, 230(2), 302 - 5 Phosphoinositide signalling in nuclei of Friend cells: DMSO-induced differentiation reduces the association of phosphatidylinositol-transfer protein with the nucleus; Rubbini S et al.; Friend erythroleukemia cells have a nuclear phosphoinositide cycle which is related to both mitogen-stimulated cell growth and erythorid differentiation . Because of the important role of the phosphatidylinositol-transfer protein (PI-TP) in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis, we have analysed nuclei isolated from Friend cells for the presence of PI-TP . By Western Blotting it was demonstrated that both intact nuclei and nuclei deprived of the outer membrane contained the PI-TP alpha isoform . Upon induction of erythroid differentiation by DMSO, the amount of nuclear PI-TP alpha was greatly diminished . As shown previously, under these same conditions, nuclear phospholipase C beta1 (PLC beta1) is down-regulated as well. Biochem Biophys Res Commun, 1997 Jan 13, 230(2), 297 - 301 DNA polymerase epsilon from Drosophila melanogaster; Aoyagi N et al.; We identified a DNA polymerase species in Drosophila melanogaster embryos, and purified it . This polymerase shared some common properties with DNA polymerase epsilon from mammals and yeast as follows; it has a preference for poly(dA)/oligo(dT) as a template/primer, it is highly processive in DNA synthesis, it co-fractionates with 3'-5' exonuclease activity, it is sensitive to aphidicolin and is resistance to ddTTP . The polymerase activity was inhibited in the immuno-precipitation assay with anti-pol-epsilon antibodies, which were produced against a polypeptide coded on the cDNA of a putative Drosophila pol-epsilon we isolated previously . Using these antibodies, Western blot analysis revealed that this polymerase is a 250kDa polypeptide, which is the same size as observed in mammals and yeast . These results indicate that Drosophila produces the epsilon-class of DNA polymerase, and like mammals or yeast, possesses the 5 typical classes of DNA polymerases (alpha to epsilon) in its embryos. Biochem Biophys Res Commun, 1997 Jan 13, 230(2), 262 - 5 14-3-3 zeta protein binds to the carboxyl half of mouse wee1 kinase; Honda R et al.; To identify proteins which bind to mouse wee1 kinase, the yeast "two-hybrid" system was used with a mouse cDNA library . Using the carboxyl half of weel kinase, the 14-3-3 zeta protein was isolated . Recombinant 14-3-3 zeta was demonstrated to bind to wee1 kinase in vitro . The wee1 kinase phosphorylated by cdc2 kinase also bound to 14-3-3 zeta protein . When both wee1 kinase and 14-3-3 zeta were transfected into COS-1 cells, they formed a complex in a cell . The sequence of wee1 kinase necessary for the binding was tested by a two hybrid system expressing different lengths of peptides derived from wee1 kinase . Both the entire kinase domain and a sequence in the carboxyl terminus was thought to be necessary for the binding . The function of 14-3-3 zeta protein remained to be elucidated in relation to the regulation of G2 to M phase transition through wee1 kinase. Cell, 1997 Jan 10, 88(1), 85 - 96 Binding of secretory precursor polypeptides to a translocon subcomplex is regulated by BiP; Lyman SK et al.; The translocation of a secretory precursor protein across the ER membrane comprises three phases: docking of the precursor at the membrane, insertion into the translocation pore, and exit from the pore into the ER lumen . We demonstrate that Sec62p, Sec71p and Sec72p form a translocon subcomplex that engages secretory precursors at the membrane site of the ER translocation machinery . Binding of a precursor to the subcomplex depends on the presence of an intact signal sequence and occurs only in the absence of ATP . In the presence of ATP, the precursor is released from the subcomplex in a reaction mediated by the lumenal hsp70, BiP . This release reaction, which is specific to BiP and requires interaction between BiP and the DnaJ homolog Sec63p, defines a role for BiP and Sec63p early in the ER translocation process. J Biol Chem, 1997 Jan 10, 272(2), 1101 - 9 Conservation and diversity of eukaryotic translation initiation factor eIF3; Asano K et al.; The largest of the mammalian translation initiation factors, eIF3, consists of at least eight subunits ranging in mass from 35 to 170 kDa . eIF3 binds to the 40 S ribosome in an early step of translation initiation and promotes the binding of methionyl-tRNAi and mRNA . We report the cloning and characterization of human cDNAs encoding two of its subunits, p110 and p36 . It was found that the second slowest band during polyacrylamide gel electrophresis of eIF3 subunits in sodium dodecyl sulfate contains two proteins: p110 and p116 . Analysis of the cloned cDNA encoding p110 indicates that its amino acid sequence is 31% identical to that of the yeast protein, Nip1 . The p116 cDNA was cloned and characterized as a human homolog of yeast Prt1, as described elsewhere (Methot, N., Rom, E., Olsen, H., and Sonenberg, N . (1997) J . Biol . Chem . 272, 1110-1116) . p36 is a WD40 repeat protein, which is 46% identical to the p39 subunit of yeast eIF3 and is identical to TRIP-1, a phosphorylation substrate of the TGF-beta type II receptor . The p116, p110, and p36 subunits localize on 40 S ribosomes in cells active in translation and co-immunoprecipitate with affinity-purified antibodies against the p170 subunit, showing that these proteins are integral components of eIF3 . Although p36 and p116 have homologous protein subunits in yeast eIF3, the p110 homolog, Nip1, is not detected in yeast eIF3 preparations . The results indicate both conservation and diversity in eIF3 between yeast and humans. J Biol Chem, 1997 Jan 10, 272(2), 1011 - 8 Determination of key structural requirements of a K+ channel pore; Nakamura RL et al.; Among the highly conserved sites in K+ channel pores, the tyrosine-glycine sequence is believed to play an important role in selectivity . Here we describe a novel approach in which comprehensive mutagenesis of the YG sites of the voltage-gated K+ channel, Kat1, is combined with phenotypic screening in Saccharomyces cerevisiae and electrophysiological analysis in Xenopus oocytes to determine the roles of these sites in K+ selectivity . We show that structural constraints necessitate a tyrosine or phenylalanine at the first position to confer full K+ selectivity . Substitution to arginine creates a channel titratable by external pH, suggesting that the side group at this position may line the channel pore . Permeation is abolished by any increase in bulk at the adjacent glycine position unless accompanied by a compensatory mutation at the tyrosine site . These results suggest a model in which the selectivity filter of the K+ channel requires an aromatic residue paired with glycine within the pore loop in order to maintain maximal K+ selectivity. J Biol Chem, 1997 Jan 3, 272(1), 680 - 7 Amino acid substitutions that convert the protein substrate specificity of farnesyltransferase to that of geranylgeranyltransferase type I; Del Villar K et al.; Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras . In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1 . All three Dpr1 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed . These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate . Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta to function as GGTase I beta . A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I beta . Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes . Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein . These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362. J Biol Chem, 1997 Jan 3, 272(1), 551 - 5 Ran-binding protein 1 (RanBP1) forms a ternary complex with Ran and karyopherin beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by karyopherin beta; Lounsbury KM et al.; The nuclear accumulation of proteins containing nuclear localization signals requires the Ran GTPase and a complex of proteins assembled at the nuclear pore . RanBP1 is a cytosolic Ran-binding protein that inhibits RCC1-stimulated release of GTP from Ran . RanBP1 also promotes the binding of Ran to karyopherin beta (also called importin beta and p97) and is a co-stimulator of RanGAP activity . Yeast karyopherin beta inhibits the GTP hydrolysis by Ran catalyzed by RanGAP . To further define the roles of RanBP1 and karyopherin beta in Ran function, we explored the effects of RanBP1 and karyopherin beta on mammalian proteins known to regulate Ran . Like RanBP1, karyopherin beta prevented the release of GTP from Ran stimulated by RCC1 or EDTA . As with the yeast protein, mammalian karyopherin beta completely blocked RanGAP activity . However, the addition of RanBP1 to this assay partially rescued the inhibited RanGAP activity . Kinetic analysis of the effects on RanGAP activity by karyopherin beta and RanBP1 revealed a combination of competitive and noncompetitive interactions . Solution binding assays confirmed the ability of RanBP1 to associate with Ran and karyopherin beta in a ternary complex, and RanBP1 binding was not competed out by the addition of karyopherin beta . These results demonstrate that RanBP1 and karyopherin beta interact with distinct sites of Ran and suggest that RanBP1 plays an essential role in nuclear transport by permitting RanGAP-mediated hydrolysis of GTP on Ran complexed to karyopherin beta. J Biol Chem, 1997 Jan 3, 272(1), 510 - 6 Folding and stability of the Z and S(iiyama) genetic variants of human alpha1-antitrypsin; Kang HA et al.; Z (Glu342 --> Lys) and S(iiyama) (Ser53 --> Phe) genetic variations of human alpha1-antitrypsin (alpha1-AT) cause a secretion blockage in the hepatocytes, leading to alpha1-AT deficiency in the plasma . Using in vitro folding analysis, we have shown previously that these mutations interfere with the proper folding of polypeptides . To understand the fundamental cause for the secretion defect of the Z and S(iiyama) variants of alpha1-AT, we investigated in vivo folding and stability of these variant alpha1-AT using the secretion system of yeast Saccharomyces cerevisiae . Various thermostable mutations suppressing the folding block of the Z variant in vitro corrected the secretion defect as well as the intracellular degradation in the yeast secretion system . Significantly, the extent of suppression in the secretion defect of Z protein was proportional to the extent of suppression in the folding defect, assuring that the in vivo defect associated with the Z variant is primarily derived from the folding block . In contrast, the folding and secretion efficiency of S(iiyama) was not much improved by the same mutations . In addition, none of the rarely secreted S(iiyama) alpha1-AT carrying the stabilizing mutations for the wild type and Z variant were active . It appears that the major defect in S(iiyama) variant is the loss of stability in contrast to the kinetic block of folding in the Z variant. J Biol Chem, 1997 Jan 3, 272(1), 48 - 50 Identification of Rox3 as a component of mediator and RNA polymerase II holoenzyme; Gustafsson CM et al.; Yeast Rox3 protein, implicated by genetic evidence in both negative and positive transcriptional regulation, is identified as a mediator subunit by peptide sequence determination and is shown to copurify and co-immunoprecipitate with RNA polymerase II holoenzyme. Science, 1997 Jan 3, 275(5296), 90 - 4 Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways; Ichijo H et al.; Mitogen-activated protein (MAP) kinase cascades are activated in response to various extracellular stimuli, including growth factors and environmental stresses . A MAP kinase kinase kinase (MAPKKK), termed ASK1, was identified that activated two different subgroups of MAP kinase kinases (MAPKK), SEK1 (or MKK4) and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and p38 subgroups of MAP kinases, respectively . Overexpression of ASK1 induced apoptotic cell death, and ASK1 was activated in cells treated with tumor necrosis factor-alpha (TNF-alpha) . Moreover, TNF-alpha-induced apoptosis was inhibited by a catalytically inactive form of ASK1 . ASK1 may be a key element in the mechanism of stress- and cytokine-induced apoptosis. Science, 1997 Jan 3, 275(5296), 86 - 8 Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins; Oliver JD et al.; Calnexin and calreticulin interact specifically with newly synthesized glycoproteins in the endoplasmic reticulum (ER) and function as molecular chaperones . The carbohydrate-specific interactions between ER components and glycoproteins synthesized in isolated canine pancreatic microsomes were analyzed using a cross-linking approach . A carbohydrate-dependent interaction between newly synthesized glycoproteins, the thiol-dependent reductase ERp57, and either calnexin or calreticulin was identified . The interaction between ERp57 and the newly synthesized glycoproteins required trimming of the N-linked oligosaccharide side chain . Thus, it is likely that ERp57 functions as part of the glycoprotein-specific quality control machinery operating in the lumen of the ER. EMBO J, 1997 Jan 2, 16(1), 163 - 72 Identification of a transcript release activity acting on ternary transcription complexes containing murine RNA polymerase I; Mason SW et al.; Termination of mammalian ribosomal gene transcription by RNA polymerase I (Pol I) requires binding of the nucleolar factor TTF-I (transcription termination factor for Pol I) to specific rDNA terminator elements . We have used recombinant murine TTF-I in an immobilized tailed template assay to analyze individual steps of the termination reaction . We demonstrate that, besides the TTF-I-DNA complex which stops elongating Pol I, an additional activity is required to release both the nascent transcript and Pol I from the template . Moreover, transcript release, but not TTF-I-directed pausing, depends on upstream sequences directly flanking the terminator element . Together, complete termination of Pol I transcription requires TTF-I bound to the terminator DNA, a stretch of thymidine residues upstream of the TTF-I-mediated pause site and an activity which releases the RNA transcript and Pol I from the DNA template. EMBO J, 1997 Jan 2, 16(1), 121 - 32 Mouse disabled (mDab1): a Src binding protein implicated in neuronal development; Howell BW et al.; Here, we identify a mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, and show it is an adaptor molecule functioning in neural development . We find that mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice . During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion . However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine . Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl . An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types . mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins . The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads . The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks. FEBS Lett, 1997 Jan 2, 400(1), 42 - 4 Substrate activation behaviour of pyruvate decarboxylase from Pisum sativum cv . Miko; Dietrich A et al.; The substrate activation behaviour of pyruvate decarboxylase from germinating seeds of Pisum sativum is characterised kinetically via stopped-flow measurements and discussed with respect to other species . The involvement of SH-groups in this process is demonstrated by reference experiments with chemically modified enzyme. FEBS Lett, 1997 Jan 2, 400(1), 37 - 41 In vitro low propensity to form nucleosomes of four telomeric sequences; Cacchione S et al.; The structural aspects of nucleosome assembly on telomeres are largely unknown . We analyzed by competitive reconstitution the affinities for the histone octamer of telomeric sequences from four different eukaryotic groups, Arabidopsis thaliana, mammals, Tetrahymena, and Saccharomyces cerevisiae . All telomeres reconstitute in nucleosomes with lower association constants than average nucleosomal DNA . DNase I digestion analysis suggests a multiple translational positioning and the lack of rotational positioning, probably due to telomeric repeats length (in most cases 6-8 bp), out of phase with the DNA helical repeat on the nucleosome (10.2 bp) . These results could partly explain the lack of nucleosomes on lower eukaryote telomeres, and suggest a high in vivo mobility of telomeric nucleosomes. Arch Insect Biochem Physiol, 1997, 36(1), 51 - 67 Light-induced vitamin deficiency in Drosophila melanogaster; Bruins BG et al.; Illumination by visible light (400 Ix) of cultures containing larvae of Drosophila melanogaster can reduce survival (Bruins et al., Insect Biochemistry 21:535-539, 1991) . Here we show that the effect of light depends on the presence of propionic or acetic acid in the food medium . We also show that survival is far more affected by illumination of the yeast food media than by direct illumination of the eggs and developing larvae . It is shown that addition of antioxidants to the food prevents light induced mortality . The action of antioxidants suggests that free radicals are important in light induced mortality . We also showed that both yeast and riboflavin (vitamin B2) solutions illuminated with visible light (400 Ix) generate hydrogen peroxide . Other vitamin and amino acid solutions do not produce peroxide in measurable amounts . However, the concentration of photogenerated hydrogen peroxide is far too low to explain the death of eggs and developing larvae upon exposure to light . A 400 Ix light treatment destroys the capability of yeast food media to support survival of larvae . Addition of vitamin C, carotene, tryptophan, nipagin, uric acid, or sucrose to the light treated medium does not restore viability . It is restored when riboflavin is added to the photo-inactivated yeast . A high concentration of pyridoxine also produced an improvement in survival . When riboflavin is treated with light, it cannot support survival on synthetic food media nor can it restore survival on light treated yeast food media . These results show that riboflavin (or a derivative) is a major light sensitive compound of yeast, which can be degraded by light . Light induced loss of riboflavin leads to mortality, because this is an essential dietary vitamin . The vitamin degradation can be prevented by dietary antioxidants . A chromatographic analysis confirms this conclusion. Fold Des, 1997, 2(3), S19 - 24 Protein structures sustain evolutionary drift; Rost B; A protein sequence folds into a unique three-dimensional protein structure . Different sequences, though, can fold into similar structures . How stable is a protein structure with respect to sequence changes? What percentage of the sequence is 'anchor' residues, that is, residues crucial for protein structure and function? Here, answers to these questions are pursued by analyzing large numbers of structurally homologous protein pairs . Most pairs of similar structures have sequence identity as low as expected from randomly related sequences (8-9%) . On average, only 3-4% of all residues are 'anchor' residues . The symmetric shape of the distribution at low sequence identity suggests that for most structures, four billion years of evolution was sufficient to reach an equilibrium . The mean identities for convergent (different ancestor) and divergent (same ancestor) evolution of proteins to similar structures are quite close and hence, in most cases, it is difficult to distinguish between the two effects . In particular, low levels of sequence identity appear not to be indicative of convergent evolution. Eur Biophys J, 1997, 25(5-6), 437 - 43 A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development; Varley PG et al.; Analytical ultracentrifugation (AUC) has reemerged as a powerful technique for protein characterisation . We report the pivotal role sedimentation equilibrium AUC has played in the development of macrophage inflammatory protein-1 alpha (MIP-1 alpha) as a protein therapeutic . MIP-1 alpha has potential clinical applications in cancer but its clinical use is limited, since it associates to form large insoluble aggregates in physiological buffers . Using AUC as a screening technique, we have produced a biologically active variant of MIP-1 alpha, BB-10010, which has a reduced tendency to aggregate in physiological buffers . The aggregation of protein based pharmaceuticals is routinely monitored by size exclusion chromatography (SEC) . Comparison of the data acquired by SEC and AUC, demonstrates that owing to the complexity of BB-10010, AUC analysis is required in addition to SEC to provide a rigorous characterisation of molecular association . This work has been extended to include the use of AUC as an analytical tool to monitor the quality of BB-10010 during formulation and stability studies. Eur Biophys J, 1997, 25(5-6), 399 - 403 An artificial HIV enhancer-binding peptide is dimerized by the addition of a leucine zipper; Liu N et al.; A 42 residue artificial peptide that binds to the HIV-1 enhancers has been described previously . The specificity of interaction of the peptide with its target DNA sequence has been demonstrated by a variety of techniques . Naturally occurring regulatory proteins frequently bind to DNA as dimers, thereby increasing the strength and specificity of the interaction, the dimer interface often being provided by a leucine zipper type coiled coil . As a suitable binding site for this kind of system is located to the 5' end of the HIV enhancer region, it was decided to design and synthesize a fusion peptide that not only contained the DNA binding sequence of the original 42 residue peptide but also incorporated a leucine zipper based on that of the GCN4 transcriptional activator, that should, therefore, be capable of dimerizing . The resultant peptide, LZ66, has now been shown to be fully active in band shift and in vitro transcription assays and to exhibit about double the inhibitory activity of the parent 42 residue peptide . Preliminary CD measurements revealed that the peptide has a high alpha-helical content and that it adopts a stable conformation down to the low micromolar peptide concentration range . Sedimentation equilibrium studies confirmed that the principles involved in the design of the peptide are valid and that the peptide is indeed dimeric in solution. Planta, 1997, 202(1), 18 - 27 Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.; Roelfsema MR et al.; Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research . Problems with the isolation of epidermal strips and the small size of A . thaliana guard cells were often prohibiting . In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes . Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted . The guard cells were found to exhibit two states . The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K+ concentrations . Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (Em) was not sensitive to the external K+ concentration . Two outward-rectifying conductances were identified in cells in the depolarized state . A slow outward-rectifying channel (s-ORC) had properties resembling the K(+)-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235-246) . The activation and inactivation times and the activation potential, all depended on the reversal potential (Erev) of the s-ORC conductance . The s-ORC was blocked by Ba2+ (K1/2 = 0.3-1.3 mM) and verapamil (K1/2 = 15-20 microM) . A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+ . Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state . The activation time and activation potential of this channel were not sensitive to the external K+ concentration . The slow activation of the IRC (t1/2 approximately 0.5 s) and its negative activation potential (Vthreshold = -155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701-2705) . The results indicate that A . thaliana guard cells provide an excellent system for the study of signal transduction processes. Clin Exp Allergy, 1997 Jan, 27(1), 87 - 95 IgE antibodies to protein and mannan antigens of pityrosporum ovale in atopic dermatitis patients; Lintu P et al.; BACKGROUND: Pityrosporum ovale is a common saprophyte on the skin capable of inducing IgE antibody production in atopic dermatitis (AD) patients . Allergens of P . ovale have been examined in several studies, but consensus on them is lacking . OBJECTIVE: This study was carried out to obtain more information about the IgE antibody response against P . ovale, including mannan . METHODS: Sera from 64 AD patients and 10 healthy controls were analysed with immunoblotting and the nitrocellulose radio allergosorbent test (RAST) method specifically developed to detect antimannan P . ovale IgE antibodies . RESULTS: In immunoblotting a total of 39 different IgE stained protein bands were seen . A high molecular weight staining was also seen especially in patients who displayed elevated mannan P . ovale RAST values . The most commonly stained protein bands in immunoblotting were 9 and 96 kD bands with antibodies in 73 and 65% of AD patients who had been positive in commercial P . orbiculare RAST with total serum IgE less than 4000 kU/l . Mannan RAST appeared positive in 77% of them . Positive immunoblotting to either of these bands was seen in 90% and, if added with staining with the 20 kD band, in 100% of these AD patients . A combination of 9 kD IgE staining and mannan P . ovale RAST was positive in 92% of the patients and 96 kD and mannan P . ovale RAST in 85% of the patients . CONCLUSION: It is evident that P . ovale has several allergens, the 9, 96 and 20 kD regions being the most important . According to our results mannan is also an important allegen of P . ovale. Exp Gerontol, 1997 Jan-Apr, 32(1-2), 11 - 22 Genetic influences on aging; Johnson TE; Genetics is an important tool for identifying key molecular events that are involved in specifying biological functions . Genetic approaches have been used repeatedly to understand diverse biological phenomena: oncogenesis, development, and the cell cycle, but have only recently been applied to the analysis of organismic aging and senescence . The power of the genetic approach stems from two facts . First, genetic analyses allow the integration of phenomena that are analyzed at many levels of observation from the molecule to the intact organism, and second, genetics has the real power to reveal causality by factors that are not dependent upon the prejudice of the investigator . I discuss several areas where genetics has been fruitfully applied to the study of the aging processes: human genes identified by "segmental progeroid" mutations, neurological diseases of the elderly, the limited proliferative life span of human somatic cells in tissue culture, studies on the life span of the mouse, and genetic analysis of life span in shorter lived metazoans (Drosophila melanogaster and Caenorhabditis elegans), and the yeast Saccharomyces cerevisiae. Lipids, 1997 Jan, 32(1), 31 - 7 Influence of diet on the kinetic behavior of hepatic carnitine palmitoyltransferase I toward different acyl CoA esters; Power GW et al.; The influence of diet on the kinetics of the overt form of rat liver mitochondrial carnitine palmitoyltransferase (CPT I; EC 2.3.1.21) was studied using rats fed either a low-fat diet (3% w/w fat), or diets which were supplemented with either olive oil (OO), safflower oil (SO) or menhaden (fish) oil (MO) to 20% w/w of fat (high fat diets) . When animals were fed each of these four diets for 10 days, the order of the apparent maximal activity (Vmax) of CPT I toward various individual fatty acyl CoA, when measured under a fixed molar ratio of acyl CoA/albumin, was 16:1 n-7 > 18:1 n-9 > 18:2 n-6 > 16:0 > 22:6 n-3, and was thus not affected by the fat composition of the diet . However, in all but one case, the SO and MO diets elicited a higher Vmax for each substrate than either the LF diet or the high fat OO diet . The apparent K0.5 for the different acyl CoA esters was generally lowest in LF-fed animals, and highest in those fed the high-fat SO diet . Moreover, when compared with the situation of animals fed high-fat diets, the K0.5 values of CPT I in LF-fed animals for palmitoyl CoA and oleoyl CoA were low . This possession by CPT I of a high "affinity" toward these nonessential fatty acyl CoAs, but a lower "affinity" toward linoleoyl CoA, the ester of an essential fatty acid, may enable this latter fatty acid to be spared from oxidation when its concentration in the diet is low . The data also emphasize that palmitoleoyl CoA, if available in the diet, is likely to be utilized by CPT I at a high rate. Chem Res Toxicol, 1997 Jan, 10(1), 85 - 90 Interaction of aflatoxin B1 with cytochrome P450 2A5 and its mutants: correlation with metabolic activation and toxicity; Pelkonen P et al.; Among members of the mouse cytochrome P450 2A family, P450 2A5 is the best catalyst of aflatoxin B1 (AFB1) oxidation to its 8,9-epoxide (Pelkonen, P., Lang, M., Wild, C . P., Negishi, M., and Juvonen, R . O . (1994) Eur . J . Pharmacol., Environ . Toxicol . Pharmacol . Sect . 292, 67-73) . Here we studied the role of amino acid residues 209 and 365 of the P450 2A5 in the metabolism and toxicity of AFB1 using recombinant yeasts . The two sites have previously been shown to be essential in the interaction of coumarin and steroids with the P450 2A5 . Reducing the size of the amino acid at position 209 or introducing a negatively charged residue at this site increased the 8,9-epoxidation of AFB1 compared to the wild type . In addition, replacing the hydrophobic amino acid at the 365 position with a positively charged lysine residue strongly decreased the metabolism of AFB1 . These mutations changed the KM values generally less than the Vmax values . The changes in AFB1 metabolism contrast with the changes in coumarin 7-hydroxylation caused by these amino acid substitutions, since reducing the size of the 209 residue strongly reduced coumarin metabolism and increased the K(M) values . On the other hand, the results with AFB1 are similar to those obtained with steroid hydroxylation . This suggests that the size of the substrate is important when interacting with the residue 209 of the protein . The catalytic parameters of AFB1 correlated generally with its toxicity to the recombinant yeasts expressing the activating enzyme and with the binding of AFB1 to yeast DNA . Furthermore high affinity substrates and inhibitors (e.g., methoxsalen, metyrapone, coumarin 311, 7-methylcoumarin, coumarin, and pilocarpine) of P450 2A5 could efficiently block the toxicity of AFB1 . It is suggested that the recombinant yeasts expressing engineered P450 enzymes are a useful model to understand the substrate protein interactions, to study the relationship of metabolic parameters to toxicity, and to test potential inhibitors of metabolism based toxicity. Curr Biol, 1997 Jan 1, 7(1), R50 - 2 Cell cycle: will the real Cdk-activating kinase please stand up; Draetta GF; Activation of a cyclin-dependent kinase (Cdk) requires its association with a cyclin subunit and phosphorylation on a conserved residue by the Cdk-activating kinase (CAK) . The discovery in budding yeast of a novel CAK that is structurally distinct from the CAKs of other eukaryotes raises questions about the 'true' CAK. Curr Biol, 1997 Jan 1, 7(1), R13 - 6 Nuclear transport: proliferating pathways; Goldfarb DS; A new pathway of nuclear import has been discovered with the identification of receptors that mediate the nuclear import of shuttling hnRNP proteins in yeast and human cells. Electrophoresis, 1997 Jan, 18(1), 150 - 5 Translationally controlled tumor protein: a protein identified in several nontumoral cells including erythrocytes; Sanchez JC et al.; The translationally controlled tumor protein (TCTP) is a growth-related protein which is regulated at the translational level . It is present in mammals, higher plants and Saccharomyces cerevisiae . This study was undertaken to localize and further characterize the TCTP in human cell lysates using two-dimensional gel electrophoresis, monoclonal antibodies, and 45Ca-gel overlay . TCTP was found in several healthy and tumoral cells including erythrocytes, hepatocytes, macrophages, platelets, keratinocytes, erythroleukemia cells, gliomas, melanomas, hepatoblastomas, and lymphomas . It could not be detected in kidney and renal cell carcinoma (RCC) . A monoclonal antibody raised against TCTP detected three isoforms likely due to post-translational modifications . A calcium binding property was found as well as heat stability and cytoplasmic localization . The high degree of homology from plants to man and its expression in many tissues suggests that TCTP most likely has a cell housekeeping function. Development, 1997 Jan, 124(2), 271 - 8 Induction of Drosophila eye development by decapentaplegic; Pignoni F et al.; The Drosophila decapentaplegic (dpp) gene, encoding a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily, controls proliferation and patterning in diverse tissues, including the eye imaginal disc . Pattern formation in this tissue is initiated at the posterior edge and moves anteriorly as a wave; the front of this wave is called the morphogenetic furrow (MF) . Dpp is required for proliferation and initiation of pattern formation at the posterior edge of the eye disc . It has also been suggested that Dpp is the principal mediator of Hedgehog function in driving progression of the MF across the disc . In this paper, ectopic Dpp expression is shown to be sufficient to induce a duplicated eye disc with normal shape, MF progression, neuronal cluster formation and direction of axon outgrowth . Induction of ectopic eye development occurs preferentially along the anterior margin of the eye disc . Ectopic Dpp clones situated away from the margins induce neither proliferation nor patterning . The Dpp signalling pathway is shown to be under tight transcriptional and post-transcriptional control within different spatial domains in the developing eye disc . In addition, Dpp positively controls its own expression and suppresses wingless transcription . In contrast to the wing disc, Dpp does not appear to be the principal mediator of Hedgehog function in the eye. Trends Biotechnol, 1997 Jan, 15(1), 15 - 9 Environmental stress response in plants: the role of mitogen-activated protein kinases; Mizoguchi T et al.; Mitogen-activated protein kinase (MAPK) cascades have essential roles in diverse intracellular signaling processes in plants, animals and yeasts . In plants, transcription of genes encoding protein kinases involved in MAPK cascades is upregulated by environmental stresses and plant hormones; in addition, MAPK-like kinase activities are transiently activated in response to environmental stresses . Consequently, MAPK cascades are now thought to have important roles in stress signal transduction pathways in higher plants. Plant J, 1997 Jan, 11(1), 83 - 92 The cloning of two Arabidopsis genes belonging to a phosphate transporter family; Smith FW et al.; Two genes, APT1 and APT2, with DNA sequences that exhibit significant sequence identity to yeast and fungal H+/orthophosphate co-transporters, have been isolated from Arabidopsis thaliana . These genes are genetically linked and map to chromosome 5 between markers g4028 and m435 . The genes encode almost identical 524 amino acid polypeptides and are predicted to contain 12 membrane-spanning domains . Both APT1 and APT2 are predominantly expressed in A . thaliana root tissues . The level of expression of both genes in roots is regulated by the phosphorus status of the plant, being considerably enhanced when the plants were deprived of an external phosphate supply . The APT1 and APT2 polypeptides are likely to be associated with the membrane transport of phosphate within A . thaliana roots. Plant Cell, 1997 Jan, 9(1), 5 - 19 Hexokinase as a sugar sensor in higher plants; Jang JC et al.; The mechanisms by which higher plants recognize and respond to sugars are largely unknown . Here, we present evidence that the first enzyme in the hexose assimilation pathway, hexokinase (HXK), acts as a sensor for plant sugar responses . Transgenic Arabidopsis plants expressing antisense hexokinase (AtHXK) genes are sugar hyposensitive, whereas plants overexpressing AtHXK are sugar hypersensitive . The transgenic plants exhibited a wide spectrum of altered sugar responses in seedling development and in gene activation and repression . Furthermore, overexpressing the yeast sugar sensor YHXK2 caused a dominant negative effect by elevating HXK catalytic activity but reducing sugar sensitivity in transgenic plants . The result suggests that HXK is a dual-function enzyme with a distinct regulatory function not interchangeable between plants and yeast. Plant Physiol, 1997 Jan, 113(1), 281 - 91 Plant 21D7 protein, a nuclear antigen associated with cell division, is a component of the 26S proteasome; Smith MW et al.; Previously, we cloned a carrot (Daucus carota L.) cDNA encoding a 45-kD protein, 21D7, located in the nuclei of proliferating cells . The 21D7 protein is similar to the partial sequence of a regulatory subunit of the bovine 26S proteasome, p58 (G . DeMartino, C.R . Moomaw, O.P . Zagnitko, R.J . Proske, M . Chu-Ping, S.J . Afendis, J.C . Swaffield, C.A . Slaughter {1994} J Biol Chem 269: 20878-20884) and to the deduced sequence encoded by the Saccharomyces cerevisiae gene SUN2 (M . Kawamura, K . Kominami, J . Takeuchi, A . Toh-e {1996} Mol Gen Genet 251: {146-152}) . In our work, the expression of plant 21D7 cDNA rescued the yeast sun2 mutant . Fractionation of carrot and spinach (Spinacia oleracea L.) crude extracts showed that the 21D7 protein sedimented with the active 26S proteasomes . The cessation of cell proliferation in carrot suspensions at the stationary phase caused 26S proteasome dissociation and, correspondingly, the 21D7 protein sedimented together with the free regulatory complexes of the 26s proteasomes . Large-scale purification of carrot 26s proteasomes resulted in co-isolation of the 21D7 protein . Polyacrylamide gel electrophoresis under nondenaturing conditions showed that the 21D7 protein had the same mobility as the 26S proteasome and that proteasome dissociation changed the mobility of the 21D7 protein accordingly . We conclude that the 21D7 protein is a subunit of the plant 26S proteasome and that it probably belongs to the proteasome regulatory complex. Mol Microbiol, 1997 Jan, 23(1), 23 - 33 The Aspergillus niger GCN4 homologue, cpcA, is transcriptionally regulated and encodes an unusual leucine zipper; Wanke C et al.; The general control transcriptional regulator gene cpcA of Aspergillus niger was cloned by complementation of a Saccharomyces cerevisiae delta gcn4 mutant strain . The encoded protein conferred resistance to amino acid analogues when expressed in yeast . Disruption of cpcA in A . niger resulted in a strain which is sensitive towards 3-aminotriazole and fails to respond to amino acid starvation, cpcA encodes a transcript of approximately 2400 nucleotides in length that includes a 5' leader region of 900 nucleotides . The 5' leader region contains two small open reading frames, suggesting translational control of gene expression . Steady-state mRNA levels of cpcA increase by a factor of three upon amino acid starvation . The coding region of cpcA is interrupted by a 57 bp intron and the deduced amino acid sequence displays an approximately 30% overall identity to yeast GCN4p and Neurospora crassa cpc1p . Critical amino acid residues of the transcriptional activation domains of GCN4p are conserved in cpcAp . The basic DNA-binding domain shows up to 70% amino acid sequence identity to other basic zipper (bZIP)-type transcriptional activators . cpcAp binds specifically to a GCN4p recognition element in gel retardation experiments . The C-terminal dimerization domain encodes a leucine zipper with only a single leucine residue. Curr Genet, 1997 Jan, 31(1), 80 - 8 Group-I introns in the cytochrome c oxidase genes of Dictyostelium discoideum: two related ORFs in one loop of a group-I intron, a cox1/2 hybrid gene and an unusually large cox3 gene; Ogawa S et al.; The DNA sequences of cytochrome oxidase (subunits 1, 2 and 3) genes of the cellular slime mold Dictyostelium discoideum mitochondria were determined . The genes for subunits 1 and 2 have a single continuous ORF (COX1/2) which contains four group-I introns . The insertion sites of the two group-I introns (DdOX1/2.2 and DdOX1/2.3) coincide with those of fungal and algal group-I introns, as well as a liverwort group-I intron, in the cytochrome oxidase subunit 1 . Interestingly, intron DdOX1/2.2 has two free-standing ORFs in a loop (L8) which have similar amino-acid sequences and are homologous to ai4 DNA endonuclease (I-Sce II) and bi4 RNA maturase found in group-I introns of Saccharomyces cerevisiae mitochondrial DNA . Two group-I introns (DdOX1/2.3 and DdOX1/2.4) also have a free-standing ORF in loop 1 and loop 2, respectively . These results show that these group-I introns and the intronic ORFs have evolved from the same ancestral origin, but that these ORFs have been propagated independently. Curr Genet, 1997 Jan, 31(1), 38 - 47 3-Hydroxy-3-methylglutaryl-CoA reductase gene of Gibberella fujikuroi: isolation and characterization; Woitek S et al.; 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the first specific enzyme of the isoprenoid pathway, which leads to several classes of primary and secondary metabolites such as sterols, quinones, carotenoids and gibberellins . The structural gene of HMG-CoA reductase was isolated from the ascomycetous fungus Gibberella fujikuroi . Additionally, the most conserved region of this gene was also isolated from another plant pathogenic fungus, Sphaceloma manihoticola . Both ascomycetous fungi use the plant hormone gibberellin to induce an elongation of infected host plants, and in the case of S . manihoticola of plant tumors . Sequence analysis revealed a high degree of similarity between the deduced amino-acid sequences in the C-terminal catalytic domains of all known HMG-CoA reductases, but the highest degree was found between the sequences of both analysed ascomycetes . In contrast to Saccharomyces cerevisiae, Ustilago maydis and plants, G . fujikuroi and S . manihoticola possess only a single copy of this gene, although the product of HMGR (mevalonate) is the precursor for essential sterol and quinone biosynthesis and secondary metabolites such as gibberellins . RNA-blot and hybridization experiments showed that gene expression is not influenced by either glucose or ammonium excess. Genes Dev, 1997 Jan 1, 11(1), 106 - 18 Meiotic cells monitor the status of the interhomolog recombination complex; Xu L et al.; During meiosis, mutations that cause defects at intermediate stages in the recombination process confer arrest at the end of prophase (e.g., pachytene) . In yeast, mutations of this type include rad50S, dmc1, rad51, and zip1 . Rad50 is likely part of a recombination initiation complex . DMC1, RAD51, and ZIP1 encode two RecA homologs and a synaptonemal complex protein, respectively . We report here the effects of mutations in two other (meiosis-specific) genes, RED1 and MEK1/MRE4, that encode a chromosome structure component and a protein kinase, respectively . A red1 or mek1/mre4 mutation alleviates completely rad50S, dmc1, rad51, and zip1 arrest . Furthermore, the red1 and mek1/mre4 mutations define a unique, previously unrecognized aspect of recombination imposed very early in the process, during DSB formation . Finally, the red1 and mek1/mre4 mutations appear to alleviate prophase arrest directly rather than by eliminating, or permitting bypass of, the rad50S, dmc1, rad51, or zip1 defects . These and other observations suggest that a meiosis-specific regulatory surveillance process monitors the status of the protein/DNA interhomolog recombination machinery as an integral entity, in its proper chromosomal context, and dependent upon its appropriate Red1 and Mek1/Mre4-promoted development . We speculate that a properly developed recombination complex emits an inhibitory signal to delay progression of meiotic cells out of prophase until or unless the recombination process has progressed, at least past certain critical steps, and perhaps to completion. Clin Immunol Immunopathol, 1997 Jan, 82(1), 43 - 8 Cell cycle checkpoints and DNA repair in Nijmegen breakage syndrome; Sullivan KE et al.; Nijmegen breakage syndrome is characterized by a variable T cell and B cell immunodeficiency, growth failure, and an increased risk of malignancy . It is inherited in an autosomal recessive manner and is biochemically related to ataxia-telangiectasia . Cells from a patient with Nijmegen breakage syndrome were unable to arrest cell cycle progression after exposure to ionizing radiation, and BrdU incorporation into newly synthesized DNA was uninhibited, demonstrating that these cells have an aberrant response to radiation exposure . Although gross chromosomal breakage was observed, dinucleotide repeat segments were stable over time, suggesting that other types of DNA stability were not affected . DNA-PK activity, which is mediated by a protein related to the ataxia-telangiectasia gene product and is intimately involved in DNA repair and VDJ recombination, was normal in cells from an NBS patient . Therefore, cells from patients with Nijmegen breakage syndrome have an abnormal response to radiation exposure similar to that seen in ataxia-telangiectasia. Biophys J, 1997 Jan, 72(1), 490 - 8 19F NMR measurements of the rotational mobility of proteins in vivo; Williams SP et al.; Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan . 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used