Biochem J, 1999 Oct 15, 343 Pt 2, 461 - 6 Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin; Mizukoshi E et al.; Fibroblast growth factor-1 (FGF-1), which lacks a signal peptide and is intracellularly localized as a result of endogenous expression or endocytosis, is thought to be involved in regulating cell growth and differentiation . In the study reported here, we purified proteins that bind intracellular FGF-1 . Affinity adsorption was used to purify FGF-1-binding proteins from rat L6 cells expressing FGF-1 . One of the isolated proteins was identified as the glucose-regulated protein GRP75/mortalin/PBP-74/mthsp70, a member of the hsp70 family of heat-shock proteins known to be involved in regulating glucose responses, antigen processing and cell mortality . The interaction of FGF-1 and GRP75/mortalin in vivo was confirmed by co-immunoprecipitation, immunohistochemical co-localization in Rat-1 fibroblasts and by using the yeast two-hybrid system . Moreover, a binding assay in vitro with the use of recombinant FGF-1 and mortalin demonstrated a direct physical interaction between the two proteins . These results reveal that GRP75/mortalin is an intracellular FGF-1-binding protein in cells and suggest that GRP75/mortalin is involved in the trafficking of and/or signalling by FGF-1. Curr Opin Neurobiol, 1999 Oct, 9(5), 571 - 7 Topics in prion cell biology; Brockes JP; Recent studies of a transmembrane form of the prion protein (PrP) have indicated its importance for neuropathogenesis in certain contexts, and have analysed the transacting factors at the endoplasmic reticulum and the mutations within PrP that regulate its appearance . A significant focus for our understanding of the normal role of PrP has emerged from its interaction with copper ions . Studies on two yeast prions have analysed the structure and phenotype of the aggregated conformers underlying the prion state, as well as the interactions regulating their formation and turnover within a dividing cell. Curr Biol, 1999 Sep 23, 9(18), 1009 - 18 NANOS-3 and FBF proteins physically interact to control the sperm-oocyte switch in Caenorhabditis elegans; Kraemer B et al.; BACKGROUND: The Caenorhabditis elegans FBF protein and its Drosophila relative, Pumilio, define a large family of eukaryotic RNA-binding proteins . By binding regulatory elements in the 3' untranslated regions (UTRs) of their cognate RNAs, FBF and Pumilio have key post-transcriptional roles in early developmental decisions . In C . elegans, FBF is required for repression of fem-3 mRNA to achieve the hermaphrodite switch from spermatogenesis to oogenesis . RESULTS: We report here that FBF and NANOS-3 (NOS-3), one of three C . elegans Nanos homologs, interact with each other in both yeast two-hybrid and in vitro assays . We have delineated the portions of each protein required for this interaction . Worms lacking nanos function were derived either by RNA-mediated interference (nos-1 and nos-2) or by use of a deletion mutant (nos-3) . The roles of the three nos genes overlap during germ-line development . In certain nos-deficient animals, the hermaphrodite sperm-oocyte switch was defective, leading to the production of excess sperm and no oocytes . In other nos-deficient animals, the entire germ line died during larval development . This germ-line death did not require CED-3, a protease required for apoptosis . CONCLUSIONS: The data suggest that NOS-3 participates in the sperm-oocyte switch through its physical interaction with FBF, forming a regulatory complex that controls fem-3 mRNA . NOS-1 and NOS-2 also function in the switch, but do not interact directly with FBF . The three C . elegans nanos genes, like Drosophila nanos, are also critical for germ-line survival . We propose that this may have been the primitive function of nanos genes. Mol Cell Endocrinol, 1999 Aug 20, 154(1-2), 151 - 9 Presence of activating transcription factor 4 (ATF4) in the porcine anterior pituitary; Kato Y et al.; The two-hybrid system that identifies protein-protein interactions in a yeast expression system was used to investigate porcine anterior pituitary transcription factors . Four cDNA clones of a protein interacting with the leucine zipper domain of porcine cJun were obtained . Their nucleotide sequences revealed that they encode activating transcription factor 4 (ATF4) . A full-length cDNA of porcine ATF4 was obtained by the polymerase chain reaction, and its deduced amino acid sequence showed 88 and 83% identity to human and mouse ATF4, respectively . Reverse transcription-polymerase chain reaction analysis of mRNAs prepared from 11 porcine tissues demonstrated that ATF4 is ubiquitous . Immunohistochemistry showed that ATF4 is present in the hormone producing cells of the anterior pituitary, but absent in some cells of the anterior pituitary . Further binding analysis revealed that ATF4 also interacts with itself and cFos . This evidence of ATF4 homodimerization, as well as heterodimerization with cJun and cFos in the anterior pituitary suggests a novel mechanism for the regulation of gene expression in this tissue. Am J Med Genet, 1999 Oct 29, 86(5), 416 - 9 Interstitial deletion of bands 11q21-->22.3 in a three-year-old girl defined using fluorescence in situ hybridization on metaphase chromosomes; Horelli-Kuitunen N et al.; A 3-year-old girl has a de novo deletion of 11q21-22.3 . The patient was studied because of minor anomalies, disproportionate short stature, and developmental delay . The deletion was first detected by conventional cytogenetic analysis and defined further by using chromosome 11-specific YAC clones by fluorescent in situ hybridization (FISH) on metaphase chromosomes . Three YAC clones, 11H7, 4A5, and IH4, were lacking from one of the patient's chromosome 11 . Trigonocepahly, hypertelorism, apparently low-set ears, mild renal abnormality, and delay in speech development found in our patient are similar findings in other published interstitial deletion cases . Our study shows that a molecular cytogenetic approach is useful in defining the specific location and the extent of an interstitial deletion in cytogenetically difficult areas such as 11q . Biopolymers, 1999 Nov, 50(6), 569 - 78 Microfibrillar structure of PGG-glucan in aqueous solution as triple-helix aggregates by small angle x-ray scattering; Gawronski M et al.; The conformation of polysaccharide PGG-Glucan, isolated from yeast cell walls, in aqueous solution was investigated by small angle x-ray scattering (SAXS) and multidetector gel permeation chromatography coupled with postcolumn delivery (GPC/PCD) techniques in comparison with scleroglucan . It was shown that both polysaccharides exhibit a rigid rod-like conformation in aqueous solution by SAXS experiments . The mass per unit length (M/L) and radius (R) of rod cross section of PGG-Glucan were measured to be 6300 daltons/nm and 1.89 nm, while those of scleroglucan are 2300 and 0.83, respectively . Utilizing a GPC/light scattering technique, the average aggregation number of PGG-Glucan is 9, while that of scleroglucan is around 3 . From the comparison of the M/L and R of the respective rod cross sections as well as their aggregation number data, it is concluded that PGG-Glucan is composed of triple helices, which tend to aggregate as triplets in solution, whereas scleroglucan is composed of a single triple helix . The aggregation number distribution of PGG-Glucan was found to range from 1 to about 25 determined by GPC/PCD . From the observation of a Debye-Scherrer ring type of peak in the macroscopic scattering cross section of PGG-Glucan by SAXS, the existence of a small amount of ordered clusters of PGG-Glucan can be deduced . The "lattice parameter" of these ordered fasces-like clusters is consistent with the radius of the individual triple-helical rods forming a microfibrillar superstructure . These results indicate that higher aggregated forms of PGG-Glucan containing up to 8 triple helices behave as ordered fasces-like clusters . We conclude that PGG-Glucan is triple-helix aggregates formed by rigid rods stacking together side by side . We propose a molecular structural model for PGG-Glucan conformations . FEBS Lett, 1999 Oct 1, 459(1), 69 - 74 A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit; Ingley E et al.; We report here the isolation of a new member of the ADP-ribosylation factor (ARF)-like family (ARL-6) present in the J2E erythroleukemic cell line, but not its myeloid variants . Consistent with this lineage-restricted expression, ARL-6 mRNA increased with erythropoietin-induced maturation of J2E cells, and decreased with interleukin 6-induced differentiation of M1 monoblastoid cells . In tissues, ARL-6 mRNA was most abundant in brain and kidney . While ARL-6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP-gammaS, like many members of the ARF/ARL family . Using the yeast two-hybrid system, six molecules which interact with ARL-6 were identified including SEC61beta, a subunit of the heterotrimeric protein conducting channel SEC61p . Co-immunoprecipitation of ARL-6 confirmed a stable association between ARL-6 and SEC61beta in COS cells . These results demonstrate that ARL-6, a novel member of the ADP-ribosylation factor-like family, interacts with the SEC61beta subunit. Chem Biol, 1999 Oct, 6(10), 679 - 87 Engineering temperature-sensitive SH3 domains; Parrini MC et al.; BACKGROUND: The ability to control specific protein-protein interactions conditionally in vivo would be extremely helpful for analyzing protein-protein interaction networks . SH3 (Src homology 3) modular protein binding domains are found in many signaling proteins and they play a crucial role in signal transduction by binding to proline-rich sequences . RESULTS: Random in vitro mutagenesis coupled with yeast two-hybrid screening was used to identify mutations in the second SH3 domain of Nck that render interaction with its ligand temperature sensitive . Four of the mutants were functionally temperature sensitive in mammalian cells, where temperature sensitivity was correlated with a pronounced instability of the mutant domains at the nonpermissive temperature . Two of the mutations affect conserved residues in the hydrophobic core (Val133 and Val160), suggesting a general strategy for engineering temperature-sensitive SH3-containing proteins . Indeed mutagenesis of the corresponding positions in another SH3 domain, that of Crk-1, rendered the full-length Crk-1 protein temperature sensitive for function and stability in mammalian cells . CONCLUSIONS: Construction of temperature-sensitive SH3 domains is a novel approach to regulating the function of SH3 domains in vivo . Such mutants will be valuable in dissecting SH3-mediated signaling pathways . Furthermore, the methodology described here to isolate temperature-sensitive domains should be widely applicable to any domain involved in protein-protein interactions. Nat Genet, 1999 Oct, 23(2), 241 - 4 Functional screening of an asthma QTL in YAC transgenic mice; Symula DJ et al.; Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified . This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions . Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits . Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs . To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene . This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes . Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells . Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo . This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo. Biochemistry, 1999 Sep 28, 38(39), 12943 - 9 Regulation of neurabin I interaction with protein phosphatase 1 by phosphorylation; McAvoy T et al.; Neurabin I is a brain-specific actin-binding protein . Here we show that neurabin I binds protein phosphatase 1 (PP1) and inhibits PP1 activity . Neurabin I interacted with PP1alpha in an overlay assay, in yeast two-hybrid interaction analysis, and in coprecipitation and co-immunoprecipitation experiments . Neurabin I also copurified with both the alpha and gamma isoforms of PP1 . A glutathione S-transferase (GST)-neurabin I fusion protein (residues 318-661) containing the putative PP1 binding domain (residues 456-460) inhibited PP1 activity (K(i) = 2.7 +/- 1.2 nM) . This fusion protein was also rapidly phosphorylated in vitro by PKA (K(m) = 6 microM) to a stoichiomtry of 1 mol/mol . The phosphorylated residue was identified as serine 461 by HPLC-MS analysis of a tryptic digest . Phosphorylation of GST-neurabin I (residues 318-661) by PKA significantly reduced its binding to PP1 by overlay and by glutathione-Sepharose coprecipitation assays . A 35-fold decrease in inhibitory potency was also observed using a S461E mutant, which mimics phosphorylation of S461 . These findings identify a signaling mechanism involving the regulation of PP1 activity and localization mediated by the cAMP pathway. EMBO J, 1999 Oct 1, 18(19), 5380 - 8 Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins; Hittelman AB et al.; The hormone-activated glucocorticoid receptor (GR), through its N- and C-terminal transcriptional activation functions AF-1 and AF-2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors . Utilizing a modified yeast two-hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor-interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF-1 surface . In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF-1-mediated transactivation . Thus, GR AF-1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement . In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone-dependent manner and facilitates GR transactivation in concert with DRIP150 . These results suggest that DRIP150 and DRIP205 functionally link GR AF-1 and AF-2, and represent important mediators of GR transcriptional enhancement. J Med Genet, 1999 Sep, 36(9), 708 - 10 An interstitial deletion of 6p24-p25 proximal to the FKHL7 locus and including AP-2alpha that affects anterior eye chamber development; Davies AF et al.; The FKHL7 gene has been implicated in the pathogenesis of glaucoma/autosomal dominant iridogoniodysgenesis (IGDA) (IRID1) . This has been supported by mutations in some glaucoma and IGDA patients and the development of anterior eye chamber anomalies in patients with 6p deletions affecting the 6p25 region . We report a case with anterior eye chamber anomalies and an interstitial deletion of 6p24-p25 that does not include the FKHL7 gene, suggesting the possible additional involvement of another locus, within 6p24-6p25, in anterior eye chamber development . A candidate gene is AP-2alpha, which is contained within the deleted segment and plays a role in anterior eye chamber development. J Med Genet, 1999 Sep, 36(9), 683 - 6 Genetic heterogeneity of gingival fibromatosis on chromosome 2p; Shashi V et al.; Gingival fibromatosis (GF) occurs in several genetic forms as a simple Mendelian trait, in malformation syndromes, and in some chromosomal disorders . Specific genes responsible for GF have not been identified . An autosomal dominant form of hereditary gingival fibromatosis (HGF, MIM 135300) was recently mapped to chromosome 2p21 in a large Brazilian family and there was an earlier report of GF in a boy with a cytogenetic duplication involving 2p13-->p21 . We thus hypothesised that a common gene locus may be responsible for GF in both the Brazilian family and the boy with the chromosome 2p duplication . We performed additional genetic linkage studies on the Brazilian family and molecular cytogenetic studies on the patient with the cytogenetic duplication to correlate more precisely the genetic interval of the HGF phenotype with the duplicated 2p interval . Additional linkage analysis of new family members resulted in refinement of the candidate region for HGF to an 8 Mb region . Molecular cytogenetic analysis of the 2p13-->p21 duplication associated with GF showed that the duplicated region was proximal to the candidate interval for HGF . Thus, our results support the presence of two different gene loci on chromosome 2p that are involved in GF. J Med Genet, 1999 Sep, 36(9), 678 - 82 Mononucleotide microsatellite instability and germline MSH6 mutation analysis in early onset colorectal cancer; Verma L et al.; Germline mutations in the MSH2 and MLH1 mismatch repair genes account for most cases of hereditary non-polyposis colon cancer syndrome (HNPCC) . In addition, germline MSH2 and MLH1 mutations have been detected in patients with non-HNPCC early onset colorectal cancer . Germline MSH6 mutations appear to be rare in classical HNPCC families, but their frequency in young colorectal cancer cases has not been studied previously . In a population based study of early onset colorectal cancer (<50 years) investigated for tumour microsatellite instability (MSI), we identified a subgroup of tumours with MSI for mono- but not dinucleotide repeat markers (m-MSI+ group) . In contrast to tumours with classical MSI for dinucleotide markers (d-MSI+), the m-MSI+ group cancers were mainly left sided (6/7) . As MSH6 mutations in yeast and human cell lines are associated with weak (and preferential mononucleotide) MSI, the complete MSH6 gene coding region was sequenced in blood DNA from the five m-MSI+ cases available for analysis . A germline nonsense mutation was identified in an isolated case of early onset colorectal cancer (age 43 years) . These results support previous findings that germline MSH6 mutations may not be associated with classical MSI and suggest a role for germline MSH6 mutations in isolated early onset colorectal cancer. J Biol Chem, 1999 Oct 8, 274(41), 29366 - 75 Identification of nuclear orphan receptors as regulators of expression of a neurotransmitter receptor gene; Chew LJ et al.; Nuclear orphan receptors are known to be important mediators of neurogenesis, but the target genes of these transcription factors in the vertebrate nervous system remain largely undefined . We have previously shown that a 500-base pair fragment in the first intron of the GRIK5 gene, which encodes the kainate-preferring glutamate receptor subunit KA2, down-regulates gene expression . In our present studies, mutation of an 11-base pair element within this fragment resulted in a loss of nuclear protein binding and reverses negative regulation by the intron . Using yeast one-hybrid screening, we have identified intron-binding proteins from rat brain as COUP-TFI, EAR2, and NURR1 . Gel shift studies with postnatal day 2 rat brain extract indicate the presence of COUP-TFs, EAR2, and NURR1 in the DNA-protein complex . Competition assays with GRIK5-binding site mutations show that the recombinant clones exhibit differential binding characteristics and suggest that the DNA-protein complex from postnatal day 2 rat brain may consist primarily of EAR2 . The DNA binding activity was also observed to be enriched in rat neural tissue and developmentally regulated . Co-transfection assays showed that recombinant nuclear orphan receptors function as transcriptional repressors in both CV1 cells and rat CG4 oligodendrocyte cells . Direct interaction of the orphan receptors with and relief of repression by TFIIB indicate likely role(s) in active and/or transrepression . Our findings are thus consistent with the notion that multiple nuclear orphan receptors can regulate the transcription of a widely expressed neurotransmitter receptor gene by binding a common element in an intron and directly modulating the activity of the transcription machinery. J Biol Chem, 1999 Oct 8, 274(41), 29260 - 5 Tyrosine versus serine/threonine phosphorylation by protein kinase casein kinase-2 . A study with peptide substrates derived from immunophilin Fpr3; Marin O et al.; Protein kinase casein kinase-2 (CK2) is a spontaneously active, ubiquitous, and pleiotropic enzyme that phosphorylates seryl/threonyl residues specified by multiple negatively charged side chains, the one at position n + 3 being of crucial importance (minimum consensus S/T-x-x-E/D/S(P)/T(P) . Recently CK2 has been reported to catalyze phosphorylation of the yeast nucleolar immunophilin Fpr3 at a tyrosyl residue (Tyr(184)) fulfilling the consensus sequence of Ser/Thr substrates (Wilson, L.K., Dhillon, N., Thorner, J., and Martin, G.S . (1997) J . Biol . Chem . 272, 12961-12967) . Here we show that, by contrast to other tyrosyl peptides fulfilling the consensus sequence for CK2, a peptide reproducing the sequence around Fpr3 Tyr(184) (DEDADIY(184)DEEDYDL) is phosphorylated by CK2, albeit with much higher K(m) (384 versus 4 . 3 microM) and lower V(max) (8.4 versus 1,132 nmol.min(-1).mg(-1)) than its derivative with Tyr(184) replaced by serine . The replacement of Asp at position n + 1 with alanine and, to a lesser extent, of Ile at n - 1 with Asp are especially detrimental to tyrosine phosphorylation as compared with serine phosphorylation, which is actually stimulated by the Ile to Asp modification . In contrast the replacement of Glu at n + 3 with alanine almost suppresses serine phosphorylation but not tyrosine phosphorylation . It can be concluded that CK2 is capable to phosphorylate, under special circumstances, tyrosyl residues, which are specified by structural features partially different from those that optimize Ser/Thr phosphorylation. J Biol Chem, 1999 Oct 8, 274(41), 28991 - 8 The multifunctional herpes simplex virus IE63 protein interacts with heterogeneous ribonucleoprotein K and with casein kinase 2; Wadd S et al.; Herpes simplex virus type 1 (HSV-1), the prototype alpha-herpesvirus, causes several prominent diseases . The HSV-1 immediate early (IE) protein IE63 (ICP27) is the only regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far . IE63 is a multifunctional protein affecting transcriptional and post-transcriptional processes, and it can shuttle from the nucleus to the cytoplasm . To identify interacting cellular proteins, a HeLa cDNA library was screened in the yeast two-hybrid system using IE63 as bait . Several interacting proteins were identified including heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional protein like IE63, and the beta subunit of casein kinase 2 (CK2), a protein kinase, and interacting regions were mapped . Confirmation of interactions was provided by fusion protein binding assays, co-immunoprecipitation from infected cells, and CK2 activity assays . hnRNP K co-immunoprecipitated from infected cells with anti-IE63 serum was a more rapidly migrating subfraction than hnRNP K immunoprecipitated by anti-hnRNP K serum . Using anti-IE63 serum, both IE63 and hnRNP K were phosphorylated in vitro by CK2, while in immunoprecipitates using anti-hnRNP K serum, IE63 but not hnRNP K was phosphorylated by CK2 . These data provide important new insights into how this key viral regulatory protein exerts its functions. Nat Biotechnol, 1999 Oct, 17(10), 1030 - 2 A generic protein purification method for protein complex characterization and proteome exploration; Rigaut G et al.; We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag . The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function . Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein . The TAP method has been tested in yeast but should be applicable to other cells or organisms. Plant J, 1999 Sep, 19(5), 533 - 41 Isolation of a novel SUMO protein from tomato that suppresses EIX-induced cell death; Hanania U et al.; Challenging tomato or tobacco varieties with ethylene-inducing xylanase (EIX) from the fungus Trichoderma viride causes rapid induction of plant defence responses leading to programmed cell death . Using the yeast two-hybrid system, we isolated a novel protein, tomato small ubiquitin-related modifier protein (T-SUMO), which specifically interacts with EIX . T-SUMO, a cytoplasmic protein, is a member of the ubiquitin-like protein family . It shows homology to human protein sentrin/SUMO1, which suppresses tumour necrosis factor-induced cell death . Transgenic plants that express T-SUMO in the sense orientation suppress EIX induction of ethylene biosynthesis and cell death, while in the antisense orientation they enhance EIX-induced ethylene biosynthesis . These results indicate that T-SUMO is involved in mediating the signal generated by EIX that leads to induction of plant defence responses. Plant J, 1999 Aug, 19(4), 433 - 9 Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5'-AMP; Sugden C et al.; Members of the SNF1-related protein kinase-1 (SnRK1) subfamily of protein kinases are higher plant homologues of mammalian AMP-activated and yeast SNF1 protein kinases . Based on analogies with the mammalian system, we surmised that the SnRK1 kinases would be regulated by phosphorylation on a threonine {equivalent to Thr175 in Arabidopsis thaliana SnRK1 (AKIN10)} within the 'T loop' between the conserved DFG and APE motifs . We have raised an antibody against a phosphopeptide based on this sequence, and used it to show that inactivation of two spinach SnRK1 kinases by protein phosphatases, and reactivation by a mammalian upstream protein kinase, is associated with changes in the phosphorylation state of this threonine . We also show that dephosphorylation of this threonine by protein phosphatases, and consequent inactivation, is inhibited by low concentrations of 5'-AMP, via binding to the substrate (i.e . the kinase) . This is the first report showing that the plant SnRK1 kinases are regulated by AMP in a manner similar to their mammalian counterparts . The possible physiological significance of these findings is discussed. Eur J Biochem, 1999 Oct, 265(2), 680 - 7 Structural versatility of bovine ribonuclease A . Distinct conformers of trimeric and tetrameric aggregates of the enzyme; Gotte G et al.; Lyophilization of bovine ribonuclease A (RNase A; Sigma, type XII-A) from 40% acetic acid solutions leads to the formation of approximately 14 aggregated species that can be separated by ion-exchange chromatography . Several aggregates were identified, including two variously deamidated dimeric subspecies, two distinct trimeric and two distinct tetrameric RNase A conformers, besides the two forms of dimer characterized previously {Gotte, G . & Libonati, M . (1998) Two different forms of aggregated dimers of ribonuclease A . Biochim . Biophys . Acta 1386, 106-112} . We also have possible evidence for the existence of two forms of pentameric RNase A . The two forms of trimers and tetramers are characterized by: (a) slightly different gel filtration patterns; (b) different retention times in ion-exchange chromatography; and (c) different mobilities in cathodic gel electrophoresis under nondenaturing conditions . Therefore, they appear to have distinct structural organizations responsible for a different availability of their positively charged amino acid residues . All RNase A oligomers, in particular the two distinct trimeric and tetrameric conformers, degrade poly(A).poly(U), viral double-stranded RNA and polyadenylate with a catalytic efficiency that is in general higher for the more basic species . On the contrary, the activity of the RNase A oligomers, from dimer to pentamer, on yeast RNA and poly(C) (Kunitz assay) is lower than that of monomeric RNase A. J Cell Sci, 1999 Oct, 112 ( Pt 19), 3299 - 308 A FERM domain governs apical confinement of PTP-BL in epithelial cells; Cuppen E et al.; PTP-BL is a cytosolic multidomain protein tyrosine phosphatase that shares homologies with several submembranous and tumor suppressor proteins . Here we show, by transient expression of modular protein domains of PTP-BL in epithelial MDCK cells, that the presence of a FERM domain in the protein is both necessary and sufficient for its targeting to the apical side of epithelial cells . Furthermore, immuno-electron microscopy on stable expressing MDCK pools, that were obtained using an EGFP-based cell sorting protocol, revealed that FERM domain containing fusion proteins are enriched in microvilli and have a typical submembranous location at about 10-15 nm from the plasma membrane . Immunofluorescence microscopy suggested colocalization of the FERM domain moiety with the membrane-cytoskeleton linker ezrin . However, at the electron microscopy level this colocalization cannot be confirmed nor can we detect a direct interaction by immunoprecipitation assays . Fluorescence recovery after photobleaching (FRAP) experiments show that PTP-BL confinement is based on a dynamic steady state and that complete redistribution of the protein may occur within 20 minutes . Our observations suggest that relocation is mediated via a cytosolic pool, rather than by lateral movement . Finally, we show that PTP-BL phosphatase domains are involved in homotypic interactions, as demonstrated by yeast two-hybrid assays . Both the highly restricted subcellular compartmentalization and its specific associative properties may provide the appropriate conditions for regulating substrate specificity and catalytic activity of this member of the PTP family. J Cell Sci, 1999 Oct, 112 ( Pt 20), 3487 - 96 Interaction of the universal mRNA-binding protein, p50, with actin: a possible link between mRNA and microfilaments; Ruzanov PV et al.; We have shown previously that p50 is the most abundant protein associated with a variety of eukaryotic mRNAs and exhibits about 98% amino acid sequence identity to mammalian Y-box binding transcription factors . The dual function of p50 in the cell as a regulator of both transcription and translation has been suggested . To gain insight into the role of p50 in these processes, we performed the yeast two-hybrid screen to identify p50 molecular partners . Here we report the identification of actin as a p50-interacting protein . Coimmunoprecipitation of p50 and actin from HeLa extracts as well as in vitro binding studies indicate specificity and a high affinity for the interaction between p50 and actin . Interestingly, p50 binding to actin is affected by mRNA; binding was observed at a low p50/mRNA ratio and was greatly reduced at higher ratios . Since the p50/mRNA ratio appears to be important for mRNA translatability, we speculate that p50 can regulate the attachment of mRNA to the actin network depending on its translational activity . Using immunofluorescence, we show that p50 binds to actin filaments in permeabilized cells and causes actin fibers to bundle in vitro . Together, these findings support the view that p50 may play an important role in mRNA transport, anchoring, and localization on actin filaments in the cell. Biochemistry, 1999 Sep 28, 38(39), 12735 - 46 Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/Ferredoxin/Ferredoxin:NADP(+) reductase in a cyanobacterium; van Thor JJ et al.; A mutant of Synechocystis PCC 6803, deficient in psaE, assembles photosystem I reaction centers without the PsaE subunit . Under conditions of acceptor-side rate-limited photoreduction assays in vitro (with 15 microM plastocyanin included), using 100 nM ferredoxin:NADP(+) reductase (FNR) and either Synechocystis flavodoxin or spinach ferredoxin, lower rates of NADP(+) photoreduction were measured when PsaE-deficient membranes were used, as compared to the wild type . This effect of the psaE mutation proved to be due to a decrease of the apparent affinity of the photoreduction assay system for the reductase . In the psaE mutant, the relative petH (encoding FNR) expression level was found to be significantly increased, providing a possible explanation for the lack of a phenotype (i.e., a decrease in growth rate) that was expected from the lower rate of linear electron transport in the mutant . A kinetic model was constructed in order to simulate the electron transfer from reduced plastocyanin to NADP(+), and test for possible causes for the observed change in affinity for FNR . The numerical simulations predict that the altered reduction kinetics of ferredoxin, determined for the psaE mutant {Barth, P., et al., (1998) Biochemistry 37, 16233-16241}, do not significantly influence the rate of linear electron transport to NADP(+) . Rather, a change in the dissociation constant of ferredoxin for FNR does affect the saturation profile for FNR . We therefore propose that the PsaE-dependent transient ternary complex PSI/ferredoxin/FNR is formed during linear electron transport . Using the yeast two-hybrid system, however, no direct interaction could be demonstrated in vivo between FNR and PsaE fusion proteins. Chin J Biotechnol, 1998, 14(4), 213 - 9 Construction of a YAC contig encompassing G200 locus of rice via chromosome walking; Huang W et al.; Map-based gene cloning is now widely used to isolate genes of unknown products . After fine mapping of a target gene is completed, the key step to head forward is to construct series of contigs covering the target gene through chromosome walking . To approach the final goal of cloning a wide compatible gene of rice (S5 locus), the flanking G200 marker was used as a starting point to walk . Four positive clones ranging from 240 approximately 650 kb were obtained from the rice genomic YAC library (RGP, Japan) . With the ends of YAC inserts isolated by inverse-PCR, a YAC contig of 3 cM was initially built . Next, chromosome walking was performed with the two distal ends of the contig and another seven positive YAC clones were screened out, leading to the contig extending to about 8 cM. Mol Gen Genet, 1999 Aug, 262(1), 65 - 72 A light-inducible Myb-like gene that is specifically expressed in red Perilla frutescens and presumably acts as a determining factor of the anthocyanin forma; Gong ZZ et al.; The Myb-p1 gene was isolated by screening for differentially expressed Myb-related genes in red (anthocyanin-producing) and green (anthocyanin nonproducing) forms of Perilla frutescens . Expression of Myb-p1 is increased 10-fold in the red relative to the green form of P . futescens, and the gene is induced by light . MYB-P1 has only one DNA-binding region, which corresponds to repeat III in the general structure of MYB proteins . In the yeast two-hybrid system, it was shown that MYB-P1 interacted with MYC-RP, a MYC-related transcriptional regulatory protein involved in the control of anthocyanin biosynthesis in P . frutescens . In yeast, MYB-P1 was able to bind to a dihydroflavonol reductase (DFR) gene promoter isolated from red P . frutescens . These data suggest that Myb-p1 may be involved in the regulation of anthocyanin biosynthesis and could therefore be responsible for determining anthocyanin formation in red P . frutescens. Mol Gen Genet, 1999 Aug, 262(1), 35 - 45 The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source; Saloheimo M et al.; The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence . It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene . The putative T . reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL . The mature protein shows strong conservation relative to other fungal protein disulphide isomerases . The T . reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway . Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR . The level of T . reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes . The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1 . The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly. Virology, 1999 Sep 30, 262(2), 277 - 97 Sequence analysis of the Xestia c-nigrum granulovirus genome; Hayakawa T et al.; The nucleotide sequence of the Xestia c-nigrum granulovirus (XcGV) genome was determined and found to comprise 178,733 bases with a G+C content of 40.7% . It contained 181 putative genes of 150 nucleotides or greater that showed minimal overlap . Eighty-four of these putative genes, which collectively accounted for 43% of the genome, are homologs of genes previously identified in the Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) genome . These homologs showed on average 33% amino acid sequence identity to those from AcMNPV . Several genes reported to have major roles in AcMNPV biology including ie-2, gp64, and egt were not found in the XcGV genome . However, open reading frames with homology to DNA ligase, two DNA helicases (one similar to a yeast mitochondrial helicase and the other to a putative AcMNPV helicase), and four enhancins (virus enhancing factors) were found . In addition, several ORFs are repeated; there are 7 genes related to AcMNPV orf2, 4 genes related to AcMNPV orf145/150, and a number of repeated genes unique to XcGV . Eight major repeated sequences (XcGV hrs) that are similar to sequences found in the Trichoplusia ni GV genome (TnGV) were found . Exp Cell Res, 1999 Oct 10, 252(1), 224 - 35 Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain; Law SF et al.; HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion . HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells . While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition . In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif . This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47 . We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth . Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins . These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation . Cell Tissue Res, 1999 Oct, 298(1), 1 - 9 Signal transfer by Eph receptors; Kalo MS et al.; The Eph receptors are a unique family of receptor tyrosine kinases that enforce cellular position in tissues through mainly repulsive signals generated upon cell-cell contact . Together, Eph receptors and their membrane-anchored ligands . the ephrins, are key molecules for establishing tissue organization through signaling pathways that control axonal projection, cell migration, and the maintenance of cellular boundaries . Through their SH2 (Src Homology 2) and PDZ (postsynaptic density protein, disks large, zona occludens) domains, several signaling molecules have been demonstrated to interact with the activated cytoplasmic domain of Eph receptors by using the yeast two-hybrid system and in vitro biochemical assays . Most proteins found to interact with Eph receptors are well-known regulators of cytoskeletal organization and cell adhesion, and also cell proliferation . Promoting growth, however, does not appear to be a primary role of Eph receptors . Explaining which signaling interactions identified for the Eph receptors have physiological significance, how Eph receptor signaling cascades are propagated, and characterizing the intrinsic signaling properties of the ephrins are all exciting questions currently being investigated. Cell Tissue Res, 1999 Oct, 298(1), 1 - 9 Signal transfer by eph receptors Kalo MS, Pasquale EB. The Eph receptors are a unique family of receptor tyrosine kinases that enforce cellular position in tissues through mainly repulsive signals generated upon cell-cell contact . Together, Eph receptors and their membrane-anchored ligands, the ephrins, are key molecules for establishing tissue organization through signaling pathways that control axonal projection, cell migration, and the maintenance of cellular boundaries . Through their SH2 (Src Homology 2) and PDZ (postsynaptic density protein, disks large, zona occludens) domains, several signaling molecules have been demonstrated to interact with the activated cytoplasmic domain of Eph receptors by using the yeast two-hybrid system and in vitro biochemical assays . Most proteins found to interact with Eph receptors are well-known regulators of cytoskeletal organization and cell adhesion, and also cell proliferation . Promoting growth, however, does not appear to be a primary role of Eph receptors . Explaining which signaling interactions identified for the Eph receptors have physiological significance, how Eph receptor signaling cascades are propagated, and characterizing the intrinsic signaling properties of the ephrins are all exciting questions currently being investigated. Curr Genet, 1999 Sep, 36(3), 137 - 46 Inactivation of the Neurospora crassa mitochondrial outer membrane protein TOM70 by repeat-induced point mutation (RIP) causes defects in mitochondrial protein import and morphology; Grad LI et al.; Mitochondrial biogenesis requires the efficient import of hundreds of different cytosolically translated preproteins into existing organelles . Recognition and translocation of preproteins at the mitochondrial outer membrane is achieved by the TOM complex (translocase of the outer mitochondrial membrane) . The largest component of this complex is TOM70, an integral outer membrane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins . To investigate the functional role of TOM70 in Neurospora crassa the tom70 gene was inactivated using the natural phenomenon of repeat-induced point mutation (RIP) . Mutant strains were identified that harbored RIPed tom70 alleles and contained no immunologically detectable TOM70 . Strains that lack TOM70 grow more slowly than wild-type strains, conidiate poorly, and contain enlarged mitochondria . In vitro preprotein import studies using TOM70-deficient mitochondria revealed a defect in the uptake of the ADP/ATP carrier . Other preproteins tested were imported at wild-type rates with the exception of the precursor of the mitochondrial-processing peptidase (MPP) which was imported more efficiently by TOM70-deficient mitochondria . These data support the view that TOM70 plays a role as a specific receptor for carrier proteins in mitochondrial-preprotein import . The presence of tetratricopeptide repeats (TPRs) in the TOM70 sequence and the enlarged shape of mitochondria lacking TOM70 raise the possibility that the protein also plays a role in the maintenance of mitochondrial morphology. J Gen Virol, 1999 Sep, 80 ( Pt 9), 2411 - 5 Two-hybrid analysis of the interaction between the UL52 and UL8 subunits of the herpes simplex virus type 1 helicase-primase; Constantin N et al.; Herpes simplex virus type 1 expresses a heterotrimeric helicase-primase, the subunits of which are encoded by the viral UL5, UL8 and UL52 genes . The interactions of the UL52 protein with the UL8 and UL5 proteins were analysed by using the yeast two-hybrid system . The UL52-UL5 interaction gave a specific but weak signal in the two-hybrid system . In contrast, the UL52-UL8 interaction gave a strong signal in the two-hybrid system . Deletion analysis showed that a 548 amino acid fragment of UL52 (amino acids 366-914) retains the ability to interact with UL8 and that the N-terminal 349 amino acids are dispensable for the interaction . A fragment library screen and co-immunoprecipitation experiments confirmed the deletion analysis results. Plant Cell Physiol, 1999 Jul, 40(7), 733 - 42 Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction; Imamura A et al.; His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction . Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters" . In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin) . In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them . The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles . Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B) . It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not . Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs) . These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana. Biochim Biophys Acta, 1999 Oct 6, 1447(1), 51 - 6 Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism; Gobl AE et al.; Recently the multiple endocrine neoplasia type 1 (MEN-1) tumor suppressor gene was cloned . MEN-1 encodes a nuclear protein, called menin, of hitherto unknown function . In order to investigate the biological function of menin we employed the yeast two-hybrid system to identify menin-interacting proteins . Here we report that menin functions as a transcriptional repressor through interaction with the transcription factor JunD . The interaction is mediated via the N-terminal transcription activation domain of JunD, and the C-terminal part of menin . In transient co-transfection experiments, expression of menin leads to specific repression of JunD transcriptional activity, which is dependent on the integrity of the menin C-terminal region . C-Terminal truncations of the protein not only abolish repression, but increase JunD transcriptional activity, implying the existence of a functional domain separate from the JunD-binding region . Menin-mediated repression is relieved by the histone deacetylase inhibitor trichostatin A, indicating that deacetylation of histones is an essential component of this repression mechanism, as has recently been demonstrated for the retinoblastoma protein . Missense, in-frame deletions, frameshift and nonsense mutations lead to inactivation of menin or possibly to truncated proteins . This would result in loss of repression of menin/JunD target genes, as well as non-target genes through indirect mechanisms, deregulation of cellular growth control and endocrine tumorigenesis. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11492 - 5 Deficit of in vivo mitochondrial ATP production in patients with Friedreich ataxia; Lodi R et al.; Friedreich ataxia (FRDA), the most common of the inherited ataxias, is an autosomal recessive degenerative disorder, characterized clinically by onset before the age of 25 of progressive gait and limb ataxia, absence of deep tendon reflexes, extensor plantar responses, and loss of position and vibration sense in the lower limbs . FRDA is caused by a GAA triplet expansion in the first intron of the FRDA gene on chromosome 9q13 in 97% of patients . The FRDA gene encodes a widely expressed 210-aa protein, frataxin, which is located in mitochondria and is severely reduced in FRDA patients . Frataxin function is still unknown but the knockout of the yeast frataxin homologue gene (YFH1) showed a severe defect of mitochondrial respiration and loss of mtDNA associated with elevated intramitochondrial iron . Here we report in vivo evidence of impaired mitochondrial respiration in skeletal muscle of FRDA patients . Using phosphorus magnetic resonance spectroscopy we demonstrated a maximum rate of muscle mitochondrial ATP production (V(max)) below the normal range in all 12 FRDA patients and a strong negative correlation between mitochondrial V(max) and the number of GAA repeats in the smaller allele . Our results show that FRDA is a nuclear-encoded mitochondrial disorder affecting oxidative phosphorylation and give a rationale for treatments aimed to improve mitochondrial function in this condition. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11434 - 9 Position effect of human telomeric repeats on replication timing; Ofir R et al.; Telomeres are distinct structures, composed of short, repeated sequences, at the ends of all eukaryotic chromosomes . Telomeres have been shown in yeast to induce late replication in S phase and to silence transcription of neighboring genes . To examine the possibility of similar effects in human chromosomes, we studied cells from a subject with a microdeletion of 130 kb at the end of one copy of chromosome arm 22q, repaired by the addition of telomere repeats . Using fluorescence in situ hybridization of S phase nuclei, a distinct difference was found in the replication timing of the breakpoint region between the intact and truncated copies of chromosome 22 . This difference was evident as a shift from middle to late replication time of the breakpoint region adjacent to the repaired telomere . This finding suggests that the human telomere sequence influences activation of adjacent replication origin(s) . The difference in replication timing between the two chromosomes was not associated with differences in sensitivity to digestion by DNase I or with methylation of regions immediately adjacent to the breakpoint . Furthermore, both alleles of arylsulfatase A, a gene located at a distance of approximately 54 kb from the breakpoint, were expressed . We conclude that as in yeast, the proximity of telomeric DNA may induce a positional effect that delays the replication of adjacent chromosomal regions in humans. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11416 - 21 The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation; Yang M et al.; The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis . Nine Arabidopsis SKP1 homologues have been uncovered and are named ASK1 through ASK9 . Here, we report the isolation and characterization of a male sterile Arabidopsis mutant and show that the mutant defect was caused by a Ds transposon insertion into the ASK1 gene . In the ask1-1 mutant, abnormal microspores exhibit a range of sizes . Furthermore, during mutant male meiosis, although homologous chromosome pairing appeared normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended . Therefore, in ask1-1, at least some homologues remain associated after metaphase I . In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect . Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, we propose that ASK1 controls homologue separation by degrading or otherwise removing a protein that is required directly or indirectly for homologue association before anaphase I. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11370 - 5 Autoproteolysis in nucleoporin biogenesis; Rosenblum JS et al.; We have molecularly characterized a proteolytic cleavage in conserved nuclear pore complex proteins . This cleavage, previously demonstrated to be essential for the biogenesis of two nuclear pore complex proteins in mammals (Nup98 and Nup96) and yeast (Nup145-N and Nup145-C), occurs between Phe and Ser residues within a highly conserved domain in a polyprotein precursor . Here, we show that a protease is not involved in the cleavage event . By using a combination of domain mapping and site-directed mutagenesis, we demonstrate that the human nuclear pore complex protein Nup98 specifically cleaves itself between F863 and S864 . A region of Nup98, amino acids 715-920, is able to cleave, whereas a smaller region, amino acids 772-920, does not cleave . In addition, we have generated a Nup98 mutant that cleaves under defined conditions in vitro . Further, the two cleaved fragments of Nup98 form a complex, providing a possible mechanism whereby specific, yet low-affinity, binding between Nup98 and Nup96 is responsible for the nuclear targeting of Nup96 . Although apparently unrelated evolutionarily, Nup98 has converged on an autoproteolytic biogenesis mechanism similar to that of hedgehog proteins, the inteins, and the N-terminal nucleophile proteins. Genes Dev, 1999 Sep 15, 13(18), 2365 - 8 Ubiquitous expression and embryonic requirement for RNA polymerase II coactivator subunit Srb7 in mice; Tudor M et al.; Mammalian RNA polymerase II complexes and coactivators containing homologs of yeast Srb/Med proteins have been isolated recently from tissue culture cells . The yeast Srb/Med complex is involved in global gene expression and is essential, but it is not yet known if its mammalian counterparts are broadly expressed in tissues or if they are essential . We have isolated the murine gene encoding Srb7, an Srb/Med complex protein whose sequence and function is highly conserved between yeast and humans . The mouse Srb7 gene is single copy, and Northern analysis showed that it is expressed in all tissues examined . Disruption of the gene in embryonic stem cells revealed that it is essential for cell viability and murine embryonic development . These results, together with evidence that murine Srb7 is associated exclusively with high molecular weight forms of RNA polymerase II in extracts, suggest that Srb7-containing polymerase complexes occur in most tissues and have essential roles in expression of protein coding genes. Endocrinology, 1999 Oct, 140(10), 4713 - 24 Regulation of insulin-like growth factor I receptor promoter activity by wild-type and mutant versions of the WT1 tumor suppressor; Tajinda K et al.; The insulin-like growth factor I (IGF-I) receptor is a transmembrane tyrosine kinase that mediates the growth-promoting effects of IGF-I and IGF-II . Changes in IGF-I receptor messenger RNA levels are reflected in cell surface receptor number, and modulation of IGF-I receptor levels affects tumorigenicity in numerous cellular models; thus, control of IGF-I receptor gene expression appears to be an important level at which cellular proliferation and tumorigenic potential may be regulated . We have previously shown that the product of the WT1 Wilms' tumor suppressor gene represses IGF-I receptor gene expression both in vitro and in vivo, and that decreased WT1 levels are correlated with up-regulation of IGF-I receptor gene expression in Wilms' tumor, benign prostatic hyperplasia, and breast cancer . Gene regulation by WT1 is complex, in that the WT1 gene encodes a variety of products as a result of alternative splicing and RNA editing, and a number of missense point mutations have been characterized in Wilms' tumor-associated syndromes . Additionally, the WT1 protein has been demonstrated to self-associate through its N-terminal domain, although the role of this intermolecular interaction in transcriptional regulation by WT1 is unclear . In this report, we analyze the relative activity of wild-type and mutant versions of the WT1 protein with respect to IGF-I receptor promoter activity in transient transfection assays and assess the potential contribution of WT1 self-association to IGF-I receptor regulation using the yeast two-hybrid system . Of the naturally occurring variations in WT1 structure, only the presence of a three-amino acid KTS insert in the zinc finger domain introduced by alternative splicing of exon 9 had a significant effect on WT1 repression of IGF-I receptor promoter activity . The N- and C-terminal domains of WT1 also exhibited partial repression, as did the most common mutant version of the WT1 protein associated with Denys-Drash syndrome . Mutations in the WT1 N-terminus attenuated WT1 self-association in the yeast two-hybrid system, but did not impair transcriptional repression . Our results suggest that 1) the DNA-binding capacity of WT1 is critical for maximal repression of the IGF-I receptor promoter, but some effects may be mediated through protein-protein interactions involving the N-terminal domain; 2) WT1 self-association may not be required for repression of the IGF-I receptor promoter; and 3) the Denys-Drash syndrome version of the WT1 protein may exhibit residual or possible gain of function activity in some contexts rather than exerting dominant negative effects, as has been proposed previously. Comb Chem High Throughput Screen, 1998 Dec, 1(4), 171 - 83 Homogeneous pharmacologic and cell-based screens provide diverse strategies in drug discovery: somatostatin antagonists as a case study; Zysk JR et al.; The development of high throughput, homogeneous pharmacologic and functional assays and their implementation in screening combinatorial libraries has increased the pace of stochastic drug discovery in recent years . New, noninvasive approaches involving radiometric proximity assays, an array of fluorescence-based technologies, and reporter gene constructs in mammalian and nonmammalian systems are providing more options for the selection of specific therapeutic targets . The increasing sophistication of homogeneous assay designs has also served as a springboard to better lead validation in drug discovery initiatives . This review examines these approaches in the context of new drug discovery strategies which combine a growing repertoire of high throughput screening techniques . The utility and importance of cell-based assays vis-a-vis pharmacologic (cell-free) assays is considered with specific reference given to yeast-based functional screens and homogeneous binding methodologies used to search for somatostatin antagonists and other potential growth hormone secretagogues . Also considered is the custom tailoring of specific chemical libraries which provide yet another level of target selectivity . The advantages and shortcomings of these various technologies are discussed in light of emerging trends toward higher throughput and nanoscale formats which are pushing the limits of measurable response at the cellular and molecular level. Oncogene, 1999 Sep 23, 18(39), 5464 - 72 Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic effect of this cytokine: relationship with c-myc and Rb; Ameyar M et al.; Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis . We have previously shown that the MCF7 resistance to the cytotoxic action of TNF correlates with p53 mutations . In the present study, we used a recombinant adenovirus carrying a wild-type p53 gene (Adwtp53) in order to investigate the effect of wt p53 transfer on modulation of cell resistance to the cytotoxic action of TNF . Our data indicate that infection of TNF resistant MCF7 cells (1001 and MCF7/Adr) with Adwtp53 resulted in the restoration of wt p53 expression and function as respectively revealed by the yeast assay and the induction of p53 inducible genes MDM2 and p21 . Furthermore, the restoration of p53 function significantly sensitized TNF resistant cells to TNF cytotoxic action . This correlated with a significant down-regulation of c-myc in both TNF-resistant cell lines and a decrease of Retinoblastoma protein (Rb) in 1001 clone . In contrast, the effect of p53 seems to be independent from Bcl-2 and Bax protein level regulation . The present study suggests that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine. Nucleic Acids Res, 1999 Oct 15, 27(20), 4114 - 20 Expression specificity of the mouse exonuclease 1 (mExo1) gene; Lee BI et al.; Genetic recombination involves either the homo-logous exchange of nearly identical chromosome regions or the direct alignment, annealing and ligation of processed DNA ends . These mechanisms are involved in repairing potentially lethal or mutagenic DNA damage and generating genetic diversity within the meiotic cell population and antibody repertoire . We report here the identification of a mouse gene, termed mExo1 for mouse exonuclease 1, which encodes a approximately 92 kDa protein that shares homology to proteins of the RAD2 nuclease family, most notably human 5' to 3' exonuclease Hex1/hExo1, yeast exonuclease 1 (Exo1) proteins and Drosophila melanogaster Tosca . The mExo1 gene maps to distal chromosome 1, consistent with the recent mapping of the orthologous HEX1 / hEXO1 gene to chromosome 1q42-q43 . mExo1 is expressed prominently in testis, an area of active homologous recombination, and spleen, a prominent lymphoid tissue . An increased level of mExo1 mRNA was observed during a stage of testis development where cells that are actively involved in meiotic recombination arise first and represent a significant proportion of the germ cell population . Comparative evaluation of the expression patterns of the human and mouse genes, combined with previous biochemical and yeast genetic studies, indicate that the Exo1-like proteins are important contributors to chromosome processing during mammalian DNA repair and recombination. Hepatology, 1999 Oct, 30(4), 1064 - 76 Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates; Sabile A et al.; Several lines of evidence suggest that hepatitis C virus (HCV) core protein may modulate cellular transduction signals and alter lipid metabolism . We have investigated the binding of HCV core protein to cellular proteins by combining 2 yeast hybrid, confocal, and surface plasmon resonance assays . Our results show the direct binding of the viral protein to apolipoprotein AII (apoAII) and map the interaction domain to the C-terminal of HCV core protein . To investigate the biological relevance of the interaction between HCV core and lipid metabolism, we took advantage of the well-established increase in apoAII expression caused by fibrates in HepG2 cells . After fenofibric acid treatment, we show a parallel increase in apoAII and core protein secretion, this effect being abolished by brefeldin A . Our study identifies apoAII as one of the cellular targets for HCV core protein . We also show that the intervention of fenofibric acid in cellular lipid metabolism directly affects the expression pattern of HCV core protein. J Biol Chem, 1999 Oct 1, 274(40), 28497 - 504 Characterization of the interaction between the Wilson and Menkes disease proteins and the cytoplasmic copper chaperone, HAH1p; Larin D et al.; Wilson disease (WD) and Menkes disease (MNK) are inherited disorders of copper metabolism . The genes that mutate to give rise to these disorders encode highly homologous copper transporting ATPases . We use yeast and mammalian two-hybrid systems, along with an in vitro assay to demonstrate a specific, copper-dependent interaction between the six metal-binding domains of the WD and MNK ATPases and the cytoplasmic copper chaperone HAH1 . We demonstrate that several metal-binding domains interact independently or in combination with HAH1p, although notably domains five and six of WDp do not . Alteration of either the Met or Thr residue of the HAH1p MTCXXC motif has no observable effect on the copper-dependent interaction, whereas alteration of either of the two Cys residues abolishes the interaction . Mutation of any one of the HAH1p C-terminal Lys residues (Lys(56), Lys(57), or Lys(60)) to Gly does not affect the interaction, although deletion of the 15 C-terminal residues abolishes the interaction . We show that apo-HAH1p can bind in vitro to copper-loaded WDp, suggesting reversibility of copper transfer from HAH1p to WD/MNKp . The in vitro HAH1/WDp interaction is metalospecific; HAH1 preincubated with Cu(2+) or Hg(+) but not with Zn(2+), Cd(2+), Co(2+), Ni(3+), Fe(3+), or Cr(3+) interacted with WDp . Finally, we model the protein-protein interaction and present a theoretical representation of the HAH1p.Cu.WD/MNKp complex. J Biol Chem, 1999 Oct 1, 274(40), 28491 - 6 Bcl3, an IkappaB protein, stimulates activating protein-1 transactivation and cellular proliferation; Na SY et al.; Bcl3, an IkappaB protein, was originally isolated as a putative proto-oncogene in a subset of B cell chronic lymphocytic leukemias . Bcl3 was subsequently shown to associate tightly with and transactivate the NFkappaB p50 or p52 homodimer . Herein, we show that Bcl3 stimulates the activating protein-1 (AP-1) transactivation, either alone or in conjunction with transcription integrators steroid receptor coactivator-1 and CREB-binding protein/p300 . The C-terminal 158 residues of Bcl3 exhibited an autonomous transactivation function and interacted with specific subregions of the AP-1 components c-Jun and c-Fos, CREB-binding protein/p300, and steroid receptor coactivator-1, as demonstrated by the yeast and mammalian two-hybrid tests as well as glutathione S-transferase pull-down assays . In addition, anti-HA antibody co-precipitated c-Jun from HeLa cells co-expressing c-Jun and HA-tagged Bcl3, consistent with the idea that Bcl3 directly associates with AP-1 in vivo . Furthermore, microinjection of Bcl3 expression vector into Rat-1 fibroblast cells significantly enhanced DNA synthesis and expression of c-jun, one of the cellular target genes of AP-1 . These results suggest that Bcl3 may directly participate in the tumorigenesis processes as a novel transcription coactivator of the mitogenic transcription factor AP-1 in vivo. J Biol Chem, 1999 Oct 1, 274(40), 28256 - 63 Identification of regulatory sequences and binding proteins in the type II sodium/phosphate cotransporter NPT2 gene responsive to dietary phosphate; Kido S et al.; Dietary phosphate (P(i)) is a most important regulator for renal P(i) reabsorption . The type II sodium-dependent phosphate (Na/P(i)) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal P(i) reabsorption . The consumption of a low-P(i) diet induces the synthesis of NPT2, whereas a high P(i) diet decreases it . The molecular mechanisms of regulation by dietary P(i) are not yet known . In this report, in weaning mice fed a low-P(i) diet for 4 days, the NPT2 mRNA level was increased 1.8-fold compared with mice fed a normal P(i) diet . This increase was due to an elevation of the transcriptional activity . In the NPT2 gene promoter, the DNA footprint analysis showed that six regions were masked by the binding protein, but at the position -1010 to -985 upstream of the transcription start site, the binding clearly responded to the levels of dietary P(i) . The phosphate response element (PRE) of the NPT2 gene was found to consist of the motif related to the E box, 5'-CACGTG-3' . A yeast one-hybrid system was used to clone a transcription factor that binds to the PRE sequences in the proximal promoter of the NPT2 gene . Two cDNA clones that encoded protein of the mouse transcription factor muE3 (TFE3) were isolated . This is a DNA-binding protein that activates transcription through the muE3 site of the immunoglobulin heavy chain enhancer . TFE3 antibody completely inhibited the binding to the PRE . The coexpression of TFE3 in COS-7 cells transfected with the NPT2 gene promoter markedly stimulated the transcriptional activity . The feeding of a low P(i) diet significantly increased the amount of TFE3 mRNA in the kidney . These findings suggest that TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary P(i). Virology, 1999 Sep 1, 261(2), 357 - 66 Second site mutations in the N-terminus of the major capsid protein (VP5) overcome a block at the maturation cleavage site of the capsid scaffold proteins of herpes simplex virus type 1; Desai P et al.; VP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames . Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process . In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins . A previously isolated virus (KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth . The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site . Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800 . Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site . The second site mutations were extragenic . Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5' region of the gene encoding VP5 . DNA sequence analysis was performed for the N-terminal 571 codons encoding VP5 for all of the revertant viruses . Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 78 of VP5 . Three of these were identical and changed Ala to Val at residue 78 . The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site . The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5 . One revertant gave no detectable interaction by this assay . The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus . The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated . Mol Pharmacol, 1999 Oct, 56(4), 797 - 806 Dissociated glucocorticoids with anti-inflammatory potential repress interleukin-6 gene expression by a nuclear factor-kappaB-dependent mechanism; Vanden Berghe W et al.; Synthetic glucocorticoids (GCs) remain among the most effective agents for the management of chronic inflammatory diseases . However, major side effects severely limit their therapeutic use . Physiologic and therapeutic activities of GCs are mediated by a nuclear receptor belonging to a superfamily of ligand-inducible transcription factors that, in addition to directly regulating their cognate gene programs, can also mutually interfere with other signaling pathways . We recently identified selective ligands of the glucocorticoid receptor that dissociate transactivation from activator protein 1 transrepression, and most importantly retain in vivo anti-inflammatory activity . To further document the mechanisms of action sustaining the observed in vivo activity, we report here on the interference of dissociated GCs with nuclear factor kappaB (NF-kappaB)-driven gene activation . We show that dissociated GCs repress tumor necrosis factor-induced interleukin-6 gene expression by an NF-kappaB-dependent mechanism, without changing the expression level of inhibitor kappaB . The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with the various compounds . Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines constitutively expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB, which was able to efficiently repress a Gal4-dependent luciferase reporter gene upon addition of the dissociated compounds . We therefore conclude that, in addition to dissociating transactivation from activator protein 1 transrepression, dissociated GCs mediate inhibition of NF-kappaB signaling by a mechanism that is independent of inhibitor kappaB induction. J Biomol Struct Dyn, 1999 Aug, 17(1), 41 - 50 Monoclonal antibody against DNA adducts with osmium structural probes; Buzek J et al.; Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure . A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice . OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins . The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid) . The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy . The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen) . A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane. RNA, 1999 Sep, 5(9), 1191 - 9 Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains; Ashraf SS et al.; Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA-ribosomal RNA (rRNA) interactions . To better understand these interactions, U33-substituted yeast tRNA(Phe) anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome . Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs . Chemical footprints of native yeast tRNA(Phe), ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared . Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially . Ribosomal binding of yeast tRNA(Phe) enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not . Two residues, G926 and G1338 with KpS approximately 50-60 nM, were afforded significantly greater protection by both yeast tRNA(Phe) and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (KpS approximately 100-200 nM) . In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33 . ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33 . However, protection of G926 and G1338 (KpS between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively) . Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (KpS = 242 and 347 nM, respectively) . Thus, G926 and G1338 are important to efficient P-site binding of tRNA . More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other. Biochem Genet, 1999 Apr, 37(3-4), 109 - 17 CAG repeats in various organisms studied by Southern blot analysis; Grinde B et al.; A 90-nucleotide (CAG)30, single-stranded DNA was used to probe Southern blots in order to indicate the quantity and distribution of long CAG repeats in selected genomes . Bovine and rat genomes were found to contain a particularly high content of CAG repeats, while the repeats were comparatively rare in the human genome . A particularly strong signal in the bovine genome was due to a CAG repeat associated with the 1.709 satellite . A similar element was found in goat and musk, but not in the other artiodactyls tested, suggesting that this particular CAG repeat developed some 10-20 million years ago within a 3.8-kb unit presently belonging to the satellite element and that this unit has later multiplied in the genome . Single-copy repeats could be discerned in yeast, but not in mammals . Thus the probe did not detect specific repeats in patients with CAG repeat diseases. Eur J Cell Biol, 1999 Aug, 78(8), 533 - 8 Direct interaction between the ARF-specific guanine nucleotide exchange factor msec7-1 and presynaptic Munc13-1; Neeb A et al.; Msec7-1, a mammalian homologue of yeast sec7p, is a specific GDP/GTP exchange factor for small G-proteins of the ARF family . Overexpression of msec7-1 in Xenopus neuromuscular junctions leads to an increase in synaptic transmitter release that is most likely caused by an increase in the pool of readily releasable vesicles . However, the molecular mechanisms by which msec7-1 is targeted to presynaptic compartments and enhances neurotransmitter release are not known . In the present study, we demonstrate that msec7-1 interacts directly with Munc13-1, a phorbol ester-dependent enhancer of neurotransmitter release that is specifically localized to presynaptic transmitter release zones . Given that Munc13-1 and msec7-1 participate in very similar presynaptic processes and because Munc13-1 is specifically targeted to presynaptic active zones, we suggest that the msec7-1/Munc13-1 interaction serves to colocalize the two proteins at the active zone, a subcellular compartment with extremely high membrane turnover. Biol Chem, 1999 Jul-Aug, 380(7-8), 937 - 44 Sugars as signal molecules in plant seed development; Wobus U et al.; Higher plants as sessile organisms react very flexible to environmental changes and stresses and use metabolites like glucose, sucrose and nitrate not only as nutrients but also as signals as part of their life strategies . The role of metabolites as signal molecules has attracted considerable interest during recent years . Data reviewed here for developing plant seeds suggest a trigger function of especially sugars also in development in that metabolic regulatory control can override developmental regulation, i.e., the developmental programme only continues normally if a certain metabolic state is sensed at a given time point in a given cell or tissue . Several experimental strategies have provided mainly correlative evidence that certain sugar levels and/or the resulting changes in osmotic values are necessary within defined tissues or cells to maintain a distinct stage of differentiation or to proceed with the developmental programme . In young legume seeds, but certainly also in other tissues, a high hexose (probably mainly glucose) level seems to maintain the capacity of cells to divide whereas - later in seed development - a certain sucrose level is necessary to induce storage-associated cell differentiation . A major determinant of embryo hexose levels in young legume seeds is an apoplastic invertase preferentially expressed in the inner cell layers of the seed coat . The enzyme cleaves the incoming photoassimilate sucrose into glucose and fructose . During development the tissue harbouring the invertase is degraded in a very specific spatial and temporal pattern as part of the developmental programme and is thus creating steep glucose gradients within the cotyledons . These gradients can be measured at nearly cellular resolution and were found to be correlated positively with cell division rate and negatively with cell differentiation and storage activities . A hexose and a sucrose transporter accumulating only in the epidermal cell layer of the cotyledons seem to be essential in creating and maintaining these gradients . To gain further insights into the role of metabolites, especially sugars, as triggers of developmental processes we foremost have to identify receptor molecules already characterised in yeast, and to describe and understand the signal transduction networks involved. Cancer Res, 1999 Sep 15, 59(18), 4662 - 7 Detailed genetic and physical mapping of tumor suppressor loci on chromosome 3p in ovarian cancer; Fullwood P et al.; Hemizygosity and homozygosity mapping studies show that many common sporadic cancers including lung, breast, kidney, cervical, ovarian, and head and neck cancer display deletions on the short arm of chromosome 3 . For ovarian cancer, monochromosomal transfer suppression studies have identified three candidate regions for chromosome 3p ovarian cancer tumor suppressor genes (OCTSGs) . To accurately map OCTSG candidate regions, we analyzed 70 ovarian tumors for loss of heterozygosity (LOH) at 20 loci on chromosome 3p that were selected to target those regions proposed to contain tumor suppressor genes for common sporadic cancers . All samples were informative for at least five markers . In 33 (52%) tumors without microsatellite instability, LOH was observed for at least one 3p marker . Analysis of 27 ovarian tumors demonstrating both loss and retention of 3p markers enabled us to define four nonoverlapping minimal deletion regions (OCLOHRs): (a) OCLOHR-1 mapped distal to D3S3591 at 3p25-26; (b) OCLOHR-2 mapped between D3S1317 and D3S1259 at 3p24-25; (c) OCLOHR-3 mapped between D3S1300 and D3S1284, an area that includes the FHIT locus at 3p14.2; and (d) OCLOHR-4 mapped between D3S1284 and D3S1274 at 3p12-13, a region known to contain overlapping homozygous deletions in lung and breast tumor cell lines . However, microsatellite markers from the chromosome 3p21.3 interval homozygously deleted in lung cancer cell lines did not identify a distinct OCLOHR . The frequency and extent of 3p LOH correlated with tumor stage such that LOH at two or more OCLOHRs was present in 53% (16 of 30) of stage III tumors but only 26% (5 of 19) of stage I/II tumors (P = 0.08) . To determine the relationship between the OCLOHRs and the three candidate ovarian cancer suppression regions (OCSRs) identified previously by monochromosome transfer studies, we performed detailed genetic and physical mapping studies to define the extent of the three candidate OCSRs and to establish YAC contigs covering each region . OCSR-A at 3p25-26 and OCSR-B at 3p24 were shown to overlap with OCLOHR-1 and OCLOHR-2, respectively, providing further evidence for OCTSGs in these regions . We also show that OCSR-C overlaps with a locus at 3p21.3 previously implicated in lung and breast cancer. DNA Res, 1999 Aug 31, 6(4), 247 - 53 Evaluation of a cDNA scanning method concerning the fidelity and efficiency of cDNA selection using the YAC CIC3B1-S region of Arabidopsis thaliana chromosome 5; Motohashi R et al.; We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155 161) . We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1-S DNA were concentrated . We isolated 545 cDNA clones from the sublibrary, and determined partial sequence of them to produce expressed sequence tags (ESTs) derived from the YAC region . In total, 74 nonredundant groups of cDNAs were obtained from 545 cDNA clones . Forty-seven percent of these EST clones had significant homology to functional proteins such as protein kinases, LON protease, nucleic acid binding protein and chloride channel protein . We compared the cDNA sequences isolated by the cDNA scanning method to the Arabidopsis genomic sequence corresponding to the YAC CIC3B1-S region, and found that 69% of the selected cDNAs are located in the region . We discuss the fidelity and efficiency of the cDNA scanning method for cloning region-specific cDNAs and its useful application in positional cloning. DNA Res, 1999 Aug 31, 6(4), 227 - 33 Significant differences in the frequency of transcriptional units, types and numbers of repetitive elements, GC content, and the number of CpG islands between a 1010-kb G-band genomic segment on chromosome 9q31.3 and a 1200-kb R-band genomic segment on chromosome 3p21.3; Daigo Y et al.; We determined the nucleotide sequence of the entire 1,010,525-bp insert contained in CEPH YAC clone 867e8 . This human genomic segment was derived from chromosome 9q31.3 and corresponds to a G-band region . We compared this segment, in terms of structure, with a previously characterized 1,201,033-bp sequence in CEPH YAC936c1 that had come from a portion of human chromosome 3p21.3 corresponding to an R-band region . The two segments were significantly different with respect to the frequency of transcriptional units, the types and numbers of repetitive elements present, their GC content, and the number of CpG islands . Alu elements, GC content, and CpG islands all showed positive correlations with the abundance of exons, but the distribution of LINE1s did not . These observations might reflect an influence of the first three of these features on the functions or expression of genes in the respective regions . In addition to a novel gene (F36) lying at the centromeric end of the 9q segment, we found a cluster of placenta-specific genes within a small section (about 400 kb) on the telomeric side of YAC867e8 . This cluster consisted of four apparently unrelated ESTs and two genes, pregnancy-associated plasma protein-A (PAPP-A) and a novel gene (tentatively named EST-YD1) . Our characterization of the two chromosomal regions provided evidence that genes are not evenly distributed throughout the human genome, and that gene richness is correlated with the GC content and with the frequency of either Alu elements or CpG islands. J Mass Spectrom, 1999 Sep, 34(9), 930 - 41 Analysis of phytochelatin-cadmium complexes from plant tissue culture using nano-electrospray ionization tandem mass spectrometry and capillary liquid chromatography/electrospray ionization tandem mass spectrometry; Yen TY et al.; Phytochelatins (PCs, also known as class III metallothioneins), a family of sulfhydryl-rich peptides with the formula (gamma-GluCys)(n)Gly(Pc(n), n = 2-11), are induced in plants, yeast and fungi exposed to heavy metals, and are thought to detoxify metals by forming PC- metal complexes . Although PCs have been detected, PC- metal complexes have not been well characterized . In this work, nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and capillary liquid chromatography/electrospray ionization tandem mass spectrometry (capillary LC/ESI-MS/MS) methods were used to analyze PC - Cd complexes isolated from Datura innoxia, also known as Jimsonweed, cell culture exposed to Cd . With nano-ESI-MS/MS and capillary LC/ESI-MS/MS we could simultaneously detect the presence of PCs and PC - Cd complexes from plant cell extracts, unambiguously identify these species and elucidate the nature of individual PC - Cd complexes . Phytochelatins with n = 3-6 were detected, as were PC - Cd complexes with PC(3), PC(4) and PC(5) . This is the first study to report the size and nature of native PC - Cd complexes from plant tissue samples . These results demonstrate that the direct analysis of plant extracts using nano-ESI-MS/MS and capillary LC/ESI-MS/MS methods is simple and sensitive to the range of PCs and PC - Cd complexes in plants . Hence these methods open up new opportunities for further quantitative analysis of PCs and PC - metal complexes in cell culture and plant systems to understand the relationship between the biosynthesis of these compounds and metal tolerance . J Natl Cancer Inst, 1999 Sep 15, 91(18), 1563 - 8 Novel tumor suppressor locus in human chromosome region 3p14.2; Julicher K et al.; BACKGROUND: Alterations of chromosome region 3p14 are observed in numerous human malignancies . Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation . METHODS: YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice . RESULTS: We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro . Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs . Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3 . CONCLUSION: These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2 . Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations. Biochem Biophys Res Commun, 1999 Sep 24, 263(2), 482 - 90 Cloning and characterization of the murine histone deacetylase (HDAC3); Mahlknecht U et al.; Histone acetylation modifiers have been described to participate as cofactors in mammalian transcriptional complexes involved in the regulation of cellular proliferation and differentiation . The acetylation of core histone proteins is reversible and regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs) . Increasing evidence suggests a connection between histone acetylation and the development of cancer and leukemia . We have recently mapped HDAC3 to mouse chromosome 18B3, a region which is syntenic with human chromosome 5q31, where HDAC3 is imbedded in a group of potential tumor suppressor genes and which has been reported to be the smallest commonly deleted segment in malignant myeloid disease . We report herein the identification and characterization of HDAC3, a yeast RPD3 ortholog in the mouse . Studies on murine HDAC3 may yield important insights on the understanding of myeloproliferative disease in humans . Dev Biol, 1999 Oct 1, 214(1), 87 - 101 EMK protein kinase-null mice: dwarfism and hypofertility associated with alterations in the somatotrope and prolactin pathways; Bessone S et al.; Gene trapping was used in embryonic stem (ES) cells in an attempt to inactivate genes involved in development . The Emk (ELKL motif kinase) gene has been disrupted and a mutant mouse line derived . Previous work had shown that EMK kinases, called MARK in the rat, exert a major control on microtubule stability by phosphorylating microtubule-associated proteins and that genes homologous to Emk in yeast or Caenorhabditis elegans are essential for cell and embryonic polarity . Although we found the Emk gene to be active in the preimplantation mouse embryo and then to show a widespread expression, Emk-null mice had no embryonic defect and were viable . They show an overall proportionate dwarfism and a peculiar hypofertility: homozygotes are not fertile when intercrossed, but are fertile in other types of crosses . Insulin-like growth factor I (IGF I) and IGF-binding protein 3 (IGFBP3) were reduced in the plasma of homozygotes of both sexes . A direct implication of the EMK kinase in IGF I plasmatic production is unlikely because the Emk gene does not seem to be expressed in hepatocytes . Nevertheless, GH assayed at arbitrary times in plasma did not show differences between genotypes and GH concentrations in pituitary extracts were not found to be altered in homozygotes . Our results, though, do not exclude the possibility that in the mutants the overall quantity of GH secreted daily is reduced . Our observation of a smaller size of the pituitaries of the mutants is in favor of this hypothesis . The prolactin concentration in the pituitaries was much lowered in homozygous females, but it was normal in males . The possible involvement of EMK protein kinase in hormone secretion in the pituitary and/or the hypothalamus, via the microtubule network, is discussed . Mol Cell Biol, 1999 Oct, 19(10), 7138 - 46 Interacting regions in Stat3 and c-Jun that participate in cooperative transcriptional activation; Zhang X et al.; Independent but closely spaced DNA binding sites for Stat3 and c-Jun are required for maximal enhancer function in a number of genes, including the gene encoding the interleukin-6 (IL-6)-induced acute-phase response protein, alpha(2)-macroglobulin . In addition, a physical interaction of Stat3 with c-Jun, based on yeast two-hybrid interaction experiments, has been reported . Here we confirm the existence of an interaction between Stat3 and c-Jun both in vitro, with recombinant proteins, and in vivo, during transient transfection . Using fragments of both proteins, we mapped the interactive sites to the C-terminal region of c-Jun and to two regions in Stat3, within the coiled-coil domain and in a portion of the DNA binding domain distant from DNA contact sites . In transient-transfection experiments with the alpha(2)-macroglobulin enhancer, Stat3 and c-Jun cooperated to yield maximal enhancer function . Point mutations of Stat3 within the interacting domains blocked both physical interaction of Stat3 with c-Jun and their cooperation in IL-6-induced transcription directed by the alpha(2)-macroglobulin enhancer . While the amino acid sequences and the three-dimensional structures of Stat3 and Stat1 cores are very similar, fragments of Stat1 failed to bind c-Jun in vitro . Although Stat1 binds in vitro to the gamma interferon gene response (GAS) element in the alpha(2)-macroglobulin enhancer, Stat1 did not stimulate transcription, nor did Stat1 and c-Jun cooperate in driving transcription controlled by the alpha(2)-macroglobulin enhancer. Mol Cell Biol, 1999 Oct, 19(10), 7050 - 60 Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins; Adler HT et al.; One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins . Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein . We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis . The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis . Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis . In contrast, expression of wild-type HRX gave rise to an increase in apoptosis . The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis . We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription . These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein . We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression. Mol Cell Biol, 1999 Oct, 19(10), 6509 - 22 Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1; Sladek FM et al.; Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events . Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators . The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants . In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 alpha1 (HNF4alpha1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction . In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III . The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4alpha2) abrogates that inhibition in vivo and in vitro . A series of protease digestion assays indicates that there may be structural differences between HNF4alpha1 and HNF4alpha2 in the F domain as well as in the ligand binding domain (LBD) . The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4alpha1 and -alpha2 and that that contact is different in the HNF4alpha1 and HNF4alpha2 isoforms . Finally, we propose a model in which the F domain of HNF4alpha1 acts as a negative regulatory region for transactivation and in which the alpha2 insert ameliorates the negative effect of the F domain . A conserved repressor sequence in the F domains of HNF4alpha1 and -alpha2 suggests that this model may be relevant to other nuclear receptors as well. Eur J Biochem, 1999 Oct 1, 265(1), 361 - 6 Direct interaction of EEA1 with Rab5b; Callaghan J et al.; The early endosomal autoantigen EEA1 is essential for early endosomal membrane fusion . It binds to endosomes via a C-terminal domain (EEA1-CT) . To identify proteins interacting with EEA1-CT, we screened a human brain library in the yeast two-hybrid system . Fourteen clones reacted strongly with EEA1-CT . Sequencing of these clones revealed that they all contained the ORF of the small GTPase, Rab5b . Further two-hybrid analysis suggested that Rab5b also interacts with the N-terminus of EEA1 (EEA1-NT) . The interaction of both EEA1-CT and EEA1-NT with Rab5b was confirmed biochemically, and was found to be GTP dependent . Confocal immunofluorescence microscopy indicated that EEA1 colocalizes with Rab5b on early endosomes . Although EEA1-CT and EEA1-NT interacted strongly with wild-type Rab5b in the two-hybrid system, we detected no interaction with wild-type Rab5a, even though GTPase-deficient mutants of both Rab5a and Rab5b interacted equally well with EEA1 . This difference could not be explained by differences in intrinsic GTPase activities, as these were found to be very similar . Instead, we speculate that yeast may contain a GTPase-activating protein (GAP) activity that stimulates Rab5a but not Rab5b . In contrast, pig brain cytosol was found to contain a GAP activity that stimulates the GTPase activity of Rab5b in preference to that of Rab5a . These data provide evidence that EEA1 interacts with both Rab5a and Rab5b, and that the GTPase activities of the two proteins are differentially regulated in vivo. Eur J Biochem, 1999 Oct 1, 265(1), 145 - 51 Purification and properties of a basic endo-beta-1,6-glucanase (BGN16.1) from the antagonistic fungus Trichoderma harzianum; de la Cruz J et al.; The antagonistic fungus Trichoderma harzianum CECT 2413 produces at least two extracellular beta-1,6-glucanases, among other hydrolases acting on polysaccharides from fungal cell walls, when grown in chitin as the sole carbon source . We have previously reported on the purification and biochemical characterization of the major activity, which corresponds to an acidic enzyme named BGN16.2 {de la Cruz, J., Pintor-Toro, J.A., Benitez, T . & Llobell, A . (1995) J . Bacteriol . 177, 1864-1871} . In this paper, we report on the purification to electrophoretical homogeneity of BGN16.1, the second beta-1, 6-glucanase enzyme . BGN16.1 was purified by ammonium sulfate precipitation followed by adsorption and digestion of pustulan (a beta-1,6-glucan), chromatofocusing and gel-filtration chromatography . BGN16.1 is a non-glycosylated protein with an apparent molecular mass of 51 kDa and a basic isoelectric point (pI 7.4-7.7) . The enzyme was active toward substrates containing beta-1,6-glycosidic linkages, including yeast cell walls . The Km was 0.8 mg x mL-1 with pustulan as the substrate . Reaction product analysis by HPLC clearly indicated that BGN16.1 has an endo-hydrolytic mode of action . The probable role of this enzyme in the antagonistic action of T . harzianum is also discussed. Oncogene, 1999 Sep 9, 18(36), 5126 - 30 Association of RACK1 and PKCbeta with the common beta-chain of the IL-5/IL-3/GM-CSF receptor; Geijsen N et al.; Granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5 belong to a family of cytokines that regulate proliferation, differentiation and function of haematopoietic cells . Their receptor consists of a ligand specific alpha-chain and a signal transducing beta-chain (betac) . While, the role of phosphotyrosine residues in the betac as mediators of downstream signalling cascades has been established, little is known about non-phosphotyrosine mediated events . To identify proteins interacting with betac, we screened a yeast two-hybrid library with the intracellular domain of betac . We found that RACK1, a molecule associating with activated PKC, PLCgamma and Src kinases, associated with the membrane proximal region of betac in both yeast two-hybrid, immunoprecipitation and GST-pull-down assays . The association of RACK1 was constitutive, demonstrating no alteration upon cellular stimulation . Furthermore, upon stimulation of cells with IL-5 or PMA, a complex of betac and PKCbeta was found . Together, these findings suggest a novel role for RACK1 as a possible adapter molecule associating with the intracellular domain of cytokine receptors. Oncogene, 1999 Sep 2, 18(35), 4891 - 8 Identification of a chromosome 3p14.3-21.1 gene, APPL, encoding an adaptor molecule that interacts with the oncoprotein-serine/threonine kinase AKT2; Mitsuuchi Y et al.; AKT2 is a serine/threonine kinase implicated in human ovarian and pancreatic cancers . AKT2 is activated by a variety of growth factors and insulin via phosphatidylinositol 3-kinase (PI3K) . However, its normal cellular role is not well understood . To gain insight into the function of AKT2, we performed yeast two-hybrid system to screen for interacting proteins . Using this technique, we identified a novel interactor, designated APPL, which contains a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a leucine zipper, classes of motifs defined in signaling molecules as functional interaction domains with specific targets . The PH domain of APPL shows similarity to those found in GTPase-activating proteins such as oligophrenin-1 and Graf, whereas its PTB domain exhibits homology with CED-6, an adaptor protein that promotes engulfment of apoptotic cells, and IB1, a transactivator of the GLUT2 gene . APPL is highly expressed in skeletal muscle, heart, ovary and pancreas, tissues in which AKT2 mRNA is abundant . APPL interacts with the inactive form of AKT2; moreover, APPL binds to the PI3K catalytic subunit, p110alpha . These data suggest that APPL is an adaptor that may tether inactive AKT2 to p110alpha in the cytoplasm and thereby may expedite recruitment of AKT2 and p110alpha to the cell membrane upon mitogenic stimulation . Furthermore, the APPL gene was mapped to human chromosome 3p14.3-p21.1, where deletions and other rearrangements have often been reported in a variety of tumor types . The identification of APPL may facilitate further analysis of the physiological and oncogenic activities of AKT2. Gene Ther, 1999 Sep, 6(9), 1634 - 7 Transfer of YACs up to 2.3 Mb intact into human cells with polyethylenimine; Marschall P et al.; The transfer of large YAC DNA into human cells is a laborious procedure . High quality pulsed field gel purified DNA is required, which is easily sheared during manipulation before transfection or degraded in the endosome of the cell following transfection . NaCl and polyamines compact and prevent DNA from shearing, but may not consistently protect DNA after transfection . We investigated if other polycations such as poly-L-lysine (PLL) and polyethylenimine (PEI) could condense and protect large YAC DNA (up to 2.3 Mb) from being degraded after lipofection . DNA condensation was monitored by a gel retardation assay, and atomic force microscopy (AFM) . DNA was retarded in the gel when complexed with high concentrations of PLL and PEI, indicating that DNA had condensed . However, AFM images of PLL-DNA complexes showed aggregates of DNA molecules resulting from incomplete condensation, whereas PEI-DNA complexes produced condensed particles approximately 30-60 nm . Exogenous PLL-DNA remained intact in 36% of positive clones after lipofection, whereas PEI-DNA was intact in 100% of positive clones . PEI is a better condensing reagent than PLL, protecting DNA from shearing and endosomal degradation, and assists in delivering YACs up to 2.3 Mb intact into human cells. Mol Cell Biol, 1999 Oct, 19(10), 7276 - 86 De novo synthesis of sphingolipids is required for cell survival by down-regulating c-Jun N-terminal kinase in Drosophila imaginal discs; Adachi-Yamada T et al.; Mitogen-activated protein kinase (MAPK) is a conserved eukaryotic signaling factor that mediates various signals, cumulating in the activation of transcription factors . Extracellular signal-regulated kinase (ERK), a MAPK, is activated through phosphorylation by the kinase MAPK/ERK kinase (MEK) . To elucidate the extent of the involvement of ERK in various aspects of animal development, we searched for a Drosophila mutant which responds to elevated MEK activity and herein identified a lace mutant . Mutants with mild lace alleles grow to become adults with multiple aberrant morphologies in the appendages, compound eye, and bristles . These aberrations were suppressed by elevated MEK activity . Structural and transgenic analyses of the lace cDNA have revealed that the lace gene product is a membrane protein similar to the yeast protein LCB2, a subunit of serine palmitoyltransferase (SPT), which catalyzes the first step of sphingolipid biosynthesis . In fact, SPT activity in the fly expressing epitope-tagged Lace was absorbed by epitope-specific antibody . The number of dead cells in various imaginal discs of a lace hypomorph was considerably increased, thereby ectopically activating c-Jun N-terminal kinase (JNK), another MAPK . These results account for the adult phenotypes of the lace mutant and suppression of the phenotypes by elevated MEK activity: we hypothesize that mutation of lace causes decreased de novo synthesis of sphingolipid metabolites, some of which are signaling molecules, and one or more of these changes activates JNK to elicit apoptosis . The ERK pathway may be antagonistic to the JNK pathway in the control of cell survival. Mol Cell Biol, 1999 Oct, 19(10), 7191 - 202 NRIF3 is a novel coactivator mediating functional specificity of nuclear hormone receptors; Li D et al.; Many nuclear receptors are capable of recognizing similar DNA elements . The molecular event(s) underlying the functional specificities of these receptors (in regulating the expression of their native target genes) is a very important issue that remains poorly understood . Here we report the cloning and analysis of a novel nuclear receptor coactivator (designated NRIF3) that exhibits a distinct receptor specificity . Fluorescence microscopy shows that NRIF3 localizes to the cell nucleus . The yeast two-hybrid and/or in vitro binding assays indicated that NRIF3 specifically interacts with the thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion but does not bind to the retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor . Functional experiments showed that NRIF3 significantly potentiates TR- and RXR-mediated transactivation in vivo but has little effect on other examined nuclear receptors . Domain and mutagenesis analyses indicated that a novel C-terminal domain in NRIF3 plays an essential role in its specific interaction with liganded TR and RXR while the N-terminal LXXLL motif plays a minor role in allowing optimum interaction . Computer modeling and subsequent experimental analysis suggested that the C-terminal domain of NRIF3 directly mediates interaction with liganded receptors through an LXXIL (a variant of the canonical LXXLL) module while the other part of the NRIF3 protein may still play a role in conferring its receptor specificity . Identification of a coactivator with such a unique receptor specificity ma