Yeasts

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 may provide new insight into the molecular mechanism(s) of receptor-mediated transcriptional activation as well as the functional specificities of nuclear receptors.

Mol Cell Biol, 1999 Oct, 19(10), 6906 - 17
Nucleolar factors direct the 2'-O-ribose methylation and pseudouridylation of U6 spliceosomal RNA; Ganot P et al.; The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place . In this study, we demonstrate that the nucleolus contains all the trans-acting factors that are responsible for the accurate and efficient synthesis of the eight 2'-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA . Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus . For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2'-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified . This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2'-O-methylation of the U6 spliceosomal RNA (K . T . Tycowski, Z.-H . You, P . J . Graham, and J . A . Steitz, Mol . Cell 2:629-638, 1998) . We demonstrate that a novel 2'-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2'-O-methylation small nucleolar guide RNA . We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2'-O-methylated and pseudouridylated . These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.

Mol Cell Biol, 1999 Oct, 19(10), 6815 - 24
Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya; Ohto H et al.; Drosophila sine oculis and eyes absent genes synergize in compound-eye formation . The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development . We hypothesized that Six and Eya proteins cooperate to regulate their target genes . Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites . A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya . To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells . Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm . In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins . Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver . Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya . In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation . A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis . Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins . This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.

Nature, 1999 Sep 9, 401(6749), 173 - 7
Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP; Yeung K et al.; Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK) . This kinase cascade controls the proliferation and differentiation of different cell types . Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen . This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein) . In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras . RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy . RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases . RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase . Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription . RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

J Histochem Cytochem, 1999 Oct, 47(10), 1315 - 22
High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation; El Mansouri S et al.; To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed . In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation . P53 mutations were screened by functional assay in yeast in 14 samples . With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern . However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples . P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors . In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry . These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)

Cell, 1999 Sep 3, 98(5), 629 - 40
Measles virus infection in a transgenic model: virus-induced immunosuppression and central nervous system disease; Oldstone MB et al.; Measles virus (MV) infects 40 million persons and kills one million per year primarily by suppressing the immune system and afflicting the central nervous system (CNS) . The lack of a suitable small animal model has impeded progress of understanding how MV causes disease and the development of novel therapies and improved vaccines . We tested a transgenic mouse line in which expression of the MV receptor CD46 closely mimicked the location and amount of CD46 found in humans . Virus replicated in and was recovered from these animals' immune systems and was associated with suppression of humoral and cellular immune responses . Infectious virus was recovered from the CNS, replicated primarily in neurons, and spread to distal sites presumably by fast axonal transport . Thus, a small animal model is available for analysis of MV pathogenesis.

Mol Cell, 1999 Aug, 4(2), 167 - 74
Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint; Esashi F et al.; DNA damage induces cell cycle arrest (called the damage checkpoint), during which cells carry out actions for repair . A fission yeast protein, Crb2/Rhp9, which resembles budding yeast Rad9p and human BRCA1, promotes checkpoint by activating Chk1 kinase, which restrains Cdc2 activation . We show here that phosphorylation of the T215 Cdc2 site of Crb2 is required for reentering the cell cycle after the damage-induced checkpoint arrest . If this site is nonphosphorylatable, irradiated cells remain arrested, though damage is repaired, and maintain the phosphorylated state of Chk1 kinase . The T215 site is in vitro phosphorylated by purified Cdc2 kinase . Phosphorylation of T215 occurs intensely in response to DNA damage at a late stage, suggesting an antagonistic role of Cdc2 phosphorylation toward checkpoint.

Plant Cell, 1999 Sep, 11(9), 1755 - 68
MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope; Gindullis F et al.; The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells . MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA . Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix . MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope . Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays . MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins . MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix . These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.

Plant Cell, 1999 Sep, 11(9), 1623 - 34
Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis; Couteau F et al.; In yeast, the DMC1 gene is required for interhomolog recombination, which is an essential step for bivalent formation and the correct partition of chromosomes during meiosis I . By using a reverse genetics approach, we were able to identify a T-DNA insertion in AtDMC1, the Arabidopsis homolog of DMC1 . Homozygotes for the AtDMC1 insertion failed to express AtDMC1, and their residual fertility was 1.5% that of the wild type . Complete fertility was restored in mutant plants when a wild-type copy of the AtDMC1 gene was reintroduced . Cytogenetical analysis points to a correlation of the sterility phenotype with severely disturbed chromosome behavior during both male and female meiosis . In this study, our data demonstrate that AtDMC1 function is crucial for meiosis in Arabidopsis . However, meiosis can be completed in the Arabidopsis dmc1 mutant, which is not the case for mouse or some yeast mutants.

J Biol Chem, 1999 Sep 24, 274(39), 27981 - 8
ERK MAP kinase links cytokine signals to activation of latent HIV-1 infection by stimulating a cooperative interaction of AP-1 and NF-kappaB; Yang X et al.; Human immunodeficiency virus type 1 (HIV-1) can establish latent infection following provirus integration into the host genome . NF-kappaB plays a critical role in activation of HIV-1 gene expression by cytokines and other stimuli, but the signal transduction pathways that regulate the switch from latent to productive infection have not been defined . Here, we show that ERK1/ERK2 mitogen-activated protein kinase (MAPK) plays a central role in linking signals at the cell surface to activation of HIV-1 gene expression in latently infected cells . MAPK was activated by cytokines and phorbol 12-myristate 13-acetate in latently infected U1 cells . The induction of HIV-1 expression by these stimuli was inhibited by PD98059 and U0126, which are specific inhibitors of MAPK activation . Studies using constitutively active MEK or Raf kinase mutants demonstrated that MAPK activates the HIV-1 long terminal repeat (LTR) through the NF-kappaB sites . Most HIV-1 inducers activated NF-kappaB via a MAPK-independent pathway, indicating that activation of NF-kappaB is not sufficient to explain the activation of HIV-1 gene expression by MAPK . In contrast, all of the stimuli activated AP-1 via a MAPK-dependent pathway . NF-kappaB and AP-1 components c-Fos and c-Jun were shown to physically associate by yeast two-hybrid assays and electrophoretic mobility shift assays . Coexpression of NF-kappaB and c-Fos or c-Jun synergistically transactivated the HIV-1 LTR through the NF-kappaB sites . These studies suggest that MAPK acts by stimulating AP-1 and a subsequent physical and functional interaction of AP-1 with NF-kappaB, resulting in a complex that synergistically transactivates the HIV-1 LTR . These results define a mechanism for signal-dependent activation of HIV-1 replication in latently infected cells and suggest potential therapeutic strategies for unmasking latent reservoirs of HIV-1.

J Biol Chem, 1999 Sep 24, 274(39), 27674 - 81
The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein; Torii KU et al.; The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness . The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins . To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger . This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay . In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster . The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association . Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction . Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain . The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.

J Biol Chem, 1999 Sep 24, 274(39), 27439 - 47
Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity; Kester HA et al.; TGF-beta-stimulated clone-22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved during evolution . Two homologues are known that share a similar leucine zipper domain and another conserved domain (designated the TSC box) . Only limited data are available on the function of TSC-22 and its homologues . TSC-22 is transcriptionally up-regulated by many different stimuli, including anti-cancer drugs and growth inhibitors, and recent data suggest that TSC-22 may play a suppressive role in tumorigenesis . In this paper we show that TSC-22 forms homodimers via its conserved leucine zipper domain . Using a yeast two-hybrid screen, we identified a TSC-22 homologue (THG-1) as heterodimeric partner . Furthermore, we report the presence of two more mammalian family members with highly conserved leucine zippers and TSC boxes . Interestingly, both TSC-22 and THG-1 have transcriptional repressor activity when fused to a heterologous DNA-binding domain . The repressor activity of TSC-22 appears sensitive for promoter architecture, but not for the histone deacetylase inhibitor trichostatin A . Mutational analysis showed that this repressor activity resides in the non-conserved regions of the protein and is enhanced by the conserved dimerization domain . Our results suggest that TSC-22 belongs to a family of leucine zipper-containing transcription factors that can homodimerize and heterodimerize with other family members and that at least two TSC-22 family members may be repressors of transcription.

EMBO J, 1999 Sep 15, 18(18), 5085 - 98
MEF-2 function is modified by a novel co-repressor, MITR; Sparrow DB et al.; The MEF-2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types . In skeletal muscle cells, MEF-2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression . Similar interactions with tissue or lineage-specific cofactors may also underlie MEF-2 function in other cell types . In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two-hybrid screen . This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A . The MEF-2 interacting transcription repressor (MITR) protein binds to the N-terminal MADS/MEF-2 region of the MEF-2 proteins but does not bind to the related Xenopus MADS protein serum response factor . In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle . In functional assays, MITR negatively regulates MEF-2-dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1 . Thus, we propose that MITR acts as a co-repressor, recruiting a specific deacetylase to downregulate MEF-2 activity.

EMBO J, 1999 Sep 15, 18(18), 4949 - 60
GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system; Shorter J et al.; We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery . GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system . GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae . GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system . Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi . Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65 . These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.

Plant Mol Biol, 1999 Jul, 40(5), 825 - 34
Molecular cloning, expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage; Cheong NE et al.; A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage . The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members . It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%) . Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome . RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots . The expression of C2C-Prx was slightly induced by treatment with H2O2 (100 microM) or Fe3+/O2/DTT oxidation system, but not by ABA (50 microM) or GA3 (10 microM) . The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus . The N-terminally truncated recombinant protein (deltaC2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition . The deltaC2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa . The deltaC2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system . In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the deltaC2C-Prx exhibits peroxidase activity on H2O2.

Biochem Biophys Res Commun, 1999 Sep 16, 263(1), 187 - 91
Isolation and characterization of angiogenin-1 and a novel protein designated lactogenin from bovine milk; Ye XY et al.; This paper reports the isolation and characterization from bovine milk of two proteins: angiogenin-1, a recently discovered angiogenin, and lactogenin, a novel protein . Both proteins were adsorbed on and eluted closely from CM-Sepharose and Mono S . Lactogenin possessed a molecular weight (17 kDa) slightly higher than that of angiogenin-1 (15 kDa) . Lactogenin had a higher ribonucleolytic (RNase) activity than angiogenin-1 towards yeast transfer RNA (tRNA) . The Km values estimated for the RNase activities of angiogenin-1 and lactogenin were 51 microM and 40 microM respectively . Both were specific for poly C . The optimal pH for the RNase activities of angiogenin-1 and lactogenin was 7.75 and 7.5 respectively . Comparison of the amino acid sequences of cyanogen bromide fragments and the pyroglutaminase-treated N-terminal fragment of lactogenin with the sequence of bovine liver RNase (RNase BL4) revealed identity in residues 3-22, 24, 26-27, 37, 41-44, 46-50, 54, 56, 63, 72-80, and 83 . Considerable similarity to the N-terminal sequence of angiogenin-2 was also noted . Both lactogenin and angiogenin-1 inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) below 100 nM .

Biochem Biophys Res Commun, 1999 Sep 16, 263(1), 149 - 55
Tip60 interacts with human interleukin-9 receptor alpha-chain; Sliva D et al.; Interleukin-9 (IL-9) exerts its pleiotropic effects through the IL-9 receptor (IL-9R) complex that consists of the ligand specific IL-9R alpha-chain, and the IL-2R gamma-chain . In this study, we used a modified yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the intracellular domain of the human IL-9R alpha-chain (hIL-9Ralpha) . We have identified Tip60, an HIV-1 Tat transcription cofactor, as an hIL-9Ralpha interacting protein . The interaction between hIL-9Ralpha and Tip60 was confirmed by coimmunoprecipitation and colocalization studies . This is the first demonstration that Tip60 associates with a membrane receptor . We also mapped amino acids 411-423 in hIL-9Ralpha and amino acids 100-147 in Tip60 to be important for interaction . Interestingly, the region in hIL-9alpha that binds Tip60 is adjacent to the site previously shown to interact with Stat3 . Tip60 binds HIV-Tat and mediates Tat-dependent transactivation possibly through its histone acetyltransferase activity . Our results therefore suggest that Tip60 may act as a cofactor of Stat3 or as an adaptor protein for molecules that are important for IL-9 signaling .

Genomics, 1999 Sep 1, 60(2), 161 - 71
Molecular analysis of the human chromosome 5q13.3 region in patients with hairy cell leukemia and identification of tumor suppressor gene candidates; Wu X et al.; The pathogenesis of hairy cell leukemia (HCL) remains largely unknown since no specific genetic lesion has been identified in this disease . Previous cytogenetic analysis from our group has shown that chromosome abnormalities involving the 5q13 band are common in HCL, occurring in approximately 1/3 of patients . The data suggest that the 5q13.3 band is likely to harbor a gene involved in the transformational events of this disease . We have recently found two cosmids flanking the 5q13.3 breakpoint in patients with HCL, and the distance between them is approximately 35 kb, as analyzed by fiber-FISH . The two cosmids have been located between the markers SGC34998 and WI-15505/WI-6897 by radiation hybrid mapping . Five of 11 patients with HCL had a hemizygous deletion of the two cosmids, indicating that the function of a tumor suppressor gene may be lost . With the aim of delineating the critical region of 5q13.3 loss in patients with HCL, we have constructed an integrated contig of YAC, BAC, PAC, P1, and cosmid clones that covers the region . Within this area, three expressed sequences were identified as candidates for the putative 5q13.3 tumor suppressor gene involved in the pathogenesis of HCL .

Genomics, 1999 Sep 1, 60(2), 111 - 20
Testis-specific murine centrin, Cetn1: genomic characterization and evidence for retroposition of a gene encoding a centrosome protein; Hart PE et al.; Centrin is a centrosome component in species from yeast to humans . Here, the mouse centrin 1 gene (Cetn1) is analyzed with respect to its genomic structure, chromosome localization, tissue-specific expression, and phylogenetic relationship to the other mouse centrin genes and their human orthologs . Cetn1 is an intronless gene located on chromosome 18A2 that encodes a 172-amino-acid protein with a predicted molecular mass of 19,696 Da (pI 4.61) and all of the structural features common to centrin . Cetn1 possesses the sequence features of an expressed retroposon: the gene lacks introns, the open reading frame is not interrupted by stop codons, and the coding region is flanked by a pair of direct repeats . Reverse transcriptase-polymerase chain reaction and Northern blot analysis demonstrate that Cetn1 expression is limited exclusively to the testis in adult male mice . Cetn1 expression is first seen in the neonatal testis at 14 days postpartum, reaching adult levels by day 17 . These observations provide new insight into the regulation, function, and evolutionary history of centrin in higher eukaryotes .

Arch Virol, 1999, 144(8), 1653 - 7
The C-terminal region of the P3 structural protein of rice dwarf phytoreovirus is important for P3-P3 interaction; Uyeda S et al.; Using a random peptide library designed for the yeast two-hybrid system, we identified a peptide that binds strongly to the P3 structural protein of rice dwarf phytoreovirus (RDV) . The amino acid sequence of the peptide showed a high homology to the C-terminal region of P3 . C-terminally truncated P3 lost its ability to interact with authentic P3 . Our observations suggest that the C-terminal region of P3 is important for the P3-P3 interaction, which forms the core shell structure of RDV.

Genes Dev, 1999 Sep 1, 13(17), 2301 - 14
A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila; Prokopenko SN et al.; Cytokinesis ensures the successful completion of the cell cycle and distribution of chromosomes, organelles, and cytoplasm between daughter cells . It is accomplished by formation and constriction of an actomyosin contractile ring that drives the progression of a cleavage furrow . Microinjection experiments and in vitro transfection assays have suggested a requirement for small GTPases of the Rho family in cytokinesis . Yet, the identity of proteins regulating Rho signaling pathways during cytokinesis remains unknown . Here we show that in Drosophila, Pebble (Pbl), a putative exchange factor for Rho GTPases (RhoGEF), is required for the formation of the contractile ring and initiation of cytokinesis . The dynamics of Pbl expression and its distribution during mitosis, as well as structure-function analysis, indicate that it is a key regulatory component of the pathway . pbl interacts genetically with Rho1, but not with Rac1 or Cdc42, and Pbl and Rho1 proteins interact in vivo in yeast . Similar to mutations in pbl, loss of Rho1 or expression of a dominant-negative Rho1 blocks cytokinesis . Our results identify Pbl as a RhoGEF specifically required for cytokinesis and linked through Rho1 activity to the reorganization of the actin cytoskeleton at the cleavage furrow.

Genes Dev, 1999 Sep 1, 13(17), 2258 - 70
Synapsis and chiasma formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome core component that functions in chromosome segregation; Zetka MC et al.; Meiotic chromosomes are organized about a proteinaceous core that forms between replicated sister chromatids . We have isolated a Caenorhabditis elegans gene, him-3, which encodes a meiosis-specific component of chromosome cores with some similarity to the yeast lateral element protein Hop1p . Antibodies raised against HIM-3 localize the protein to condensing chromosomes in early prophase I and to the cores of both synapsed and desynapsed chromosomes . In RNA interference experiments, chromosomes appear to condense normally in the absence of detectable protein but fail to synapse and form chiasmata, indicating that HIM-3 is essential for these processes . Hypomorphs of him-3, although being synapsis proficient, show severe reductions in the frequency of crossing-over, demonstrating that HIM-3 has a role in establishing normal levels of interhomolog exchange . Him-3 mutants also show defects in meiotic chromosome segregation and the persistence of the protein at the chromosome core until the metaphase I-anaphase I transition suggests that HIM-3 may play a role in sister chromatid cohesion . The analysis of him-3 provides the first functional description of a chromosome core component in a multicellular organism and suggests that a mechanistic link exists between the early meiotic events of synapsis and recombination, and later events such as segregation.

Cancer Genet Cytogenet, 1999 Sep, 113(2), 183 - 7
Deletion 7q in uterine leiomyoma: fluorescence in situ hybridization characterization on primary cytogenetic preparations; Vanni R et al.; Deletions of the long arm of chromosome 7 constitute one of the most common clonal chromosomal changes associated with uterine leiomyoma cells . Recently, the molecular cytogenetic refinement of 7q deletions in two myoma-derived cell lines, with the use of a panel of 39 ordered 7q DNA probes corresponding to 87 genetic markers, showed results in line with those obtained by loss of heterozygosity (LOH) analysis . Referring to this panel, we extended fluorescence in situ hybridization (FISH) analysis on primary cytogenetic preparations from three myomas with del(7q), thereby avoiding cell passages . This was fundamental in the maintenance of cells with del(7q) in the two cases showing mosaicism (i.e., the presence of an extra normal clone), which are prone to lose the abnormal clone in the very early passages . The data obtained, together with previously published findings on the two leiomyoma-derived cell lines, indicated a commonly deleted region of 11 cM . If the fact that the presence of normal cells may interfere with LOH analysis is taken into account, the FISH approach seems to be a reliable complementing tool for refining the deletion and analyzing the smallest overlapping region in cases with both normal and del(7q) cells.

Endocrine, 1999 Jun, 10(3), 281 - 9
Ligand-free RAR can interact with the RNA polymerase II subunit hsRPB7 and repress transcription; Shen XQ et al.; Upon binding retinoic acid (RA), the retinoic acid receptors (RARs) are able to positively and negatively regulate transcription . It has been shown that the DNA-binding domain and carboxy terminus of RARs are necessary for the ligand-dependent ability of the receptor to repress AP-1 transcriptional activity . A fusion of these two regions, shown to constitutively inhibit AP-1 activity, was used in a yeast two-hybrid screen to identify a novel hRARalpha-interacting protein . This protein, hsRPB7, a subunit of RNA polymerase II, interacts with hRARalpha in the absence of RA and addition of RA disrupts the interaction . Truncation analysis indicates that hsRPB7 specifically interacts with the hRARalpha DNA-binding domain . This interaction appears to compromise transcription, since overexpressed hRARalpha, in the absence of RA, is able to repress the activity of several RNA polymerase II-dependent activators, including AP-1 and the glucocorticoid receptor . This repression is relieved by transfected hsRPB7, strongly suggesting that ligand-free hRARalpha can block AP-1 activity by sequestering hsRPB7 . The repression is dependent on the integrity of the hRARalpha DBD, since a mutation within the DBD blocks both the hRARalpha-hsRPB7 interaction and ligand-free hRARalpha repression of AP-1 . These results provide evidence that non-liganded hRARalpha can regulate transcription by directly interacting with RNA polymerase II, and thus suggest a novel pathway by which hRARalpha can cross-talk with AP-1 and perhaps other families of transcriptional activators.

Plant Cell Physiol, 1999 Jun, 40(6), 624 - 39
Regional insertional mutagenesis of specific genes on the CIC5F11/CIC2B9 locus of Arabidopsis thaliana chromosome 5 using the Ac/Ds transposon in combination with the cDNA scanning method; Seki M et al.; We have recently developed a novel cDNA selection method (the cDNA scanning method) to select cDNAs for expressed genes in specific regions of the genome {Hayashida et al . (1995) Gene 165: 155, Seki et al . (1997) Plant J . 12: 481} . The gene Ds is known to transpose mainly in its neighborhood . By combining the cDNA scanning method with this trait of Ds, we started functional analysis of region-specific expressed genes on the Arabidopsis thaliana genome . DNA fragments of yeast artificial chromosome (YAC) clones CIC5F11 and CIC2B9 on A . thaliana chromosome 5 were used for the selection of region-specific cDNAs . In total, 50 and 68 cDNA clones were selected from CIC5F11 and CIC2B9, respectively . In parallel, we transposed Ds from a donor T-DNA line, which was mapped on the CIC5F11/CIC2B9 locus of chromosome 5, and obtained Ds-transposed lines . To isolate Ds insertion mutants in the 10 specific genes identified by the cDNA scanning method, we carried out PCR-based screening of 100 Ds-transposed lines and found that 2 lines contain Ds mutations in the genes isolated . We also isolated Ds-flanking genomic DNAs by thermal asymmetric interlaced PCR (TAIL-PCR) in 153 Ds transposon-tagged lines . Southern blot analysis showed that 14% of the lines contained the transposed Ds in the CIC5F11/2B9 region . This suggests that this Ac/Ds transposon system is effective for region-specific insertional mutagenesis.

J Biotechnol, 1999 May 28, 71(1-3), 245 - 9
Characterization of cell population growth by cell cycle parameters; Cazzador L; The quantitative description of the relationships between global properties, defined at the cellular population level, and individual properties, defined at the single cell level, is considered in this communication along with the analysis of some segregated models of yeast and hybridoma cell cultures.

Biochim Biophys Acta, 1999 Aug 4, 1412(3), 282 - 7
Primary structure and characterisation of a 64 kDa NADH dehydrogenase from the inner membrane of Neurospora crassa mitochondria; Melo AM et al.; A cDNA clone encoding a mitochondrial NADH dehydrogenase from Neurospora crassa was sequenced . The total DNA sequence encompasses 2570 base pairs and contains an open reading frame of 2019 base pairs coding for a precursor polypeptide of 673 amino acid residues . The protein is encoded by a single-copy gene located to the right side of the centromere in linkage group IV of the fungal genome . The N-terminus of the precursor protein has characteristics of a mitochondrial targeting pre-sequence . The protein displays homology with mitochondrial NADH dehydrogenases from yeast . In contrast to these polypeptides, however, analysis of its primary structure revealed that it contains a well-conserved calcium-binding domain . Rabbit antiserum against the protein expressed in an heterologous system recognises a mitochondrial protein of N . crassa with an apparent molecular mass of 64 kDa . Analysis of the fungal mitochondria by swelling, digitonin fractionation and alkaline treatment indicate that the protein is located in the inner membrane of the organelles, possibly facing the matrix side.

Plant Physiol, 1999 Sep, 121(1), 291 - 300
The relationship between ethylene binding and dominant insensitivity conferred by mutant forms of the ETR1 ethylene receptor; Hall AE et al.; Ethylene responses in Arabidopsis are mediated by a small family of receptors, including the ETR1 gene product . Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity . To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast . The etr1-1 and etr1-4 mutations completely eliminated ethylene binding, while the etr1-3 mutation severely reduced binding . Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants . By contrast, the etr1-2 mutation did not disrupt ethylene binding in yeast . These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor . Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors.

J Virol, 1999 Oct, 73(10), 8884 - 9
Walleye dermal sarcoma virus: OrfA N-terminal end inhibits the activity of a reporter gene directed by eukaryotic promoters and has a negative effect on the growth of fish and mammalian cells; Zhang Z et al.; Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a skin tumor termed walleye dermal sarcoma, which develops and regresses on a seasonal basis . The WDSV genome contains three short open reading frames designated orfA, orfB, and orfC in addition to the viral structural genes, gag, pol, and env . orfA and orfB transcripts are detected in tumors by reverse transcription-PCR . Recently, OrfA, whose amino acid sequence is similar to that of cyclins A and D, has been shown to complement a cyclin-deficient yeast strain . We report that expression of the accessory gene orfA inhibited nonspecifically the activity of a reporter gene directed by various eukaryotic promoters . In addition, stable transfection with the wild-type orfA generated substantially fewer G418-resistant colonies in both fish and mammalian cells than the parent vector . An orfA mutant expressing only the first N-terminal 49 residues of the full-length protein had the same negative effect on the activity of the reporter gene and on the number of stably transfected colonies as the full-length OrfA . Thus, OrfA inhibits cell growth and/or causes cell death, and the first 49 N-terminal residues of this protein are sufficient to cause these negative effects.

J Virol, 1999 Oct, 73(10), 8384 - 92
Casein kinase 2-mediated phosphorylation of respiratory syncytial virus phosphoprotein P is essential for the transcription elongation activity of the viral polymerase; phosphorylation by casein kinase 1 occurs mainly at Ser(215) and is without effect; Dupuy LC et al.; The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser(232) in the P proteins of human, bovine, and ovine strains of respiratory syncytial virus (RSV) . Enzymatic removal of this phosphate group from the P protein instantly halted transcription elongation in vitro . Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no full-length transcripts . A recombinant P protein in which Ser(232) was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function . Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser(215) . In vitro, P protein could be phosphorylated by purified CK1 at Ser(215) but this phosphorylation did not result in transcriptionally active P protein . A triple mutant P protein in which Ser(215), Ser(232), and Ser(237) were all mutated to Ala was completely defective in phosphorylation in vitro as well as ex vivo . The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex vivo . Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase.

Biosystems, 1999 Aug, 51(2), 95 - 100
Specific amino acid content and codon usage account for the existence of overlapping ORFS; Boldogkoi Z et al.; Here we present a novel hypothesis for the origin of overlapping open reading frames (O-ORFs) observed in the 'non-coding frames' of several genes of yeast chromosome II . By computer analysis it was found that the specific amino acid content and base distribution pattern at certain genomic locations and the presence of O-ORFs were related . This observation prompt us to conclude that these O-ORFs are mere statistical curiosities without any biological function, which is in contrast to the hypotheses proposed by other authors.

J Lab Clin Med, 1999 Sep, 134(3), 275 - 82
Despite structural similarities between gp91phox and FRE1, flavocytochrome b558 does not mediate iron uptake by myeloid cells; DeLeo FR et al.; Superoxide (O2-) generated by the phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is dependent on electron transfer by flavocytochrome b558 (flavocytochrome b), a transmembrane heterodimer that forms the redox center of the oxidase at the plasma or phagosomal membrane . The larger of its two subunits, gp91phox, is homologous to the yeast iron reductase subunit FRE1, and these two proteins share many structural and functional characteristics . Because FRE1 is required for iron uptake in yeast, we hypothesized that flavocytochrome b might serve a similar function in human phagocytes and thus provide a mechanism for the transferrin-independent iron acquisition observed in myeloid cells . To determine whether flavocytochrome b was required for iron uptake, we compared iron acquisition by polymorphonuclear neutrophils (PMNs) or Epstein-Barr virus (EBV)-transformed B lymphocytes derived from individuals with X-linked chronic granulomatous disease (CGD) with iron acquisition by normal cells . Our results indicate that all cells acquired iron to the same extent and that uptake could be significantly enhanced in the presence of the trivalent metal gallium . The gallium enhancement of iron uptake observed in PMNs or in EBV-transformed B lymphocytes derived from healthy individuals was mirrored by those derived from individuals deficient in flavocytochrome b . Furthermore, both normal and CGD-derived EBV-transformed B lymphocytes had similar iron reductase activity, suggesting that flavocytochrome b is not a biologically significant iron reductase . In contrast to previously suggested hypotheses, these results show conclusively that flavocytochrome b is not necessary for cellular iron acquisition, despite structural and functional similarities between yeast iron reductases and flavocytochrome b.

J Biochem Biophys Methods, 1999 Jul 28, 40(1-2), 39 - 44
Stabilization of alginate beads using radiation polymerized polyacrylamide; Gupte A et al.; A technique has been described for the stabilization of calcium alginate beads using radiation polymerized acrylamide . The technique involved dropping a mixture containing the cells (20%), sodium alginate (2%), acrylamide (2.5%) and N-N'-methylene-bis-acrylamide (0.1%) through a syringe needle into cold (-75 degrees C) toluene . The frozen beads obtained were exposed to 60Co gamma-rays (0.5 KGy) and were then thawed in 0.1 M CaCl2 solution . Unlike the calcium alginate beads the conjugate beads were not found to be dissolved when incubated in 3% trisodium citrate solution . Stabilized beads containing entrapped yeast cells could be reused for over 15 batches for the inversion of sucrose without loss in activity or chemical integrity of the beads.

FEBS Lett, 1999 Sep 17, 458(2), 197 - 203
Structure of the human VIPR2 gene for vasoactive intestinal peptide receptor type 2; Lutz EM et al.; The VPAC(2) (vasoactive intestinal peptide (VIP)(2)) receptor is a seven-transmembrane spanning G protein-coupled receptor which responds similarly to VIP and pituitary adenylate cyclase activating polypeptide (PACAP) in stimulating cAMP production . Recently, we reported the localisation of the human VPAC(2) receptor gene (VIPR2) to chromosome 7q36.3 (Mackay, M . et al . (1996) Genomics 37, 345-353) . Here, we describe the characterisation of the VIPR2 gene structure and promoter region . The VIPR2 gene is encoded by 13 exons, the initiator codon of the 438 amino acid open reading frame is located in exon 1 and the termination signal and a poly-adenylation signal sequence are located in exon 13 . The 5' untranslated region extends 187 bp upstream of the initiator codon and is extremely GC-rich (80%) . The poly-adenylation signal is located 2416 bp downstream of the stop codon . Intron sizes range from 68 bp (intron 11) to 45 kb (intron 4) and the human gene spans 117 kb.

J Biol Chem, 1999 Sep 17, 274(38), 26878 - 84
Characterization and chromosomal localization of USP3, a novel human ubiquitin-specific protease; Sloper-Mould KE et al.; Conjugation to the small eukaryotic protein ubiquitin can functionally modify or target proteins for degradation by the proteasome . Removal of the ubiquitin modification, or deubiquitination, is performed by ubiquitin-specific proteases and is an important mechanism regulating this pathway . Here we describe a novel human ubiquitin-specific protease, USP3, initially identified as a partial cDNA clone similar to one of two highly conserved sequence regions common to all ubiquitin-specific proteases . We have isolated a complete USP3 cDNA clone containing both of these conserved sequence regions . The USP3 gene appears to be single copy and maps to human chromosome 15q22.3 . A USP3 probe detects two mRNA transcripts, one of which corresponds in length to the cDNA . Both are expressed at low levels in all tissues examined, with highest expression in pancreas . The USP3 protein is a functional ubiquitin-specific protease in vitro, and is able to inhibit ubiquitin-dependent degradation of both an N-end Rule substrate and abnormal endogenous proteins in yeast . USP3 is also only the second known ubiquitin-specific protease capable of efficiently cleaving a ubiquitin-proline bond.

J Biol Chem, 1999 Sep 17, 274(38), 26810 - 4
Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein; Sambamurti K et al.; The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase . beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta . Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha . In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases . To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC) . PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha . A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+) . When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC . These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells . The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.

J Biol Chem, 1999 Sep 17, 274(38), 26647 - 53
Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase delta; Zhang P et al.; The formation of a complex between DNA polymerase delta (pol delta) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork . In this study, the ability of the p125 catalytic subunit of DNA polymerase delta to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods . p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells . Because pol delta is a multimeric protein, this interaction could be indirect . Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA . To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means . p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)) . An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system . Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA . The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol delta and provides evidence for a site on pol delta that is involved in the protein-protein interactions between PCNA and pol delta . This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins . These studies provide an unequivocal demonstration that the p125 subunit of pol delta interacts with PCNA.

J Infect Dis, 1999 Oct, 180(4), 1064 - 71
The safety and efficacy of granulocyte-macrophage colony-stimulating factor (Sargramostim) added to indinavir- or ritonavir-based antiretroviral therapy: a randomized double-blind, placebo-controlled trial; Skowron G et al.; Sargramostim is a yeast-derived, recombinant human granulocyte-macrophage colony-stimulating factor with therapeutic potential in human immunodeficiency virus (HIV) infection . Its safety and activity when used in combination with protease inhibitors were evaluated in a randomized, double-blind trial in which 20 HIV-infected subjects on stable antiretroviral regimens, including indinavir or ritonavir, received sargramostim or placebo 3 times a week for 8 weeks . Analysis of HIV virus load excluded any 0 . 5 log10 increase due to sargramostim (95% confidence interval, -0.68 to 0.44) . Sargramostim was well tolerated, and inflammatory cytokines and surrogate markers of disease progression, such as serum levels of interleukin-10 and soluble tumor necrosis factor receptors types Iota and IotaIota, remained stable in subjects receiving sargramostim . Sargramostim treatment was associated with a trend toward decreased HIV RNA (>0.5 log10) and increased CD4+ cell count (>30%) . These results became statistically significant only when subjects with baseline virus loads within the limits of detection or baseline CD4 cell count >50 were analyzed . No difference in indinavir pharmacokinetics was observed before or after sargramostim therapy.

J Cell Biol, 1999 Sep 6, 146(5), 1005 - 18
mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila; Cullen CF et al.; We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant . Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells . Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected . The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast . Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro . In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase . The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

J Cell Biol, 1999 Sep 6, 146(5), 993 - 1004
Gamma-synergin: an EH domain-containing protein that interacts with gamma-adaptin; Page LJ et al.; The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles . To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the gamma-adaptin subunit of the AP-1 complex as bait . Two novel, ubiquitously expressed proteins were found: p34, which interacts with both gamma-adaptin and alpha-adaptin, and gamma-synergin, an alternatively spliced protein with an apparent molecular mass of approximately 110-190 kD, which only interacts with gamma-adaptin . gamma-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles . It binds directly to the ear domain of gamma-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the gamma-adaptin binding site . In cells expressing alpha-adaptin with the gamma-adaptin ear, a construct that goes mainly to the plasma membrane, much of the gamma-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there . The presence of an EH domain suggests that gamma-synergin links the AP-1 complex to another protein or proteins.

J Cell Biol, 1999 Sep 6, 146(5), 941 - 54
Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC; Chan GK et al.; Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1 . hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T . Chan, C.A . Cooke, W.C . Earnshaw, and T.J . Yen . 1998 . Chromosoma . 107:386-396) . In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C . Lengauer, J . Yu, G.J . Riggins, J.K . Willson, S.D . Markowitz, K.W . Kinzler, and B . Vogelstein . 1998 . Nature . 392:300-303; Taylor, S.S., and F . McKeon . 1997 . Cell . 89:727-735) . We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint . hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis . Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase . We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E . hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis . hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells . Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells . The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity.

J Cell Biol, 1999 Sep 6, 146(5), 917 - 28
The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase; Kickhoefer VA et al.; Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size . The 100-kD major vault protein (MVP) accounts for >70% of the particle mass . We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis . Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP) . PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage . The catalytic domain of p193 was expressed and purified from bacterial extracts . Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP . Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity . Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP . Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s) . A portion of p193 is nuclear and localizes to the mitotic spindle.

Blood, 1999 Sep 15, 94(6), 2039 - 47
The human platelet alphaIIb gene is not closely linked to its integrin partner beta3; Thornton MA et al.; alphaIIbb3 integrin is a heterodimeric receptor facilitating platelet aggregation . Both genes are on chromosome 17q21.32 . Intergenic distance between them has been reported to be 125 to 260 kilobasepairs (kb) by pulsed-field gel electrophoresis (PFGE) genomic analysis, suggesting that they may be regulated coordinately during megakaryopoiesis . In contrast, other studies suggest these genes are greater than 2.0 megabasepairs (mb) apart . Because of the potential biological implications of having these two megakaryocytic-specific genes contiguous, we attempted to resolve this discrepancy . Taking advantage of large kindreds with mutations in either alphaIIb or beta3, we have developed a genetic linkage map between the thyroid receptor hormone-1 gene (THRA1) and beta3 as follows: cen-THRA1-BRCA1-D17S579/alphaIIb-beta3-qte r, with a distance of 1.3 centiMorgans (cM) between alphaIIb and beta3 and the two genes being oriented in the same direction . PFGE genomic and YAC clone analysis showed that the beta3 gene is distal and >/=365 kb upstream of alphaIIb . Additional restriction mapping shows alphaIIb is linked to the erythrocyte band 3 (EPB3) gene, and beta3 to the homeobox HOX2b gene . Analysis of alphaIIb(+)-BAC and P1 clones confirm that the EPB3 gene is approximately 110 kb downstream of the alphaIIb gene . Sequencing the region surrounding the human alphaIIb locus showed the Granulin gene approximately 18 kb downstream to alphaIIb, and the KIAA0553 gene approximately 5.7 kb upstream . This organization is conserved in the murine sequence . These studies show that alphaIIb and beta3 are not closely linked, with alphaIIb flanked by nonmegakaryocytic genes, and imply that they are unlikely to share common regulatory domains during megakaryopoiesis.

J Immunol, 1999 Sep 15, 163(6), 3295 - 303
An activation-responsive element in single C motif-1/lymphotactin promoter is a site of constitutive and inducible DNA-protein interactions involving nuclear factor of activated T cell; Yoshida T et al.; Single C motif-1 (SCM-1)/lymphotactin is a C-type chemokine whose expression is activation dependent, cyclosporin A sensitive and restricted to CD8+ T cells, double-negative thymocytes, gammadelta-type T cells, and NK cells . In humans, there are two highly homologous genes encoding SCM-1alpha and SCM-1beta . Here we examined the regulatory mechanism of the SCM-1 genes . The luciferase reporter gene under the control of the 5' flanking region of 0.7 kb was strongly induced upon activation with anti-CD3 or PHA plus PMA only in SCM-1-producer T cell lines through a cyclosporin A-sensitive mechanism . An element termed E1 located at -108 to -95 nt relative to the major transcription start site was found to be critical for the promoter activity . In electrophoretic mobility shift assays using the E1 oligonucleotide as probe, nuclear extracts from unstimulated T and B cell lines formed a constitutive complex termed complex I, while nuclear extracts from stimulated SCM-1-producer T cell lines formed a higher mobility complex termed complex II with a concomitant decrease in complex I . The shift from complex I to complex II seen only in SCM-1-producer T cell lines upon activation was completely suppressed by cyclosporin A . Both complexes were critically dependent on the NF-AT core sequence TTTCC in the E1 element and were partially supershifted by anti-NF-ATp . One-hybrid assays in yeast isolated NF-ATp as an E1 binding protein, and transfection of NF-ATp into T and B cell lines strongly enhanced the activation-dependent SCM-1 promoter activity . Collectively, a unique mechanism involving NF-ATp appears to regulate the cell type-specific and activation-dependent expression of the SCM-1 genes.

Mutat Res, 1999 Jul 21, 444(1), 49 - 60
Illumination of human keratinocytes in the presence of the sunscreen ingredient Padimate-O and through an SPF-15 sunscreen reduces direct photodamage to DNA but increases strand breaks; Gulston M et al.; On illumination with simulated sunlight, the UVB-absorbing sunscreen chemical 2-ethylhexyl-4-dimethylaminobenzoate (Padimate-O) generates excited species which inflict non-ligatable strand breaks on DNA in vitro and it also becomes mutagenic to yeast in vivo . Padimate-O is known to penetrate human skin but its effects on human cells are not clear . Here, we first simulate the sunlight which penetrates human skin and use it to illuminate human keratinocytes . The DNA damage observed in terms of UV-endonuclease-sensitive sites (ESS) and direct strand breaks per kilobase (kb) of DNA per joule per square metre agrees well with that predicted from action spectra based on monochromatic light . Using plasmid DNA in vitro, we find a very similar pattern of results . Next, we simulate the spectrum that results when the incident light is first attenuated by a film of sunscreen (SPF-15; 2 mg/cm(2)) containing benzophenone-3 (a UVA absorber), octyl methoxycinnamate (a UVB absorber), and Padimate-O . If the sunscreen is not in contact with keratinocytes it reduces direct DNA damage from sunlight (ESS) . However, any Padimate-O in contact with the cells substantially increases indirect damage (strand breaks) even though the film of sunscreen reduces direct photodamage . We estimate that applying an SPF-15 sunscreen which contains Padimate-O to human skin followed by exposure to only 5 minimum erythemal doses (MED) of sunlight could, while suppressing the formation of ESS, increase strand breaks in cells under the epidermis by at least 75-fold compared to exposure to 1 MED in the absence of sunscreen.

Biochem J, 1999 Sep 15, 342 Pt 3, 729 - 35
Calcium-dependent properties of CIB binding to the integrin alphaIIb cytoplasmic domain and translocation to the platelet cytoskeleton; Shock DD et al.; The alphaIIbbeta3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation . Fibrinogen binding to alphaIIbbeta3 subsequently induces a cascade of intracellular signalling events . The molecular mechanisms of this bi-directional alphaIIbbeta3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains . We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the alphaIIb cytoplasmic domain in the yeast two-hybrid system . Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed . In addition, isothermal titration calorimetry indicated that CIB binds to an alphaIIb cytoplasmic-domain peptide in a Ca(2+)-dependent manner, with moderate affinity (K(d), 700 nM) and 1:1 stoichiometry . In aggregated platelets, endogenous CIB and alphaIIbbeta3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by alphaIIbbeta3 . Thus CIB may contribute to integrin-related functions by mechanisms involving Ca(2+)-modulated binding to the alphaIIb cytoplasmic domain and changes in intracellular distribution.

Mol Biochem Parasitol, 1999 Jul 30, 102(1), 103 - 15
Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins; Ismaili N et al.; The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs . A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs . The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes . Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65) . In both procyclic and bloodstream forms of T . brucei, TSR1 was found to localize in the nucleus . In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing . Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system . These and other observations suggest that TSR1 may be involved in trans-splicing in T . brucei.

Mol Pathol, 1999 Apr, 52(2), 84 - 91
Nuclear localisation of NOVH protein: a potential role for NOV in the regulation of gene expression; Perbal B; AIMS: To identify the NOV protein detected by immunofluorescence in the nucleus of human cancer cell lines to establish whether targeting to the nucleus reflects dual paracrine and intracrine biological functions of NOV, as has been reported previously for several signalling peptides and proteins . METHODS: Nuclear and cytoplasmic fractions were prepared from 143 and HeLa cells in which nuclear NOV protein was detected . Western blotting analysis of NOV proteins in both types of fractions was performed using two NOV specific antibodies . Confocal microscopy was used to visualise the nuclear NOV protein in HeLa and 143 cells . A yeast two hybrid screening system was used to isolate cDNAs encoding proteins able to interact with the human NOV protein . RESULTS: A 31/32 kDa doublet of NOV protein was identified in the nuclear fraction of 143 and HeLa cells . Because the antibodies were directed against the C-terminus of NOV, the 31/32 kDa NOV isoform is probably truncated at the N-terminus and might correspond to the secreted 32 kDa NOV isoform detected in cell culture medium . Confocal microscopy indicated that in addition to the cytoplasmic NOV protein already identified, a nuclear NOV protein was present in both the nucleoplasm and nucleoli of Hela and 143 cells . Screening of cDNA libraries prepared from HeLa cells, Epstein-Barr virus transformed lymphocytes, and normal human brain showed that the NOV protein interacts with the rpb7 subunit of RNA polymerase in a yeast two hybrid system . CONCLUSIONS: The NOV protein detected in the nucleus of 143 and HeLa cells is probably an N-terminus truncated isoform of the secreted 48 kDa NOV protein . A growing body of evidence suggests that novH expression is closely associated with differentiation in normal human tissues and that the nov gene encodes a signalling protein that belongs to an emerging family of cell growth regulators . The nuclear localisation of a NOV isoform potentially provides an additional degree of signalling specificity . The interaction of the NOV protein and the rpb7 subunit of RNA polymerase II in the two hybrid system suggests that NOV might be involved in regulating gene expression at the transcriptional level . As has already been suggested for several other nuclearly located cytokines, the NOV protein does not contain a typical nuclear localisation signal . Therefore, it is possible that it combines with either a receptor or a chaperone during its translocation . Disruption of the balance between the secreted and nuclear NOV isoforms might affect the putative autocrine and paracrine functions of NOV and might be of considerable importance in the development of cancers in which the expression of novH has been shown to be impaired.

J Biol Chem, 1999 Sep 10, 274(37), 26529 - 36
Using a biochemical approach to identify the primary dimerization regions in human DNA topoisomerase IIalpha; Bjergbaek L et al.; Eukaryotic topoisomerase II is a nuclear enzyme essential for DNA metabolism and chromosome dynamics . The enzyme has a dimeric structure, and subunit dimerization is vital to the cellular functions and activities of the enzyme . Two biochemical approaches based on metal ion affinity chromatography and immunoprecipitation have been carried out to map the dimerization region(s) in human topoisomerase IIalpha . The results demonstrate that two regions spanning amino acids 1053-1069 and 1124-1143 are both essential for dimerization . The regions correspond to the interaction domains revealed in yeast topoisomerase II after crystallization of a central fragment of this enzyme, indicating that the overall C-terminal dimerization structure of eukaryotic topoisomerase II is conserved from yeast to human . Furthermore, linker insertion analysis has demonstrated that the two dimerization regions are located in a highly flexible part of the enzyme . Topoisomerase IIalpha mutant enzymes unable to dimerize via the C-terminal primary dimerization regions due to lack of one of the defined dimerization regions can still be forced to dimerize if DNA and an ATP analog are added to the reaction mixture . The result indicates that secondary interactions occur by ATP analog-mediated clamp closing when the subunits are brought together on DNA.

J Biol Chem, 1999 Sep 10, 274(37), 25995 - 6002
Caspase inhibition by baculovirus P35 requires interaction between the reactive site loop and the beta-sheet core; Zoog SJ et al.; Baculovirus P35 is a universal substrate-inhibitor of the death caspases . Stoichiometric inhibition by P35 is correlated with cleavage of its reactive site loop (RSL) and formation of a stable P35.caspase complex through a novel but undefined mechanism . The P35 crystal structure predicts that the RSL associates with the beta-sheet core of P35 positioning the caspase cleavage site at the loop's apex . Here we demonstrate that proper interaction between the RSL and the beta-sheet core is critical for caspase inhibition, but not cleavage . Disruption of RSL interaction with the beta-sheet by substituting hydrophobic residues of the RSL's transverse helix alpha1 with destabilizing charged residues caused loss of caspase inhibition, without affecting P35 cleavage . Restabilization of the helix/sheet interaction by charge compensation from within the beta-sheet partially restored anti-caspase potency . Mutational effects on P35 helix/sheet interactions were confirmed by measuring intermolecular helix/sheet association with the yeast two-hybrid system . Moreover, the identification of P35 oligomers in baculovirus-infected cells suggested that similar P35 interactions occur in vivo . These findings indicate that P35's anti-caspase potency depends on a distinct conformation of the RSL which is required for events that promote stable, post-cleavage interactions and inhibition of the target caspase.

FEBS Lett, 1999 Aug 27, 457(2), 255 - 61
Involvement of branched-chain amino acid aminotransferase (Bcat1/Eca39) in apoptosis; Eden A et al.; The branched-chain amino acid aminotransferase, Bcat1/Eca39, catalyzes the first step of branched-chain amino acid catabolism . Bcat1/Eca39 was originally isolated from a c-myc-induced tumor and was proven to be a direct target for c-Myc regulation . The gene is highly conserved in evolution and disruption of its yeast homolog affects cell growth . To assess the role of Bcat1/Eca39 in mammalian cells, we overexpressed Bcat1/Eca39 in murine cells and studied effects on cell growth . Overexpression of Bcat1/Eca39 had no apparent effect on the proliferation of cells grown with high serum concentrations, but under serum deprivation conditions, led to a decrease in cell viability . Cell death under these conditions displayed apoptotic features . The branched-chain keto acid, alpha-ketoisocaproate, a metabolite of leucine catabolism produced by BCAT1/ECA39, was previously found to inhibit cell growth . We show that alpha-ketoisocaproate can induce rapid apoptotic cell death . This observation suggests that the growth inhibitory effect of BCAT1/ECA39 and its apoptosis promoting effect may be mediated by the levels of the products of BCAT1/ECA39 activity, namely, branched-chain keto acids.

Exp Cell Res, 1999 Sep 15, 251(2), 492 - 9
Normal human telomeres are not late replicating; Wright WE et al.; Telomeres in yeast are late replicating . Genes placed next to telomeres in yeast can be repressed (telomere positional effects), leading to the hypothesis that telomeres may be heterochromatic and may control the expression of subtelomeric genes . In addition, yeast telomeres are processed to have a transient long overhang at the end of S phase . The applicability of the yeast data to human biology was examined by determining the timing of telomere replication and processing in normal human diploid fibroblasts . Telomeres were purified from synchronized cells that had been labeled with 5-bromodeoxyuridine (BrdU) at hourly intervals, and the fraction of labeled telomeres was analyzed by retrieval with anti-BrdU antibodies . We determined that normal human telomeres replicate throughout S phase rather than being very late replicating . Furthermore, the overall timing of replication was unaffected by telomere length in young versus old cells or cells whose telomeres had been elongated following transfection with the catalytic subunit of telomerase . Finally, the asymmetry in the length of the G-rich overhang in daughter telomeres produced by leading versus lagging strand synthesis was shown to be established within 1 h of telomere replication, indicating there is no significant delay between synthesis and the processing events that contribute to the establishment of asymmetric overhangs . Therefore, the timings of replication and processing of human telomeres are very different from those of yeast .

Biochemistry, 1999 Aug 31, 38(35), 11261 - 70
Protein-protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain; Wu XQ et al.; Numerous functions have been proposed for the testis brain RNA-binding protein (TB-RBP) and its human homologue, Translin, ranging from mRNA transport and translational regulation to DNA rearrangement and repair . To gain insight into the likely functions of this 26 kDa protein, immunoprecipitation was used to identify proteins that interact with TB-RBP in mouse cytosolic extracts . Three proteins, the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin, and Trax, were specifically immunoprecipitated with an affinity-purified antibody to recombinant mouse TB-RBP . In vitro binding assays with recombinant proteins and EM immunocytochemistry confirm that TB-RBP interacts with the TER ATPase in vitro and in vivo . Confocal microscopy has demonstrated that TB-RBP colocalizes with actin in the cytoplasm of male germ cells . The immunoprecipitation of Trax with TB-RBP confirms a published report demonstrating protein interactions between the two proteins in a yeast two-hybrid assay . These data support the hypothesis that TB-RBP serves as a link in attaching specific mRNAs to cytoskeletal structures and suggests an involvement for the ubiquitously expressed TER ATPase in intracellular and/or intercellular mRNA transport.

DNA Res, 1999 Jun 30, 6(3), 183 - 95
Structural analysis of Arabidopsis thaliana chromosome 5 . IX . Sequence features of the regions of 1,011,550 bp covered by seventeen P1 and TAC clones; Kaneko T et al.; In this series of projects sequencing the entire genome of Arabidopsis thaliana chromosome 5, non-redundant P1 and TAC clones have been sequenced according to the fine physical map, and as of May 7, 1999, the sequences of 16.2 Mb representing approximately 60% of chromosome 5 have been accumulated and released at our web site . In parallel, structural features of the sequenced regions have been analyzed by applying a variety of computer programs, and to date we have predicted a total of 2380 potential protein-coding genes in the 10,154,580 bp regions, which are covered by 142 P1 and TAC clones . In this paper, we newly analyzed the structural features of the 1,011,550 bp regions covered by additional 17 P1 and TAC clones, and predicted 298 protein-coding genes . The average density of the genes identified was 1 gene per 3394 bp . Introns were observed in 67% of the genes, and the average number per gene and the average length of the introns were 3.2 and 159 bp, respectively . The gene density became higher than the value estimated in the previously analyzed regions (1 gene per 4,267 bp), as the data in this paper were compiled based on a new standard of gene assignment including the computer-predicted hypothetical genes . The regions also contained 8 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program . The sequence data and information on the potential genes are available on the database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.

EMBO J, 1999 Sep 1, 18(17), 4856 - 64
A conformational switch at the 3' end of a plant virus RNA regulates viral replication; Olsthoorn RC et al.; 3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity . This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses . We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot . Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus . This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses . A low but specific interaction with yeast CCA-adding enzyme was found . The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa . The role of coat protein in this modulation and in genome activation is discussed.

Genes Cells, 1999 Jul, 4(7), 381 - 90
Competitive interaction of seven in absentia homolog-1A and Ca2+/calmodulin with the cytoplasmic tail of group 1 metabotropic glutamate receptors; Ishikawa K et al.; BACKGROUND: Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) are coupled to inositol trisphosphate/Ca2+ signaling via G proteins and play an important role in excitatory synaptic transmission . To explore the regulation of group 1 mGluR function, we applied the yeast two-hybrid system using the intracellular carboxy-terminal domain of group 1 mGluRs (group 1 ct-mGluRs) and attempted to identify novel protein-protein interactions of group 1 mGluRs . RESULTS: The two-hybrid screening revealed a specific interaction between group 1 ct-mGluRs and Siah-1A, the mammalian homolog of Drosophila seven in absentia which is involved in photoreceptor cell differentiation via the ubiquitin/proteasome-dependent mechanism . This interaction occurs within a homologous 27-28 amino acid stretch within group 1 ct-mGluRs and requires the latter two-thirds of Siah-1A . Following coexpression in COS-7 cells, myc-tagged Siah-1A was coimmunoprecipitated with the flag-tagged ct-mGluR1 by anti-flag antibody . Furthermore, in vitro binding revealed that Siah-1A and Ca2+/calmodulin (CaM) binding sites overlap, such that Siah-1A binding is competitively inhibited by CaM in a Ca2+-dependent manner . CONCLUSIONS: The results demonstrate a direct interaction between group 1 mGluRs and Siah-1A and suggest a novel modulatory mechanism mediated by a competitive interaction between Ca2+/CaM and Siah-1A.

Eur J Biochem, 1999 Aug, 263(3), 879 - 88
High prevalence of 2-mono- and 2,6-di-substituted manol-terminating sequences among O-glycans released from brain glycopeptides by reductive alkaline hydrolysis; Chai W et al.; Di- to heptasaccharides isolated from total nondialyzable brain glycopeptides after release by alkaline borohydride treatment have been subjected to mass spectrometric and nuclear magnetic resonance spectroscopic analyses supplemented by TLC-MS analyses of derived neoglycolipids . A family of Manol-terminating oligosaccharides has been revealed which includes novel sequences with a 2, 6-disubstituted Manol: In contrast to the Manol-terminating HNK-1 antigen-positive chains described previously that occur as a minor population {Yuen, C.-T., Chai, W., Loveless, R.W., Lawson, A.M., Margolis, R.U . & Feizi, T . (1997) J . Biol . Chem . 272, 8924-8931}, the above oligosaccharides are abundant . The ratio of these compounds to the classical N-acetylgalactosaminitol-terminating oligosaccharides is about 1 : 3 . Thus, there appears to be in higher eukaryotes a major alternative pathway related to the yeast-type protein O-mannosylation, the enzymatic basis and functional importance of which now require investigation.

Anim Genet, 1999 Aug, 30(4), 286 - 95
Catalase gene is associated with facial eczema disease resistance in sheep; Phua SH et al.; Facial eczema (FE) is a hepatogenous photosensitization disease of ruminant animals, particularly in sheep which vary widely in their susceptibility to the disease . The liver damage is caused by the mycotoxin, sporidesmin . There is evidence that the toxicity of sporidesmin is due to its ability to generate 'active oxygen' species . We evaluated the catalase gene, which encodes an enzyme with antioxidant functions, as a candidate for determining the susceptibility of sheep to the disease . Two microsatellite markers, OarSHP3 and OarSHP4, which flank the sheep catalase gene, were isolated from a Yeast Artificial Chromosome (YAC) clone . These markers mapped the catalase locus by linkage to ovine chromosome 15 . Eleven informative markers spaced throughout chromosome 15, inclusive of the catalase marker OarSHP4, gave no significant linkage with the disease traits when analysed in four outcross resource pedigrees . However, OarSHP3 and OarSHP4 allele frequencies showed significant differences between FE resistant and susceptible selection-lines . Comparison of sequences of catalase cDNAs from sheep of resistant and susceptible lines showed only two silent mutations . A single nucleotide polymorphisms (KP1) in exon 6 of the catalase gene also showed significant differences in allele frequencies between the selection lines . The lack of evidence for linkage in outcross pedigrees, but the significant association in the genetic lines, implies that catalase is involved in determining the susceptibility of sheep to facial eczema, and that the candidate gene's effect is probably recessive or minor.

Curr Genet, 1999 Jul, 35(6), 631 - 7
Polymorphism around cog extends into adjacent structural genes; Yeadon PJ et al.; The recombination hotspot cog overlaps a highly polymorphic 950-bp region of linkage group I in Neurospora crassa . The sequence of this region in the four strains, Lindegren 25a, Lindegren A, Emerson A and St . Lawrence 74A, each differs from the others by 1.4% or more . Comparison of the sequence of St . Lawrence 74A and Lindegren 25a each side of cog shows a high level of sequence heterology extending in both directions, including the coding sequences for his-3 and a putative gene lpl with homology to yeast lysophospholipase . The St . Lawrence 74A and Lindegren 25a sequences of his-3, centromere-proximal to cog, differ at 14 nucleotides, resulting in six amino-acid variations between the predicted protein sequences . In lpl, distal from cog, the sequences differ at 19 nucleotides leading to five amino-acid differences between the predicted proteins . Sequence heterology between St . Lawrence 74A and Lindegren 25a peaks either side of cog and then declines with distance . At the am locus on linkage group V, heterology is much less but peaks close to a weak recombination hotspot 5' of the coding sequence . Uneven distribution of polymorphism along chromosomes has been explained by a hitch-hiking hypothesis in which selection for advantageous mutations causes local fixation of unselected variation . We suggest that new mutations arising from errors in recombination also contribute to the uneven distribution of polymorphism.

Am J Trop Med Hyg, 1999 Mar, 60(3), 350 - 6
Testing the efficacy of a recombinant merozoite surface protein (MSP-1(19) of Plasmodium vivax in Saimiri boliviensis monkeys; Collins WE et al.; Saimiri boliviensis monkeys were immunized with the yeast-expressed recombinant protein yP2P30Pv200(19) . The antigen consisted of the C-terminus (amino acid Asn1622-Ser1729) of the merozoite surface protein 1 of the Plasmodium vivax Salvador I strain . Two universal T helper cell epitopes (P2 and P30) of tetanus toxin and six histidine residues for purification purposes were attached to the N- and C-termini, respectively . Four groups of five monkeys were given three immunizations at four-week intervals with either 250 microg of yP2P30Pv200(19) formulated with nonionic block copolymer P1005, 250 microg of antigen adsorbed to alum, 250 microg of antigen in phosphate-buffered saline (PBS), or PBS alone . Five weeks after the last immunization, each animal was inoculated with 100,000 parasitized erythrocytes of the Salvador I strain of P . vivax . Animals were splenectomized one week after challenge to increase parasite densities; after seven weeks of infection, animals were treated . Eighteen weeks later, the animals were rechallenged with the homologous parasite . Following the first challenge, three monkeys immunized with the antigen with P1005 were protected; no animals were protected from rechallenge . One monkey immunized with yP2P30Pv200(19) with alum was protected; no protection was seen after rechallenge . Two monkeys immunized with antigen alone were protected; none were protected from rechallenge . One control animal had a low parasite count following primary infection; none were protected against rechallenge . Adverse reactions were only observed with animals receiving P1005 . It is proposed that splenectomy of the monkeys prevented adequate assessment of the efficacy of this antigen . Identification of a monkey host that supports high density parasitemia without splenectomy appears needed before further testing of blood-stage vaccines against P . vivax.

J Biol Chem, 1999 Sep 3, 274(36), 25419 - 25
Mutations that increase acidity enhance the transcriptional activity of the glutamine-rich activation domain in stage-specific activator protein; Benuck ML et al.; Sea urchin stage-specific activator protein (SSAP) activates transcription of the late H1 gene at the mid-blastula stage of development . Its C-terminal 202 amino acids form a potent glycine/glutamine rich activation domain (GQ domain) that can transactivate reporter genes to levels 5-fold higher than VP16 in several mammalian cell lines . We observed that, unlike other glutamine-rich activation domains, the GQ domain activates transcription to moderate levels in yeast . We utilized this activity to screen in yeast for intragenic mutations that enhance or inhibit the transcriptional activity of the GQ domain . We identified 37 loss of function and 23 gain of function mutants . Most gain of function mutations increased the acidity of the domain . The most frequently isolated mutations conferred enhanced transcriptional activity when assayed in mammalian cells . These mutations also enhance the ability of SSAP to up-regulate the late H1 promoter in sea urchin embryos . We conclude that the GQ domain fundamentally differs from other glutamine-rich activators and may share some properties of acidic activators . The ability of acidity to enhance SSAP-mediated transcription may reflect a mechanism by which phosphorylation of SSAP activates late H1 gene transcription during embryogenesis.

J Biol Chem, 1999 Sep 3, 274(36), 25291 - 6
S100A12 is expressed exclusively by granulocytes and acts independently from MRP8 and MRP14; Vogl T et al.; Changes in cytosolic calcium concentrations regulate a wide variety of cellular processes, and calcium-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control . S100A12, a recently described member of the S100 protein family, has been shown to be coexpressed in granulocytes and monocytes together with two other S100 proteins, MRP8 (S100A8) and MRP14 (S100A9), and a functional relationship between these three S100 proteins has been suggested . Using Western blotting, calcium overlays, intracellular flow cytometry, and cytospin preparations, we demonstrate that S100A12 expression in leukocytes is specifically restricted to granulocytes and that S100A12 represents one of the major calcium-binding proteins in these cells . S100A12, MRP8, and MRP14 translocate simultaneously from the cytosol to cytoskeletal and membrane structures in a calcium-dependent manner . However, no evidence for direct protein-protein interactions of S100A12 with either MRP8 or MRP14 or the heterodimer was found by chemical cross-linking, density gradient centrifugation, mass spectrometric measurements, or yeast two hybrid detection . Thus, S100A12 acts individually during calcium-dependent signaling, independent of MRP8, MRP14, and the heterodimer MRP8/MRP14 . This granulocyte-specific signal transduction pathway may offer attractive targets for therapeutic intervention with exaggerated granulocyte activity in pathological states.

Int Immunol, 1999 Sep, 11(9), 1423 - 9
Prf, a novel Ets family protein that binds to the PU.1 binding motif, is specifically expressed in restricted stages of B cell development; Hashimoto S et al.; During the development of lymphocytes, expression of the Ig genes is strictly regulated in a tissue-specific manner and in a time-ordered fashion . We have previously shown that the PU.1 binding motif in the Igkappa 3' enhancer (kappaE3') and a novel Ets family protein other than PU.1 may be possibly involved in the control of V(kappa)-J(kappa) joining . In the attempt to isolate the novel Ets family protein, we have screened cDNA libraries with the yeast one-hybrid method and identified a new PU.1-related factor, Prf . This novel Ets family protein is shown to interact with the PU.1 binding sequences in various promoters and enhancers, including kappaE3' . It was found that expression of the prf gene is predominant in the B-lineage cells, with the exception of immature B cells . Since Prf does not exhibit functions of transcriptional activity, this novel protein may act as an antagonist against other Ets family proteins, e.g . PU.1 and Spi-B . Possible roles of Prf with respect to the B cell differentiation are discussed.

Cancer Res, 1999 Aug 15, 59(16), 3870 - 4
Frequent deletion of hSNF5/INI1, a component of the SWI/SNF complex, in chronic myeloid leukemia; Grand F et al.; During routine two-fusion fluorescence in situ hybridization analysis of patients with blast crisis of chronic myeloid leukemia (CML), we observed that yeast artificial chromosome 29GD7, which is distal to BCR at 22q11, failed to hybridize to the 9q+ derivative chromosome in 3 of 11 (27%) cases . This deleted region is close to hSNF5/INI1 (SMARCB1), a gene that encodes a widely expressed component of the SWI/SNF chromatin remodeling complex and that suffers biallelic mutations in malignant rhabdoid tumors . To determine whether hSNF5/INI1 was also deleted in patients with CML, we performed fluorescence in situ hybridization analysis with a specific cosmid probe . Deletion of hSNF5/INI1 on the 9q+ chromosome was found in 9 of 25 (36%) cases in blast crisis (lymphoid, n = 3; myeloid, n = 6) . For the three of these nine patients for whom material was available prior to transformation, deletions were also seen in chronic phase, indicating that they are early events . Analysis of an additional 21 patients in chronic phase revealed heterozygous loss of hSNF5/INI1 in 5 (24%) cases . Of the 14 patients who had hSNF5/INI1 deletions, 7 showed a mosaic pattern of hybridization in which only a proportion of CML cells that harbored both the t(9;22) derivative chromosomes had a deletion, indicating that loss of hSNF5/INI1 was acquired during the course of the disease . Single-strand conformation polymorphism analysis of all nine hSNF5/INI1 exons and splice junctions failed to reveal any mutations for 31 patients in transformation, including 8 who had deletions, although two polymorphisms were identified . We conclude that deletions of hSNF5/INI1 are frequent in patients with CML . Such deletions may be associated with reduced levels of hSNF5/INI1 expression, which could contribute to leukemogenesis by altering chromatin-mediated transcriptional control . Alternatively, the deletions could target another unidentified gene at 22q11 that plays a role in the pathogenesis of CML.

J Food Prot, 1996 Mar, 59(3), 268 - 75
Fungicidal effect of 15 disinfectants against 25 fungal contaminants commonly found in bread and cheese manufacturing; Bundgaard-Nielsen K et al.; Resistance of 19 mold and 6 yeast species to 15 commercial disinfectants was investigated by using a suspension method in which the fungicidal effect and germination time were determined at 20 degrees C . Disinfectants containing 0.5% dodecyldiethylentriaminacetic acid, 10 g of chloramine-T per 1, 2.0% formaldehyde, 0.1% potassium hydroxide, 3.0% hydrogen peroxide, or 0.3% peracetic acid were ineffective as fungicides . The fungicidal effect of quaternary ammonium compounds and chlorine compounds showed great variability between species and among the six isolates of Penicillium roqueforti var . roqueforti tested . The isolates of P roqueforti var . carneum, P . discolor, Aspergillus versicolor, and Eurotium repens examined were resistant to different quaternary ammonium compounds . Conidia and vegetative cells were killed by alcohols, whereas ascospores were resistant . Resistance of ascospores to 70% ethanol increased with age . Both P . roqueforti var . roqueforti and E . repens showed great variability of resistance within isolates of each species.

J Cell Sci, 1999 Sep, 112 Pt 18, 3137 - 46
Translational control of the cdc25 cell cycle phosphatase: a molecular mechanism coupling mitosis to cell growth; Daga RR et al.; The eukaryotic translation initiation factor 4A (eIF4A) is an RNA helicase required for translation initiation of eukaryotic mRNAs . By engineering fission yeast mutants with diminished eIF4A activity, we have found that translation of cdc25 mRNAs (a dosage-dependent activator of mitosis in all eukaryotic cells) is particularly sensitive to limitations of protein synthesis mediated by limited eIF4A activity . Genetic and biochemical analysis indicated that a rate-limited translation initiation of cdc25 mRNAs, exerted throughout its unusual 5' untranslated leader, acts as a molecular sensor to ensure that a minimum cell mass (protein synthesis) is attained before mitosis occurs . The Cdc13 cyclin B is also among the limited pool of proteins whose translation is sensitive to reduced translation initiation activity . Interestingly, the 5' leader sequences of cdc25 and cdc13 mRNAs have conserved features which are unusual in other yeast mRNAs, suggesting that common mechanisms operate in the expression of these two key mitotic activators at the translational level.

Biochem Biophys Res Commun, 1999 Aug 27, 262(2), 411 - 7
Structural analysis of the titin gene in hypertrophic cardiomyopathy: identification of a novel disease gene; Satoh M et al.; Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy accompanied by myofibrillar disarrays . Molecular genetic analyses have revealed that mutations in 8 different genes cause HCM . Mutations in these disease genes, however, could be found in about half of HCM patients, suggesting that there are other unknown disease gene(s) . Because the known disease genes encode sarcomeric proteins expressed in the cardiac muscle, we searched for a disease-associated mutation in the titin gene in 82 HCM patients who had no mutation in the known disease genes . A G to T transversion in codon 740, from CGC to CTC, replacing Arginine with Leucine was found in a patient . This mutation was not found in more than 500 normal chromosomes and increased the binding affinity of titin to alpha-actitin in the yeast two-hybrid assay . These observations suggest that the titin mutation may cause HCM in this patient via altered affinity to alpha-actinin .

Bioessays, 1999 Sep, 21(9), 713 - 7
G proteins, chemosensory perception, and the C . elegans genome project: An attractive story; Wilkie TM; Heterotrimeric G proteins, consisting of alpha, beta, and gamma subunits, couple ligand-bound seven transmembrane domain receptors to the regulation of effector proteins and production of intracellular second messengers . G protein signaling mediates the perception of environmental cues in all higher eukaryotic organisms, including yeast, Dictyostelium, plants, and animals . The nematode Caenorhabditis elegans is the first animal to have complete descriptions of its cellular anatomy, cell lineage, neuronal wiring diagram, and genomic sequence . In a recent paper, Jansen et al . used sequence searches of the C . elegans genome database to identify all heterotrimeric G protein genes (20 Galpha, 2 Gbeta, 2 Ggamma) . C . elegans encodes one ortholog of each of the four Galpha classes found in metazoans and 16 new Galpha genes . The orthologous genes are widely expressed, whereas 14 of the divergent Galpha genes are almost exclusively expressed in sensory neurons where they may regulate perception and chemotaxis .

Vaccine, 1999 Aug 6, 17(23-24), 3083 - 5
Response to HBV vaccine in relation to anti-HCV and anti-HBc positivity: a study in intravenous drug addicts; Minniti F et al.; Drug addicts represent the group of young adults with the lowest response to hepatitis B virus (HBV) vaccine . A study was carried out on 110 current intravenous heroin users attending the service providing assistance to intravenous drug users (IVDUs) (SERT) in Padua: 66.4% of them were found anti-hepatitis C virus (HCV)-positive and 33.6% were anti-HBc positive; 29.9% were positive for both . The subjects were vaccinated with 10 microg of yeast-derived vaccine at months 0, 1 and 2 (fast schedule) . The overall response rate was 66.4% . Response seems to be affected by positivity to anti-HBc, but not to HCV infection.

Trends Cell Biol, 1999 Sep, 9(9), 345 - 50
The coronin family of actin-associated proteins; de Hostos EL; Coronin was first isolated from Dictyostelium, but similar proteins have been identified in many species and individual cell types . The coronin-like protein in yeast promotes actin polymerization and also interacts with microtubules . Dictyostelium mutants lacking coronin are impaired in cytokinesis and all actin-mediated processes . Analysis of coronin-GFP (green-fluorescent protein) fusions and knockout mutants shows that coronin participates in the remodelling of the cortical actin cytoskeleton that is responsible for phagocytosis and macropinocytosis . Likewise, in mammalian neutrophils, a coronin-like protein is also associated with the phagocytic apparatus . The diversity of function in this family of actin-associated proteins is just beginning to be explored.

Parasitol Today, 1999 Sep, 15(9), 372 - 8
Genetic manipulation of kinetoplastida; Clayton CE; During the 1980s, many kinetoplastid genes were cloned and their function inferred from homology with genes from other organisms, location of the corresponding proteins or expression in heterologous systems . Up until 1990, before the availability of DNA transfection methodology, we could not analyze the function of kinetoplastid genes within the organisms themselves . Since then, it has become possible to create and complement mutants, to overexpress foreign proteins in the parasites, to knock out genes and even to switch off essential functions . However, these methods are not equally applicable in all parasites . Here, Christine Clayton highlights the differences and similarities between the most commonly used model organisms, and assesses the relative advantages of different approaches and parasites for different types of investigation.

J Neurosci, 1999 Sep 1, 19(17), 7507 - 15
Disabled-1 binds to the cytoplasmic domain of amyloid precursor-like protein 1; Homayouni R et al.; Disruption of the disabled-1 gene (Dab1) results in aberrant migration of neurons during development and disorganization of laminar structures throughout the brain . Dab1 is thought to function as an adapter molecule in signal transduction processes . It contains a protein-interaction (PI) domain similar to the phosphotyrosine-binding domain of the Shc oncoprotein, it is phosphorylated by the Src protein tyrosine kinase, and it binds to SH2 domains in a phosphotyrosine-dependent manner . To investigate the function of Dab1, we searched for binding proteins using the yeast two-hybrid system . We found that the PI domain of Dab1 interacts with the amyloid precursor-like protein 1 (APLP1) . The association of Dab1 with APLP1 was confirmed in biochemical assays, and the site of interaction was localized to a cytoplasmic region of APLP1 containing the amino acid sequence motif Asn-Pro-x-Tyr (NPxY) . NPxY motifs are involved in clathrin-mediated endocytosis, and they have been shown to bind to PI domains present in several proteins . This region of APLP1 is conserved among all members of the amyloid precursor family of proteins . Indeed, we found that Dab1 also interacts with amyloid precursor protein (APP) and APLP2 in biochemical association experiments . In transiently transfected cells, Dab1 and APLP1 colocalized in membrane ruffles and vesicular structures . Cotransfection assays in cultured cells indicated that APP family members increased serine phosphorylation of Dab1 . Dab1 and APLP1 are expressed in similar cell populations in developing and adult brain tissue . These results suggest that Dab1 may function, at least in part, through association with APLP1 in the brain.

Chest Surg Clin N Am, 1999 Aug, 9(3), 617 - 31, ix
Early complications . Esophagopleural fistula; Massard G et al.; Esophagopleural fistulae complicate the outcome of approximately 0.5% of pneumonectomies, regardless of whether performed for benign or malignant conditions . Early postoperative fistulae result from operative injury to the esophagus: both direct tears of the mucosa and devascularization with secondary necrosis have been documented . Late esophagopleural fistulae, diagnosed beyond the third postoperative month, are due to cancer recurrence or various inflammatory disorders . The usual presentation is empyema thoracis . Diagnosis is suggested by drainage of food particles or saliva, and the presence of yeast cells within the pleural fluid . Confirmation relies on direct opacification of the fistulous tract during opaque swallow studies . Treatment is initiated by clearance of empyema with either tube thoracostomy or Clagett window, and feeding gastrostomy or jejunostomy.

J Cell Biol, 1999 Aug 23, 146(4), 731 - 40
The developmental role of warthog, the notch modifier encoding Drab6; Purcell K et al.; The warthog (wrt) gene, recovered as a modifier for Notch signaling, was found to encode the Drosophila homologue of rab6, Drab6 . Vertebrate and yeast homologues of this protein have been shown to regulate Golgi network to TGN trafficking . To study the function of this protein in the development of a multicellular organism, we analyzed three different warthog mutants . The first was an R62C point mutation, the second a genomic null, and the third was an engineered GTP-bound form . Our studies show, contrary to yeast, that the Drosophila homologue of rab6 is an essential gene . However, it has limited effects on development beyond the larval stage . Only the mechanosensory bristles on the head, notum, and scutellum are affected by warthog mutations . We present models for the modifying effect of Drab6 on Notch signaling.

Genomics, 1999 Aug 15, 60(1), 105 - 9
Cloning and characterization of ZNF236, a glucose-regulated Kruppel-like zinc-finger gene mapping to human chromosome 18q22-q23; Holmes DI et al.; We report the cDNA cloning and characterization of ZNF236, a novel Kruppel-like zinc-finger gene initially identified by its glucose-regulated expression in human mesangial cells using mRNA differential display . Using the differential display fragment as a probe, we screened a human fetal kidney cDNA library and isolated several clones representing two differently spliced mRNA transcripts, designated ZNF236a and -b . Both transcripts were identical apart from the presence of an additional exon in ZNF236a that truncates the open reading frame . RT-PCR analysis confirmed the expression of both transcripts to be upregulated in human mesangial cells in response to elevated levels of d-glucose . ZNF236a and -b cDNAs encode polypeptides of 174 and 204 kDa, containing 25 and 30 C(2)H(2) zinc-finger motifs, respectively . Northern blot analysis showed that ZNF236 is ubiquitously expressed in all human tissues tested . Expression levels were highest in skeletal muscle and brain, intermediate in heart, pancreas, and placenta, and lowest in kidney, liver, and lung . Southern zoo blot analysis indicated that ZNF236 is conserved in the genomes of all mammalian species tested, but not in yeast . The mapping of ZNF236 to human chromosome 18q22-q23, close to the IDDM6 locus, coupled with the glucose-regulated expression of the gene in human mesangial cells, suggests that ZNF236 may be a candidate gene for diabetic nephropathy .

Infect Immun, 1999 Sep, 67(9), 4732 - 6
Interactions of Penicillium marneffei with human leukocytes in vitro; Rongrungruang Y et al.; Penicillium marneffei, a dimorphic fungus endemic in parts of Asia, causes disease in those with impaired cell-mediated immunity, especially persons with AIDS . The histopathology of penicilliosis marneffei features the intracellular infection of macrophages . We studied the interactions between human leukocytes and heat-killed yeast-phase P . marneffei . Monocyte-derived macrophages bound and internalized P . marneffei in the presence of complement-sufficient pooled human serum (PHS) . Binding and phagocytosis were still seen if PHS was heat inactivated or omitted altogether . The binding of unopsonized P . marneffei to monocyte-derived macrophages occurred in the absence of divalent cations and was not affected by inhibitors of mannose and beta-glucan receptors or monoclonal antibodies directed against CD14 and CD11/CD18 . Binding was profoundly inhibited by wheat germ agglutinin . A vigorous respiratory burst was seen in peripheral blood mononuclear cells (PBMC) stimulated with P . marneffei, regardless of whether the fungi were opsonized . However, tumor necrosis factor alpha (TNF-alpha) release from PBMC stimulated with P . marneffei occurred only if serum was present . These data demonstrate that (i) monocyte-derived macrophages bind and phagocytose P . marneffei even in the absence of opsonization, (ii) binding is divalent cation independent but is inhibited by wheat germ agglutinin, suggesting that the major receptor(s) recognizing P . marneffei is a glycoprotein with exposed N-acetyl-beta-D-glucosaminyl groups, (iii) P . marneffei stimulates the respiratory burst regardless of whether opsonins are present, and (iv) serum factors are required for P . marneffei to stimulate TNF-alpha release . The ability of unopsonized P . marneffei to parasitize mononuclear phagocytes without stimulating the production of TNF-alpha may be critical for the virulence of this intracellular parasite.

J Biol Chem, 1999 Aug 27, 274(35), 25061 - 8
Precursor processing of pro-ISG15/UCRP, an interferon-beta-induced ubiquitin-like protein; Potter JL et al.; Induction of the 17-kDa ubiquitin-like protein ISG15/UCRP and its subsequent conjugation to cellular targets is the earliest response to type I interferons . The polypeptide is synthesized as a precursor containing a carboxyl-terminal extension whose correct processing is required for subsequent ligation of the exposed mature carboxyl terminus . Recombinant pro-ISG15 is processed in extracts of human lung fibroblasts by a constitutive 100-kDa enzyme whose activity is unaffected by type I interferon stimulation . The processing enzyme has been purified to apparent homogeneity by a combination of ion exchange and hydrophobic chromatography and found to be stimulated 12-fold by micromolar concentrations of ubiquitin . Analysis of the products of pro-ISG15 processing enzyme demonstrates specific cleavage exclusively at the Gly(157)-Gly(158) peptide bond to generate a mature ISG15 carboxyl terminus . Irreversible inhibition of pro-ISG15 processing activity by thiol-specific alkylating agents and a pH rate dependence conforming to titration of a single group of pK(a) 8.1 indicate the 100-kDa enzyme is a thiol protease . Partial sequencing of a trypsin-derived peptide indicates the enzyme is either the human ortholog of yeast Ubp1 or a Ubp1-related protein . As yeast do not contain ISG15, these results suggest that a ubiquitin-specific enzyme was recruited for pro-ISG15/UCRP processing by adaptive divergence.

J Biol Chem, 1999 Aug 27, 274(35), 24865 - 72
Nuclear localization of protein kinase U-alpha is regulated by 14-3-3; Zhang S et al.; 14-3-3 proteins are intracellular, dimeric molecules that bind to and modify the activity of several signaling proteins . We used human 14-3-3zeta as a bait in the yeast two-hybrid system to screen a murine embryonic cDNA library . One interacting clone was found to encode the carboxyl terminus of a putative protein kinase . The coding sequence of the human form (protein kinase Ualpha, PKUalpha) of this protein kinase was found in GenBank(TM) on the basis of sequence homology . The two-hybrid clone was also highly homologous to TOUSLED, an Arabidopsis thaliana protein kinase that is required for normal flower and leaf development . PKUalpha has been found by coimmunoprecipitation to bind to 14-3-3zeta in vivo . Our confocal laser immunofluorescence microscopic experiments revealed that PKUalpha colocalizes with the cytoplasmic intermediate filament system of cultured fibroblasts in the G(1) phase of the cell cycle . PKUalpha is found in the perinuclear area of S phase cells and in the nucleus of late G(2) cells . Transfection of cells with a dominant negative form of 14-3-3eta promotes the nuclear localization of PKUalpha . These results suggest that the subcellular localization of PKUalpha is regulated, at least in part, by its association with 14-3-3.

Nucleic Acids Res, 1999 Aug 15, 27(16), 3318 - 24
Centromeres from telomeres? The centromeric region of the Y chromosome of Drosophila melanogaster contains a tandem array of telomeric HeT-A- and TART-related sequences; Agudo M et al.; Cytological and cytogenetic studies have previously defined the region needed for centromeric function in the Y chromosome of Drosophila melanogaster . We have identified a YAC clone that originated from this region . Molecular analysis of the YAC and genomic DNAs has allowed the description of a satellite DNA made of telomeric HeT-A- and TART-derived sequences and the construction of a long-range physical map of the heterochromatic region h18 . Sequences within the YAC clone are conserved in the centromeric region of the sibling species Drosophila simulans . That telomere-derived DNA now forms part of the centromeric region of the Y chromosome could indicate a telomeric origin of this centromere . The existence of common determinants for the function of both centromeres and telomeres is discussed.

Nucleic Acids Res, 1999 Aug 15, 27(16), 3300 - 9
Two 5'-ETS regions implicated in interactions with U3 snoRNA are required for small subunit rRNA maturation in Trypanosoma brucei; Hartshorne T et al.; Early pre-rRNA processing events were examined in the ancient protozoan parasite Trypanosoma brucei and found to have both distinctive and conserved features . Two 5'-ETS cleavages occur: A' and the newly discovered A0 . A' and A0 appear related to vertebrate and yeast primary pre-RNA cleavage sites, respectively . However, trypanosomatid primary rRNA transcripts can first be processed at the ITS1/5.8S boundary and 5'-ETS sequences then removed by consecutive cleavages at A', A0 and A1 at the 5'-ETS/SSU rRNA junction . 5'-ETS sequences previously crosslinked to U3 snoRNA were tested for their roles in rRNA processing using our new tagged rRNA system . Two distinct A'-adjacent sequence elements, which may pair with U3 hinge bases, were specifically required for SSU rRNA production, as was a downstream element . The latter element appears conserved with the yeast 5'-ETS U3 binding sequence, required for A0, A1 and A2 cleavages, in that they both share 10 bases complementary with U3 hinge sequences and lie upstream from A0 and A1 sites located in a potential stem-loop structure . The distinctive positioning of putative trypanosomatid U3 binding sites with respect to A" and A0 cleavages suggests that different U3-dependent mechanisms may direct each processing event.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3130 - 7
The polyoma virus enhancer cannot substitute for DNase I core hypersensitive sites 2-4 in the human beta-globin LCR; Tanimoto K et al.; The polyoma virus enhancer (PyE) is capable of conferring integration position-independent expression to linked genes in stably transfected erythroid cells after joining to DNase I hypersensitive site (HS) 5 of the human beta-globin locus control region (LCR) . In attempting to separate the chromatin opening activity of the LCR from its enhancer activity and to investigate contributions of the individual HS core elements to LCR function, the human beta-globin LCR HS2, HS3 and HS4 core elements were replaced with the PyE within the context of a yeast artificial chromosome (YAC) bearing the whole locus . We show here that, in contrast to its function in cultured cells, the PyE is unable to replace HS core element function in vivo . We found that the PyE substitution mutant LCR is unable to provide either chromatin opening or transcriptional potentiating activity at any erythroid developmental stage in transgenic mice . These data provide direct evidence that the human beta-globin LCR core elements specify unique functions that cannot be replaced by a ubiquitous enhancer activity.

Mol Cell Biol, 1999 Sep, 19(9), 6323 - 32
Activating signal cointegrator 1, a novel transcription coactivator of nuclear receptors, and its cytosolic localization under conditions of serum deprivation; Kim HJ et al.; Activating signal cointegrator 1 (ASC-1) harbors an autonomous transactivation domain that contains a putative zinc finger motif which provides binding sites for basal transcription factors TBP and TFIIA, transcription integrators steroid receptor coactivator 1 (SRC-1) and CBP-p300, and nuclear receptors, as demonstrated by the glutathione S-transferase pull-down assays and the yeast two-hybrid tests . The ASC-1 binding sites involve the hinge domain but not the C-terminal AF2 core domain of nuclear receptors . Nonetheless, ASC-1 appears to require the AF2-dependent factors to function (i.e., CBP-p300 and SRC-1), as suggested by the ability of ASC-1 to coactivate nuclear receptors, either alone or in cooperation with SRC-1 and p300, as well as its inability to coactivate a mutant receptor lacking the AF2 core domain . By using indirect immunofluorescence, we further show that ASC-1, a nuclear protein, is localized to the cytoplasm under conditions of serum deprivation but is retained in the nucleus when it is serum starved in the presence of ligand or coexpressed CBP or SRC-1 . These results suggest that ASC-1 is a novel coactivator molecule of nuclear receptors which functions in conjunction with CBP-p300 and SRC-1 and may play an important role in establishing distinct coactivator complexes under different cellular conditions.

Int J Obes Relat Metab Disord, 1999 Jun, 23 Suppl 6, S19 - 23
UCP1, UCP2 and UCP3: are they true uncouplers of respiration?
Bouillaud F.
The thermogenesis in brown adipose tissue (BAT) is due to the activity of a mitochondrial uncoupling protein (UCP1) . This protein allows the protons pumped by the respiratory chain to re-enter the matrix without ATP synthesis . Therefore respiration is dramatically increased and produces only heat . The discovery of genes showing strong similarities with the UCP1 gene and expressed in other tissues raised the possibility that these proteins participate in the proton leak observed in mitochondria, and therefore participate in the regulation of energy expenditure . The recombinant expression of UCP1, UCP2 and UCP3 in yeast allows the comparison of the coupling state of yeast mitochondria in the presence or absence of these proteins.

Int J Obes Relat Metab Disord, 1999 Jun, 23 Suppl 6, S4 - 11
The significance and mechanism of mitochondrial proton conductance; Brand MD et al.; There is a futile cycle of pump and leak of protons across the mitochondrial inner membrane . The contribution of the proton cycle to standard metabolic rate is significant, particularly in skeletal muscle, and it accounts for 20% or more of the resting respiration of a rat . The mechanism of the proton leak is uncertain: basal proton conductance is not a simple biophysical leak across the unmodified phospholipid bilayer . Equally, the evidence that it is catalysed by homologues of the brown adipose uncoupling protein, UCP1, is weak . The yeast genome contains no clear UCP homologue but yeast mitochondria have normal basal proton conductance . UCP1 catalyses a regulated inducible proton conductance in brown adipose tissue and the possibility remains open that UCP2 and UCP3 have a similar role in other tissues, although this has yet to be demonstrated.

Acta Biochim Pol, 1999, 46(1), 51 - 60
Differential regulation of signaling pathways for insulin and insulin-like growth factor I; Lopaczynski W; The insulin receptor (IR) and the insulin-like growth factor receptor I (IGF-IR) have different functions in cell growth, apoptosis, differentation, and transformation . Although some of these differences may be explained by the relative level of receptor expression and receptor structure (alpha and beta subunits), they may also be attributed to differences in intracellular signals generated by insulin and IGF-I . The presence of hybrid receptors (IR alphabeta subunits and IGF-IR alphabeta subunits) making up the heterotetramers has added a new dimension to our understanding of the functional roles of these receptors . However, to date the results of efforts to understand the differences between these two closely related receptors have indicated mostly similarities . For example, both receptors utilize IRS-1/IRS-2 and Shc as immediate downstream adaptors, leading to activation of the Ras, Raf, ERK kinases and PI-3 kinase pathways . We have used the yeast two hybrid system to identify proteins which bind to the activated IGF-IR but not to the IR . The cytoplasmic domain of the IGF-IR was used to screen a human fetal brain library and two isoforms of the 14-3-3 family were identified . 14-3-3 proteins are a highly conserved family of proteins which have recently been shown to interact with other components of the mitogenic and apoptotic signaling pathways, including Raf, BAD, Bcr/Bcr-Abl, middle-T antigen, Ksr, PKC, PI-3 kinase, ASK1 kinase, and cdc25C phosphatase . We also identified human Grb10, an adaptor protein with SH2 domain associated with the IGF-IR beta subunit . Smith's laboratory showed that Grb10 preferentially binds to the IR in intact cells . Using the interaction trap screen (active cytoplasmic domain of the IGF-IR) 55PIK and SOCS-2 proteins were also identified . However, 55PIK and SOCS-2 also interact with the IR in the yeast two hybrid system . These studies raise the possibility that 14-3-3 and Grb10 may play a role in insulin and IGF-I signal transduction and may underlie the observed differences.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1997 Jun, 19(3), 163 - 70
{Construction of human genomic YAC clones with "terminal modification"}; Zhang X et al.; A human YAC genomic pool with more than 4,000 clones was constructed by means of "terminal modification" of YAC vector and genomic inserts, as well as optimization of relevant experimental parameters . The transformation rate is approximately 400-500 transformants per microgram DNA . Preliminary characterization of randomly sampled clones demonstrates that YAC-specific inserts, in the average size of 450 kb and ranging 150-750 kb, were detected from 80% of the clones . The vector ligation experiment indicates the efficient reduction of "coligation" by terminal modification.

Cell Biol Int, 1998, 22(7-8), 493 - 7
Presence and localization of histidine decarboxylase enzyme (HDC) and histamine in Tetrahymena pyriformis; Hegyesi H et al.; Histidine decarboxylase (HDC) enzyme and its function under hormonal influences were studied in a low level of phylogeny . HDC protein is present in the unicellular ciliate Tetrahymena and its expression was not altered by insulin or histamine treatment . Starvation for 24 h enormously decreased the quantity of histamine in the cells . However, insulin influenced the activity of the HDC enzyme, demonstrated by the seven-fold quantity of histamine in the starved cells after insulin treatment . Insulin also increased the uptake of histamine from the tryptone-yeast extract medium . HDC was found in different parts of the cytoplasm, mainly in the periphery (epiplasm) of the cells . The experiments demonstrated the uptake and synthesis of histamine by Tetrahymena as well as the possibility of hormonal regulation of HDC activity .

Clin Genet, 1999 Jun, 55(6), 466 - 72
Cloning of translocation breakpoints associated with Shwachman syndrome and identification of a candidate gene; Ikegawa S et al.; Shwachman syndrome is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency, bone-marrow dysfunction, and metaphyseal chondrodysplasia . A de novo balanced translocation was recently documented in a patient with this disease . Toward isolating the gene(s) responsible for Shwachman syndrome, we cloned and sequenced the translocation breakpoints in the DNA of this patient . The nucleotide sequences around the breakpoints contained neither repetitive elements nor motifs reported to be implicated in recombination events, although we did detect gains or losses of oligonucleotides at the translocation junctions . By large-scale genomic sequencing and in silico gene trapping, we identified two novel transcripts in the vicinity of the breakpoints that might represent candidate genes for Shwachman syndrome, one on chromosome 6 and the other on chromosome 12 . The gene on chromosome 12 was actually disrupted by the translocation.

J Nutr Sci Vitaminol (Tokyo), 1999 Apr, 45(2), 223 - 9
Stereoisomers of glutathione: preparation and enzymatic reactivities; Oikawa T et al.; We synthesized a series of stereoisomers of glutathione (GSH) and glutathione disulfide (GSSG) by the solid-phase method . These peptides were used to examine their reactivities with enzymes acting on glutathione . The glutathione reductase of yeast acted only on LL-GSSG . Glutathione S-transferase catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with LL-GSH and DL-GSH (Km (mM): for LL-GSH, 0.035; and for DL-GSH, 0.62), but the DD- and LD-diastereomers were inert . gamma-Glutamyl transpeptidase catalyzed the transfer of gamma-glutamyl moiety of LL-GSH and DL-GSH to taurine forming gamma-glutamyl taurine and cysteinyl taurine (Km (mM): for LL-GSH, 0.336; and for DL-GSH, 0.628), but the other diastereomers were not the substrates . The occurrence of L-cysteinyl residue in the tripeptides is required for the glutathione analogue to be a substrate of the enzymes.

Cytogenet Cell Genet, 1999, 85(3-4), 273 - 8
Human chromosome 3 and pig chromosome 13 show complete synteny conservation but extensive gene-order differences; Sun HF et al.; A comparative map of human chromosome 3 (HSA 3) and pig chromosome 13 (SSC 13) was constructed using physically assigned pig sequence-tagged sites (STSs) . Pig STSs representing 11 HSA 3 genes, including v-Raf-1 murine leukemia viral oncogene homolog 1 (RAF1), retinoic acid beta receptor (RARB), cholecystokinin (CCK), pituitary transcription factor 1 (POU1F1), ceruloplasmin (CP), guanine nucleotide binding protein, alpha-inhibiting polypeptide 2 (GNAI2), sucrase-isomaltase (SI), rhodopsin (RHO), dopamine receptor D3 (DRD3), growth-associated protein 43 (GAP43), and somatostatin (SST), were developed . Ten pig STSs were regionally mapped using a somatic cell hybrid panel (SCHP) to SSC 13 with 80-100% concordance . Large-insert probes were obtained by screening a pig yeast artificial chromosome (YAC) library with primers for each STS . Several YACs were identified for DRD3, GAP43, POU1F1, RHO, SI, and SST for fluorescence in situ hybridization (FISH) mapping . Single gene and bi-color FISH with each pairwise combination were used to further define the gene order on SSC 13 . While these data confirm chromosome painting results showing that HSA 3 probes hybridize to a major portion of SSC 13, they also demonstrate extensive gene-order differences between man and pig within this large conserved synteny group . Interestingly, several conserved chromosomal regions have been detected between pig and mouse that are not conserved between man and mouse, suggesting that the SSC 13 gene arrangement may be the closest to that of the ancestral eutherian chromosome.

Cytogenet Cell Genet, 1999, 85(3-4), 244 - 7
Physical assignment of the bovine MHC class IIa and class IIb genes; Hess M et al.; Screening of a bovine yeast artificial chromosome (YAC) library revealed two clones which contain most of the class II genes of the major histocompatibility complex (MHC) known to date . The YACs were mapped by fluorescence in situ hybridization (FISH) and characterized for the class II genes they contain . We found that the classic class II genes BoLA- DQA, -DQB, -DRA, and -DRB3 are located at BTA 23q21 and the non-classic class II genes DYA, DIB, LMP2, LMP7, TAP2, BoLA-DOB, -DMA, -DMB, and -DNA are located at BTA 23q12-->q13 . These two different mapping locations confirm and extend previous findings of a gross physical distance between classic and non-classic MHC class II genes in cattle.

Mol Med, 1999 Jul, 5(7), 459 - 70
A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies; Soares IS et al.; BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria . A major concern in vaccine development is the polymorphism observed in different plasmodial strains . The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite . MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P . vivax . Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P . vivax MSP1(19) described thus far . RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P . vivax . Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein . CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P . vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9515 - 20
The C-terminal region of Schizosaccaromyces pombe proliferating cell nuclear antigen is essential for DNA polymerase activity; Kelman Z et al.; Proliferating cell nuclear antigen (PCNA), the processivity factor (sliding clamp) of DNA polymerases (Pols), plays essential roles in DNA metabolism . In this report, we examined the functional role of the C-terminal region of Schizosaccaromyces pombe PCNA both in vitro and in vivo . The deletion or Ala substitution of the last 9 aa (252-260A), as well as Ala replacement of only 4 aa (252-255A) at the C terminus, failed to substitute for the wild-type PCNA protein for cell growth in S . pombe . Two other PCNA mutant proteins, A251V and K253E, exhibited cold-sensitive phenotypes . Several yeast strains harboring mutations, including those at the acidic C-terminal region, showed elevated sensitivity to DNA damage . The ability of the mutant PCNA proteins to stimulate DNA synthesis by Poldelta and Polepsilon also was studied in vitro . The mutant proteins that did not support cell growth and a mutant protein containing a single amino acid substitution at position 252, where Pro is replaced by Ala, stimulated Poldelta and Polepsilon activities poorly . All mutant PCNA proteins, however, were assembled around DNA by the clamp loader, replication factor C, efficiently . Thus, the C-terminal region of PCNA is important for interactions with both Poldelta and Polepsilon and for cell survival after DNA damage . The C terminus of sliding clamps from other organisms has been shown to be important for clamp loading as well as polymerase interactions . The relationship between the conserved sequence in this region in different organisms is discussed.

J Mol Biol, 1999 Aug 20, 291(3), 531 - 6
Identification of the INO1 gene of Mycobacterium tuberculosis H37Rv reveals a novel class of inositol-1-phosphate synthase enzyme; Bachhawat N et al.; 1L-myo-inositol (inositol) is vital for the biogenesis of mycothiol, phosphatidylinositol and glycosylphosphatidylinositol anchors linked to complex carbohydrates in Mycobacterium tuberculosis . All these cellular components are thought to play important roles in host-pathogen interactions and in the survival of the pathogen within the host . However, the inositol biosynthetic pathway in M . tuberculosis is not known . To delineate the pathways for inositol formation, we employed a unique combination of tertiary structure prediction and yeast-based functional assays . Here, we describe the identification of the gene for mycobacterial INO1 that encodes inositol-1-phosphate synthase distinct in many respects from the eukaryotic analogues .

Plant Cell, 1999 Aug, 11(8), 1457 - 72
Multiubiquitin chain binding subunit MCB1 (RPN10) of the 26S proteasome is essential for developmental progression in Physcomitrella patens; Girod PA et al.; The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes . We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates . To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination . PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide . Unlike yeast Deltamcb1 strains, which grow normally, P . patens Deltamcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores . Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores . Complementation of a Deltamcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype . These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones.

EMBO J, 1999 Aug 16, 18(16), 4476 - 84
The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants; Cebolla A et al.; Plant organs develop mostly post-embryonically from persistent or newly formed meristems . After cell division arrest, differentiation frequently involves endoreduplication and cell enlargement . Factors controlling transition from mitotic cycles to differentiation programmes have not been identified yet in plants . Here we describe ccs52, a plant homologue of APC activators involved in mitotic cyclin degradation . The ccs52 cDNA clones were isolated from Medicago sativa root nodules, which exhibit the highest degree of endopolyploidy in this plant . ccs52 represents a small multigenic family and appears to be conserved in plants . Overexpression of ccs52 in yeast triggered mitotic cyclin degradation, cell division arrest, endoreduplication and cell enlargement . In Medicago, enhanced expression of ccs52 was found in differentiating cells undergoing endoreduplication . In transgenic M.truncatula plants, overexpression of the ccs52 gene in the antisense orientation resulted in partial suppression of ccs52 expression and decreased the number of endocycles and the volume of the largest cells . Thus, the ccs52 product may switch proliferating cells to differentiation programmes which, in the case of endocycles, result in cell size increments.

EMBO J, 1999 Aug 16, 18(16), 4455 - 63
A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2; Menke FL et al.; Jasmonate (JA) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites . The induction of the secondary metabolite biosynthetic gene Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by elicitor requires JA as a second messenger . A 42 bp region in the Str promoter is both necessary and sufficient for JA- and elicitor-responsive expression . This region is unlike other previously identified JA-responsive regions, and contains a GCC-box-like element . Yeast one-hybrid screening identified cDNAs encoding two AP2-domain proteins . These octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) proteins bind in a sequence-specific manner the JA- and elicitor-responsive element . ORCA2 trans-activates the Str promoter and its expression is rapidly inducible with JA and elicitor, whereas Orca1 is expressed constitutively . The results indicate that a GCC-box-like element and ORCA2 play key roles in JA- and elicitor-responsive expression of the terpenoid indole alkaloid biosynthetic gene Str.

Mol Med, 1999 Apr, 5(4), 211 - 23
In vivo analysis of DNase I hypersensitive sites in the human CFTR gene; Moulin DS et al.; BACKGROUND: The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex pattern of expression . The regulatory elements conferring tissue-specific and temporal regulation are thought to lie mainly outside the promoter region . Previously, we identified DNase I hypersensitive sites (DHS) that may contain regulatory elements associated with the CFTR gene at -79.5 and at -20.5 kb with respect to the ATG and at 10 kb into the first intron . MATERIALS AND METHODS: In order to evaluate these regulatory elements in vivo we examined these DHS in a human CFTR gene that was introduced on a yeast artificial chromosome (YAC) into transgenic mice . The 310 kb human CFTR YAC was shown to restore the pheno-type of CF-null mice and so is likely to contain most of the regulatory elements required for tissue-specific expression of CFTR . RESULTS: We found that the YAC does not include the -79.5 kb region . The DHS at -20.5 kb is present in the chromatin of most tissues of the transgenic mice, supporting its non-tissue-specific nature . The DHS in the first intron is present in a more restricted set of tissues in the mice, although its presence does not show complete concordance with CFTR expression . The intron I DHS may be important for the higher levels of expression found in human pancreatic ducts and in lung submucosal glands . CONCLUSION: These data support the in vivo importance of these regulatory elements.

Genome Res, 1999 Aug, 9(8), 751 - 62
High-resolution landmark framework for the sequence-ready mapping of Xq23-q26.1; Steingruber HE et al.; We have established a landmark framework map over 20-25 Mb of the long arm of the human X chromosome using yeast artificial chromosome (YAC) clones . The map has approximately one landmark per 45 kb of DNA and stretches from DXS7531 in proximal Xq23 to DXS895 in proximal Xq26, connecting to published framework maps on its proximal and distal sides . There are three gaps in the framework map resulting from the failure to obtain clone coverage from the YAC resources available . Estimates of the maximum sizes of these gaps have been obtained . The four YAC contigs have been positioned and oriented using somatic-cell hybrids and fluorescence in situ hybridization, and the largest is estimated to cover approximately 15 Mb of DNA . The framework map is being used to assemble a sequence-ready map in large-insert bacterial clones, as part of an international effort to complete the sequence of the X chromosome . PAC and BAC contigs currently cover 18 Mb of the region, and from these, 12 Mb of finished sequence is available.

Biochim Biophys Acta, 1999 Aug 12, 1451(1), 17 - 34
Import of proteins into peroxisomes; Hettema EH et al.; Peroxisomes are organelles that confine an important set of enzymes within their single membrane boundaries . In man, a wide variety of genetic disorders is caused by loss of peroxisome function . In the most severe cases, the clinical phenotype indicates that abnormalities begin to appear during embryological development . In less severe cases, the quality of life of adults is affected . Research on yeast model systems has contributed to a better understanding of peroxisome formation and maintenance . This framework of knowledge has made it possible to understand the molecular basis of most of the peroxisome biogenesis disorders . Interestingly, most peroxisome biogenesis disorders are caused by a failure to target peroxisomal proteins to the organellar matrix or membrane, which classifies them as protein targeting diseases . Here we review recent fundamental research on peroxisomal protein targeting and discuss a few burning questions in the field concerning the origin of peroxisomes.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3543 - 9
Structural and functional roles of the N1- and N3-protons of psi at tRNA's position 39; Yarian CS et al.; Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop domain (ASL) is highly conserved . To determine the physicochemical contributions of Psi(39)to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe)ASL and ASLs with various derivatives of U(39)and Psi(39) . Psi(39)increased the thermal stability of the ASL (Delta T (m)= 1.3 +/- 0.5 degrees C), but did not significantly affect ribosomal binding ( K (d)= 229 +/- 29 nM) compared to that of the unmodified ASL (K (d)= 197 +/- 58 nM) . The ASL-Psi(39)P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL . The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39)were comparable to that of the ASL with Psi(39) . Thus, the contribution of Psi(39)to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U . In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39)and m(1)m(3)Psi(39)destabilized the ASL by disrupting the A(31)-U(39)base pair in the stem, as confirmed by NMR . N3-methylations of both U and Psi dramatically decreased ribosomal binding ( K (d)= 1060 +/- 189 to 1283 +/- 258 nM) . Thus, canonical base pairing of Psi(39)to A(31)through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U(39)to Psi(39)may have biological roles other than tRNA's binding to the ribosomal P-site.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3527 - 33
The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G(1)/S regulators; Ramirez-Parra E et al.; Association of the retinoblastoma (Rb) protein with E2F transcription factors is central to cell cycle-specific gene expression and growth in animal cells . Whether Rb-E2F complexes are also involved in plant cell growth and differentiation is still unknown since E2F proteins have not yet been identified in plants . Here we report the isolation and characterisation of a wheat E2F (TmE2F) cDNA clone . Interestingly, the overall domain organisation of plant E2F is related to the human E2F-1/2/3 subset but its primary sequence is slightly more related to the E2F-4/5 subset . TmE2F-Rb binding depends on residues, located at the C-terminus, which are different from those of animal E2Fs . However, the acidic or hydrophobic nature of certain residues is maintained, strongly suggesting that they may have a crucial role in E2F activities . Plant E2F is expressed in proliferating cultured cells and in differentiated tissues and is up-regulated early in S phase . Our studies reinforce the idea that G(1)/S regulators in plants are unrelated to those of yeast cells but similar to those of animal cells and provide new tools to analyse the links between cell cycle regulators, plant growth and developmental signals.

J Biol Chem, 1999 Aug 20, 274(34), 24094 - 9
mGrb10 interacts with Nedd4; Morrione A et al.; We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors . We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-AP carboxyl-terminus) domain and three WW domains . The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain . The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10 . This interaction was Ca(2+)-independent . Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.

J Biol Chem, 1999 Aug 20, 274(34), 24007 - 13
Interaction of Alzheimer's presenilin-1 and presenilin-2 with Bcl-X(L) . A potential role in modulating the threshold of cell death; Passer BJ et al.; The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2, have been reported to be functionally involved in amyloid precursor protein processing, notch receptor signaling, and programmed cell death or apoptosis . However, the molecular mechanisms by which presenilins regulate these processes remain unknown . With regard to the latter, we describe a molecular link between presenilins and the apoptotic pathway . Bcl-X(L), an anti-apoptotic member of the Bcl-2 family was shown to interact with the carboxyl-terminal fragments of PS1 and PS2 by the yeast two-hybrid system . In vivo interaction analysis revealed that both PS2 and its naturally occurring carboxyl-terminal products, PS2short and PS2Ccas, associated with Bcl-X(L), whereas the caspase-3-generated amino-terminal PS2Ncas fragment did not . This interaction was corroborated by demonstrating that Bcl-X(L) and PS2 partially co-localized to sites of the vesicular transport system . Functional analysis revealed that presenilins can influence mitochondrial-dependent apoptotic activities, such as cytochrome c release and Bax-mediated apoptosis . Together, these data support a possible role of the Alzheimer's presenilins in modulating the anti-apoptotic effects of Bcl-X(L).

J Biol Chem, 1999 Aug 20, 274(34), 23850 - 7
M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6; Quilliam LA et al.; M-Ras is a Ras-related protein that shares approximately 55% identity with K-Ras and TC21 . The M-Ras message was widely expressed but was most predominant in ovary and brain . Similarly to Ha-Ras, expression of mutationally activated M-Ras in NIH 3T3 mouse fibroblasts or C2 myoblasts resulted in cellular transformation or inhibition of differentiation, respectively . M-Ras only weakly activated extracellular signal-regulated kinase 2 (ERK2), but it cooperated with Raf, Rac, and Rho to induce transforming foci in NIH 3T3 cells, suggesting that M-Ras signaled via alternate pathways to these effectors . Although the mitogen-activated protein kinase/ERK kinase inhibitor, PD98059, blocked M-Ras-induced transformation, M-Ras was more effective than an activated mitogen-activated protein kinase/ERK kinase mutant at inducing focus formation . These data indicate that multiple pathways must contribute to M-Ras-induced transformation . M-Ras interacted poorly in a yeast two-hybrid assay with multiple Ras effectors, including c-Raf-1, A-Raf, B-Raf, phosphoinositol-3 kinase delta, RalGDS, and Rin1 . Although M-Ras coimmunoprecipitated with AF6, a putative regulator of cell junction formation, overexpression of AF6 did not contribute to fibroblast transformation, suggesting the possibility of novel effector proteins . The M-Ras GTP/GDP cycle was sensitive to the Ras GEFs, Sos1, and GRF1 and to p120 Ras GAP . Together, these findings suggest that while M-Ras is regulated by similar upstream stimuli to Ha-Ras, novel targets may be responsible for its effects on cellular transformation and differentiation.

J Biol Chem, 1999 Aug 20, 274(34), 23719 - 25
Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase; Schmidt PJ et al.; The copper chaperone for superoxide dismutase (SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown mechanism . We demonstrate here that this process involves the cooperation of three distinct regions of the copper chaperone for SOD1 (CCS): an amino-terminal Domain I homologous to the Atx1p metallochaperone, a central portion (Domain II) homologous to SOD1, and a short carboxyl-terminal peptide unique to CCS molecules (Domain III) . These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies . The biological roles of the yeast CCS domains were examined in yeast cells . Surprisingly, Domain I was found to be necessary only under conditions of strict copper limitation . Domain I and Atx1p were not interchangeable in vivo, underscoring the specificity of the corresponding metallochaperones . A putative copper site in Domain II was found to be irrelevant to yeast CCS activity, but SOD1 activation invariably required a CXC in Domain III that binds copper . Copper binding to purified yeast CCS induced allosteric conformational changes in Domain III and also enhanced homodimer formation of the polypeptide . Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates target recognition, and Domain III, perhaps in concert with Domain I, mediates copper insertion into apo-SOD1.

Genes Dev, 1999 Aug 1, 13(15), 1918 - 23
Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development; Michaelson JS et al.; The mammalian Daxx gene has been identified in a diverse set of yeast interaction trap experiments . Although a facilitating role for Daxx in Fas-induced apoptosis has been suggested, Daxx's physiologic function remains unknown . To elucidate the in vivo role of Daxx, we have generated Daxx-deficient mice . Surprisingly, rather than a hyperproliferative disorder expected from the loss of a pro-apoptotic gene, mutation of Daxx results in extensive apoptosis and embryonic lethality . These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo.

Am J Hum Genet, 1999 Sep, 65(3), 722 - 7
A locus for an axonal form of autosomal recessive Charcot-Marie-Tooth disease maps to chromosome 1q21.2-q21.3; Bouhouche A et al.; Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders that affect the peripheral nervous system . Three loci are known for the autosomal dominant forms of axonal CMT (CMT2), but none have yet been identified for autosomal recessive axonal CMT (ARCMT2) . We have studied a large consanguineous Moroccan ARCMT2 family with nine affected sibs . The onset of CMT was in the 2d decade in all affected individuals who presented with a severe motor and sensory neuropathy, with proximal muscle involvement occurring in some patients . After exclusion of known loci for CMT2 and for demyelinating ARCMT2, a genomewide search was performed . Evidence for linkage was found with markers on chromosome 1q . The maximum pairwise LOD score was above the threshold value of 3.00, for markers D1S514, D1S2715, D1S2777, and D1S2721, and it reached 6.10 at the loci D1S2777, D1S2721, and D1S2624, according to multipoint LOD-score analysis . These markers defined a region of homozygosity that placed the gene in a 4.4-cM interval . Moreover, a recombination event detected in an unaffected 48-year-old individual excludes the D1S506 marker, thereby reducing the interval to 1.7 cM . In addition, the P0 gene, an attractive candidate because of both its location on chromosome 1q and its role in myelin structure, was excluded by physical mapping and direct sequencing.

Am J Hum Genet, 1999 Sep, 65(3), 621 - 34
PEX13 is mutated in complementation group 13 of the peroxisome-biogenesis disorders; Liu Y et al.; The peroxisome-biogenesis disorders (PBDs) are a genetically and phenotypically diverse group of diseases caused by defects in peroxisome assembly . One of the milder clinical variants within the PBDs is neonatal adrenoleukodystrophy (NALD), a disease that is usually associated with partial defects in the import of peroxisomal matrix proteins that carry the type 1 or type 2 peroxisomal targeting signals . Here, we characterize the sole representative of complementation group 13 of the PBDs, a patient with NALD (patient PBD222) . Skin fibroblasts from patient PBD222 display defects in the import of multiple peroxisomal matrix proteins . However, residual matrix-protein import can be detected in cells from patient PBD222, consistent with the relatively mild phenotypes of the patient . PEX13 encodes a peroxisomal membrane protein with a cytoplasmically exposed SH3 domain, and we find that expression of human PEX13 restores peroxisomal matrix-protein import in cells from patient PBD222 . Furthermore, these cells are homozygous for a missense mutation at a conserved position in the PEX13 SH3 domain . This mutation attenuated the activity of human PEX13, and an analogous mutation in yeast PEX13 also reduced its activity . The mutation was absent in >100 control alleles, indicating that it is not a common polymorphism . Previous studies have demonstrated extragenic suppression in the PBDs, but the phenotypes of patient PBD222 cells could not be rescued by expression of any other human PEX genes . Taken together, these results provide strong evidence that mutations in PEX13 are responsible for disease in patient PBD222 and, by extension, in complementation group 13 of the PBDs.

Plant Physiol, 1999 Aug, 120(4), 961 - 8
Delivery of a secreted soluble protein to the vacuole via a membrane anchor; Barrieu F et al.; To further understand how membrane proteins are sorted in the secretory system, we devised a strategy that involves the expression of a membrane-anchored yeast invertase in transgenic plants . The construct consisted of a signal peptide followed by the coding region of yeast invertase and the transmembrane domain and cytoplasmic tail of calnexin . The substitution of a lysine near the C terminus of calnexin with a glutamic acid residue ensured progression through the secretory system rather than retention in or return to the endoplasmic reticulum . In the transformed plants, invertase activity and a 70-kD cross-reacting protein were found in the vacuoles . This yeast invertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus . The microsomal fraction contained a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid of invertase activity . Our results indicate that this membrane-anchored protein proceeds in the secretory system beyond the point where soluble proteins are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.

Phys Med Biol, 1999 Jul, 44(7), 1743 - 53
The influence of a clear layer on near-infrared spectrophotometry measurements using a liquid neonatal head phantom; Wolf M et al.; It is difficult to test near-infrared spectrophotometry instruments in vivo . Therefore we constructed a liquid phantom which mimics the neonatal head . It consists of a spherical 3.5 mm thick layer of silicone rubber simulating skin and bone and a 0.5 mm thick clear layer of polypropylene imitating cerebrospinal fluid . It acts as container for a liquid solution with Intralipid, 60 micromol l(-1) haemoglobin and yeast . The solution was oxygenated using oxygen and then deoxygenated by the yeast . From the instrumental (Critikon 2020) algorithm, we found that with increasing scattering (0.5%, 1%, 1.5% and 2% Intralipid concentration) the reading was increasingly offset from the expected value of 0 micromol l(-1) by 55.7, 68.6, 76.5 and 80.4 micromol l(-1) (oxyhaemoglobin) and 16.0, 24.4, 29.6 and 31.7 micromol l(-1) (deoxyhaemoglobin) . This reduced the range of the oxygen saturation reading from the expected 100% to 31.5, 21.1, 14.3 and 11.5% . Haemoglobin concentration changes were increasingly underestimated by a factor of two to four . For a second algorithm based on the diffusion approximation the offsets were smaller: oxyhaemoglobin 11.4, 17.8, 22.5 and 25.1 micromol l(-1) and deoxyhaemoglobin 1.3, 3.4, 5.2 and 6.0 micromol l(-1) . The range of the oxygen saturation reading was higher: 41.3, 29.2, 23.4 and 16.6% . Concentration changes were underestimated by a factor of six to ten . This study demonstrates the need to develop algorithms which take into consideration anatomical structures.

Mamm Genome, 1999 Sep, 10(9), 900 - 5
Genomic organization and mapping of the human and mouse neuronal beta2-nicotinic acetylcholine receptor genes; Lueders KK et al.; As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the beta2-nAChR genes from human and mouse BAC libraries . Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene . We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene . While the sizes of exons in the mouse and human genes are the same, the introns differ in size . Both promoters have a high GC content (60-80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72% . Using a 6-Mb YAC contig of Chr 1, we mapped the human beta2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel . The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1 . We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel . Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3.

Mamm Genome, 1999 Sep, 10(9), 858 - 63
Physical mapping of the autoimmune disease susceptibility locus, Bphs: co-localization with a cluster of genes from the TNF receptor superfamily on mouse chromosome 6; Meeker ND et al.; An important approach to understanding complex diseases is to reduce them into well-characterized subphenotypes that are under monogenic control . One such example is Bordetella pertussis toxin-induced histamine sensitization in mice, a subphenotype of experimental allergic encephalomyelitis and experimental allergic orchitis . This subphenotype is controlled by a single locus, Bphs, previously mapped to a 33 cM region on mouse Chromosome (Chr) 6 . We achieved considerable reduction of this candidate region and constructed a YAC contig across the refined interval . Our results demonstrate that Bphs is located between D6Mit151 and a newly developed marker, EC108RR, a region containing a small cluster of genes belonging to the TNF receptor superfamily . Sequence and quantitative analysis of the candidate gene, tumor necrosis factor receptor 1 (Tnfr1, p55), indicates that it is unlikely to be Bphs . However, the location of Bphs, together with physiologic effects it shares with Tnfr1 activation, suggest that Bphs may prove to be another member of the TNF receptor superfamily.

Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 829 - 32
An Arabidopsis thaliana cDNA complements the N-acetylglucosaminyltransferase I deficiency of CHO Lec1 cells; Bakker H et al.; N-Acetylglucosaminyltransferase I (GlcNAcT-I, EC 2.4.1.101) is the enzyme which initiates the formation of complex N-linked glycans in eukaryotes by transforming GlcNAc to the oligo-mannosyl acceptor Man(5)GlcNAc(2)-Asn . The enzymatic activity and the structure that is synthesised by this enzyme are found in animals and plants but not in yeast . cDNAs encoding the enzyme have already been cloned from several mammals and the nematode Caenorhabditis elegans . In this article the cloning of an Arabidopsis thaliana GlcNAcT-I cDNA with homology to animal cDNAs is described . By expression of the plant cDNA in CHO Lec1 cells, a mammalian cell line deficient in GlcNAcT-I, it was shown that it encodes an active enzyme with the same enzymatic activity as the animal homologue . It has already been shown that a human GlcNAcT-I can complement an A . thaliana mutant (cgl-1) . Here it is shown that the reverse is also true, the plant glycosyltransferase is able to complement a mammalian mutant (Lec1) deficient in GlcNAcT-I .

Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 614 - 21
Human ZHX1: cloning, chromosomal location, and interaction with transcription factor NF-Y; Yamada K et al.; NF-YA, B, and C comprise the heterotrimeric transcription factor known as nuclear factor Y (NF-Y) or CCAAT-binding protein (CBF) . NF-Y binds many CCAAT and Y box (an inverted CCAAT box, ATTGG) elements . Mutations of these elements that disrupt the binding of NF-Y result in decreased transcription from various tissue-specific and inducible promoters . We employed a yeast two-hybrid system to screen a human liver cDNA library in an effort to isolate proteins that interact with NF-Y and that may play a role in tissue-specific or hormone-inducible promoter activity . Using a fragment of the NF-YA subunit as bait we isolated a cDNA that encodes most of the open reading frame of the human zinc fingers and homeobox 1 (ZHX1) protein . The complete open reading frame was subsequently isolated and found to encode a protein of 873 amino acids that contains two zinc fingers and five homeodomain motifs . Northern blot analysis of poly(A)(+) RNA isolated from various tissues revealed two major ZHX1 transcripts of about 4.5 and 5 kilobases . Both transcripts were expressed ubiquitously, although the 5-kilobase transcript is of greater abundance in most tissues examined . The human ZHX1 gene is located on chromosome 8q, between markers CHCL.GATA50B06 and CHLC . GATA7G07 .

Hum Mol Genet, 1999 Sep, 8(9), 1769 - 77
Point mutations throughout the GLI3 gene cause Greig cephalopolysyndactyly syndrome; Kalff-Suske M et al.; Greig cephalopolysyndactyly syndrome, characterized by craniofacial and limb anomalies (GCPS; MIM 175700), previously has been demonstrated to be associated with translocations as well as point mutations affecting one allele of the zinc finger gene GLI3 . In addition to GCPS, Pallister-Hall syndrome (PHS; MIM 146510) and post-axial polydactyly type A (PAP-A; MIM 174200), two other disorders of human development, are caused by GLI3 mutations . In order to gain more insight into the mutational spectrum associated with a single phenotype, we report here the extension of the GLI3 mutation analysis to 24 new GCPS cases . We report the identification of 15 novel mutations present in one of the patient's GLI3 alleles . The mutations map throughout the coding gene regions . The majority are truncating mutations (nine of 15) that engender prematurely terminated protein products mostly but not exclusively N-terminally to or within the central region encoding the DNA-binding domain . Two missense and two splicing mutations mapping within the zinc finger motifs presumably also interfere with DNA binding . The five mutations identified within the protein regions C-terminal to the zinc fingers putatively affect additional functional properties of GLI3 . In cell transfection experiments using fusions of the DNA-binding domain of yeast GAL4 to different segments of GLI3, transactivating capacity was assigned to two adjacent independent domains (TA(1)and TA(2)) in the C-terminal third of GLI3 . Since these are the only functional domains affected by three C-terminally truncating mutations, we postulate that GCPS may be due either to haploinsufficiency resulting from the complete loss of one gene copy or to functional haploinsufficiency related to compromised properties of this transcription factor such as DNA binding and transactivation.

Biochemistry, 1999 Aug 10, 38(32), 10371 - 6
Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2; Chung H et al.; The cellular location and substrate specificity of the catalytic subunit (C) of protein phosphatase 2A (PP2A) depend on its interaction with A and B subunits . The distribution of epitope-tagged wild-type or mutated C subunits was studied by transient expression in COS-7 cells . Wild-type tagged C expressed at low levels formed ABC trimer and AC dimer like the endogenous C . Single mutations of C at the site of phosphorylation (Y307F) or carboxymethylation (L309Q) resulted in recovery of only AC dimer . Double mutation of both residues resulted in association of C with alpha 4 protein (alpha 4), a novel subunit of PP2A, instead of with A and B subunits . Thus, the distribution of C between ABC trimer, AC dimer, and alpha 4C complexes can be affected by modifications of the C-terminal residues . The alpha 4 protein is a homologue of the yeast Tap42 protein that functions downstream of the TOR protein to regulate protein synthesis . Transient overexpression of FLAG-alpha 4 resulted in increased dephosphorylation of elongation factor 2, but had no effect on phosphorylation of either p70S6 kinase or PHAS-I (eIF4E-BP) . Signals that affect phosphorylation or methylation of the C subunit of PP2A may promote subunit exchange and direct phosphatase activity to specific intracellular substrates.

Nature, 1999 Jul 29, 400(6743), 461 - 4
Cohesin Rec8 is required for reductional chromosome segregation at meiosis; Watanabe Y et al.; When cells exit from mitotic cell division, their sister chromatids lose cohesion and separate to opposite poles of the dividing cell, resulting in equational chromosome segregation . In contrast, the reductional segregation of the first stage of meiotic cell division (meiosis I) requires that sister chromatids remain associated through their centromeres and move together to the same pole . Centromeric cohesion is lost as cells exit from meiosis II and sister chromatids can then separate . The fission yeast cohesin protein Rec8 is specific to and required for meiosis . Here we show that Rec8 appears in the centromeres and adjacent chromosome arms during the pre-meiotic S phase . Centromeric Rec8 persists throughout meiosis I and disappears at anaphase of meiosis II . When the rec8 gene is deleted, sister chromatids separate at meiosis I, resulting in equational rather than reductional chromosome segregation . We propose that the persistence of Rec8 at centromeres during meiosis I maintains sister-chromatid cohesion, and that its presence in the centromere-adjacent regions orients the kinetochores so that sister chromatids move to the same pole . This results in the reductional pattern of chromosome segregation necessary to reduce a diploid zygote to haploid gametes.

Nat Toxins, 1999, 7(1), 31 - 8
Oxidative deamination of hydrolyzed fumonisin B(1) (AP(1)) by cultures of Exophiala spinifera; Blackwell BA et al.; Fumonisins are mycotoxins of world-wide distribution in maize infected by the fungus Fusarium verticillioides . They are highly toxic to certain livestock and are potential carcinogens . Exophiala spinifera, a black yeast fungus found on moldy maize kernels, was identified previously as capable of growing on fumonisin B1 as a sole carbon source and thus is a potential source for fumonisin detoxifying enzymes . Pure cultures of E . spinifera transform fumonisin B(1) to the amino polyol AP(1) plus free tricarballylic acid through the activity of a soluble extracellular esterase, and further transformation is evidenced by accumulation in culture supernatant of a less polar compound(s) lacking a fluorescamine-reactive amino group . A free amine is thought to be critical for biological activity of FB(1) or AP(1) . As a first step towards characterizing this amine-modifying activity, we investigated the biotransformation of AP(1) by E . spinifera liquid cultures that had been previously grown in liquid medium containing AP(1) as a sole carbon source . Accumulation of AP(1)-derived metabolites was monitored by thin-layer chromatography of culture supernatants, and product metabolites were purified and evaluated by mass spectrometry and nuclear magnetic resonance . Two products of treatment of purified AP(1) with cultures of E . spinifera are shown to be N-acetyl AP(1) and a new compound, 2-oxo-12,16-dimethyl-3,5,10, 14,15-icosanepentol hemiketal (or 2-OP(1) hemiketal) .

J Immunol, 1999 Aug 15, 163(4), 1991 - 9
Localization on a physical map of the NKC-linked Cmv1 locus between Ly49b and the Prp gene cluster on mouse chromosome 6; Brown MG et al.; The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV . Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6 . We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters . This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice . Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus . To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples . These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of approximately 390 kb . This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.

J Virol, 1999 Sep, 73(9), 7231 - 40
Hepatitis B virus X protein is both a substrate and a potential inhibitor of the proteasome complex; Hu Z et al.; The hepatitis B virus X protein (HBX) is essential for the establishment of HBV infection in vivo and exerts a pleiotropic effect on diverse cellular functions . The yeast two-hybrid system had indicated that HBX could interact with two subunits of the 26S proteasome . Here we demonstrate an association in vivo of HBX with the 26S proteasome complex by coimmunoprecipitation and colocalization upon sucrose gradient centrifugation . Expression of HBX in HepG2 cells caused a modest decrease in the proteasome's chymotrypsin- and trypsin-like activities and in hydrolysis of ubiquitinated lysozyme, suggesting that HBX functions as an inhibitor of proteasome . In these cells, HBX is degraded with a half-life of 30 min . Proteasome inhibitors retarded this rapid degradation and caused a marked increase in the level of HBX and an accumulation of HBX in polyubiquitinated form . Thus, the low intracellular level of HBX is due to rapid proteolysis by the ubiquitin-proteasome pathway . Surprisingly, the proteasome inhibitors blocked the transactivation by HBX, and this effect was not a result of a squelching phenomenon due to HBX accumulation . Therefore, proteasome function is possibly required for the transactivation function of HBX . The inhibition of protein breakdown by proteasomes may account for the multiple actions of HBX and may be an important feature of HBV infection, possibly in helping stabilize viral gene products and suppressing antigen presentation.

Blood, 1999 Aug 15, 94(4), 1254 - 60
Dyskeratosis congenita caused by a 3' deletion: germline and somatic mosaicism in a female carrier; Vulliamy TJ et al.; X-linked dyskeratosis congenita (DC) is a bone marrow failure syndrome caused by mutations in the DKC1 gene located at Xq28 . By 20 years of age, most affected boys develop bone marrow failure, whereas female carriers show a skewed pattern of X-chromosome inactivation . The gene product, dyskerin, is homologous to a yeast protein involved in ribosomal RNA biogenesis, providing a unique insight into a cause of aplastic anemia . Whereas most causative mutations are single amino acid substitutions, and nonsense or frameshift mutations have not been observed, we present here a case of DC caused by a 2-kb deletion that removes the last exon of the gene . Normal levels of mRNA are produced from the deleted gene, with the transcripts using a cryptic polyadenylation site in the antisense strand of the adjacent MPP1 gene, normally located 1 kb downstream of DKC1 in a tail to tail orientation . The predicted truncated protein lacks a lysine-rich peptide that is less conserved than the rest of the dyskerin molecule and is dispensable in yeast, supporting the contention that it may retain some activity and that null mutations at this locus may be lethal . The affected boy had an unaffected brother with the same haplotype around the DKC1 gene and a sister who was heterozygous for the deletion . We conclude therefore that the mother must be a germline mosaic with respect to this deletion . Investigation of her blood cells and other somatic tissues showed that a small proportion of these cells also carried the deletion, making her a somatic mosaic and indicating that the deletion took place early in development.

J Biol Chem, 1999 Aug 13, 274(33), 23480 - 90
Isolation of mouse TFIID and functional characterization of TBP and TFIID in mediating estrogen receptor and chromatin transcription; Wu SY et al.; TFIID is a general transcription factor required for the assembly of the transcription machinery on most eukaryotic promoters transcribed by RNA polymerase II . Although the TATA-binding subunit (TBP) of TFIID is able to support core promoter and activator-dependent transcription under some circumstances, the roles of TBP-associated factors (TAF(II)s) in TFIID-mediated activation remain unclear . To define the evolutionarily conserved function of TFIID and to elucidate the roles of TAF(II)s in gene activation, we have cloned the mouse TAF(II)55 subunit of TFIID and further isolated mouse TFIID from a murine FM3A-derived cell line that constitutively expresses FLAG-tagged mouse TAF(II)55 . Both mouse and human TFIIDs are capable of mediating transcriptional activation by Gal4 fusions containing different activation domains in a highly purified human cell-free transcription system devoid of TFIIA and Mediator . Although TAF(II)-independent activation by Gal4-VP16 can also be observed in this highly purified human transcription system with either mouse or yeast TBP, TAF(II)s are strictly required for estrogen receptor-mediated activation independently of the core promoter sequence . In addition, TAF(II)s are necessary for transcription from a preassembled chromatin template . These findings clearly demonstrate an essential role of TAF(II)s as a transcriptional coactivator for estrogen receptor and in chromatin transcription.

J Biol Chem, 1999 Aug 13, 274(33), 23119 - 27
beta(1) integrin binds the 16-kDa subunit of vacuolar H(+)-ATPase at a site important for human papillomavirus E5 and platelet-derived growth factor signaling; Skinner MA et al.; Integrins mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction . We have identified an interaction between the beta(1) integrin and the 16-kDa subunit of vacuolar H(+)-ATPase (16K) . This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and in in vitro binding assays using bacterially expressed proteins . Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged 16K demonstrate co-localization with beta(1) integrin in focal adhesions . Deletion of the fourth of four transmembrane helices in 16K results in loss of interaction with beta(1) integrin in vitro and in the two-hybrid system, and less prominent staining in focal adhesions . This helix is also required for ligand-independent activation of platelet-derived growth factor-beta receptor signaling by the human papillomavirus E5 oncoprotein . Overexpression of 16K or expression of 16K lacking this helix alters the morphology of myoblasts and fibroblasts, suggesting that the interaction of 16K with integrins could be important for cell growth control . We also discuss the possible role 16K might play in integrin movement.

J Anim Sci, 1999 Jul, 77(7), 1702 - 9
Ubiquitin gene expression and ubiquitin conjugation in chicken muscle do not reflect differences in growth rate between broiler and layer birds; Harper JM et al.; Previous work has shown that chicken strains selected for growth (broilers) degrade muscle proteins less rapidly than those selected for egg laying . They also have decreased calpain and increased calpastatin content in breast muscle . This study aimed to test the hypothesis that these differences correlate with changes in the ATP- and ubiquitin-dependent proteolytic system . Chickens of a broiler strain (Ross 1) and a layer strain (ISABrown) were reared to the age of 4 wk under identical conditions with ad libitum access to feed and water . Mean fractional growth rates were 10.4%/d for broilers and 7.4%/d for layers . Feed intake measured in the last week of the trial was slightly greater in layer birds (.11 and .12 g x g body weight(-1) x d(-1) for broilers and layers respectively; P < .006) . Polyubiquitin (UbI) messenger RNA was abundant in the muscles of these well-fed birds, but it showed little difference between strains . Muscle did not significantly express the UbII polyubiquitin gene . The ATP-dependent system conjugating ubiquitin to endogenous proteins had greatest activity in the gastrocnemius muscle of broiler birds but was not significantly different between breeds . Proteins cross-reactive with antisera to recombinant human proteasome regulatory subunits MSS1 (multicopy suppressor of SUG 1; S7) and TBP1 (tat binding protein 1; S6') were present in muscle homogenates from both strains of bird . The chick equivalent of TBP1 was more abundant in breast muscle of broiler birds than in leg muscle, or in either muscle of layers . Antiserum to recombinant yeast subunit mts2 (mitosis temperature sensitive gene 2; S4) did not react with any protein of the expected size but detected a 30-kDa peptide that was not associated with the 26S proteasome; this was found only in muscle from the layer strain . Hence, during normal growth of chickens, rates of protein degradation are not controlled by the expression of ubiquitin mRNA or the conjugation of ubiquitin . However, the composition of the 26S proteasome may be a regulatory factor.

Gene, 1999 Aug 5, 236(1), 21 - 4
Genomic structure of the human PLZF gene; van Schothorst EM et al.; The human PLZF (promyelocytic leukaemia zinc finger) gene encodes a Kruppel-like zinc finger protein, which was identified via the reciprocal translocation t(11;17)(q23;q21) fusing it to the retinoic acid receptor alpha (RARalpha) gene in promyelocytic leukaemia . To determine its complete genomic organisation, we constructed a cosmid-map fully containing the hPLZF gene . The gene has seven exons, including a novel 5' untranslated exon, varying in size from 87 to 1358bp and spans at least 120kb . Flanking intronic sequences were identified and all splice acceptor and donor sites conformed to the gt/ag rule . Five polymorphic markers could be fine located in its vicinity . These data will facilitate mutation analysis of hPLZF in t(11;17) leukaemia cases, as well as assist mapping and loss-of-heterozygosity analysis . Here we have tested hPLZF as a possible candidate for the PGL1 locus involved in hereditary head and neck paragangliomas . However, mutation analysis revealed no aberration in 12 paraganglioma patients from different families.

Mol Biol Cell, 1999 Aug, 10(8), 2787 - 802
Characterization of a fourth adaptor-related protein complex; Hirst J et al.; Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat . To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families . We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (epsilon, beta4, mu4, and sigma4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4 . Immunofluorescence reveals that AP-4 is localized to approximately 10-20 discrete dots in the perinuclear region of the cell . This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF . Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network . The mu4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs . AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

Mol Biol Cell, 1999 Aug, 10(8), 2655 - 68
The Werner syndrome protein is involved in RNA polymerase II transcription; Balajee AS et al.; Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process . The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence . The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase . Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination . We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations . Transcription was measured in permeabilized cells by {3H}UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter . With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals . This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells . Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts . A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay . This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay . We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator . A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Mol Biol Cell, 1999 Aug, 10(8), 2631 - 45
The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells; Herbig U et al.; The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication . A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form . The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective . The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides . Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.

FEMS Immunol Med Microbiol, 1999 Jul 15, 24(4), 411 - 20
Chemical and immunochemical characterization of limulus factor G-activating substance of Candida spp; Uchiyama M et al.; The limulus test is a well-established method for the diagnosis of both gram (-) sepsis and invasive fungal infection . To diagnose deep-seated fungal infections, a (1-->3)-beta-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically . It is suggested that the limulus reactive substance was released from the fungi to the blood, however, its chemical properties were not precisely examined in detail because of the limited quantity available . In this study, we used chemically defined liquid medium to culture Candida spp . and collected the water soluble fraction, CAWS . The yield of CAWS was circa 100 mg/l, independent of the strain of Candida . CAWS reacted with limulus factor G (Fungitec G test MK) at concentrations as low as 100 ng/ml . Limulus factor G reactivity of CAWS was sensitive to (1-->3)-beta-glucanase, zymolyase and was, at least in part, bound to ConA-agarose . The ConA-bound fraction also reacted with anti-beta-glucan antibody . CAWS is mainly composed of mannan and (1-->6)-beta-glucan, in addition to protein, assessed by 1H-NMR spectroscopy . CAWS also reacted with typing sera of Candida spp., specific for cell wall mannan . Chemical, immunochemical and biochemical analyses of CAWS strongly suggested that the limulus factor G-activating substance was a mannan-beta-glucan complex, present within the architecture of the yeast cell wall.

Oncogene, 1999 Jul 15, 18(28), 4131 - 6
Allelic loss mapping and physical delineation of a region harboring a putative thymic lymphoma suppressor gene on mouse chromosome 12; Shinbo T et al.; Our previous allelic loss analysis of gamma-ray induced thymic lymphomas in F1 hybrid and backcross mice between BALB/c and MSM strains mapped the Tlsr4 region exhibiting a high frequency of allelic loss (62%) to a 2.9 cM interval between the markers D12Mit53 and D12Mit279 on mouse chromosome 12 . To narrow further the interval harboring a putative tumor suppressor gene, a high-density scan has been carried out for informative 361 thymic lymphomas . Construction of a physical map of Tlsr4 with 3 YAC and 15 BAC clones and isolation of YAC- and BAC-derived polymorphic probes lead to fine allelic loss mapping . Three successive polymorphic sites within one BAC exhibit the retention of both alleles in seven, one and four lymphomas, suggesting that a common region of allelic loss for Tlsr4 exists within the BAC region . Pulsed-field gel electrophoresis of NotI digests of this and other clones determines that the commonly lost region is a 35 kb interval with a NotI site . NotI sites are frequently associated with coding regions, and our preliminary sequencing has identified ESTs in the region . Thus, the present study facilitates the identification of genes in the Tlsr4 region that would lead to isolation of a novel tumor suppressor gene.

Gene Ther, 1999 Mar, 6(3), 393 - 402
Reduced toxicity, attenuated immunogenicity and efficient mediation of human p53 gene expression in vivo by an adenovirus vector with deleted E1-E3 and inactivated E4 by GAL4-TATA promoter replacement; Ji L et al.; A recombinant adenovirus with deleted E1 and E3, and E4-inactivated by replacing the E4 promoter with a synthetic promoter composed of a minimal TATA box and five consensus yeast GAL4-binding site elements was developed and used to express the human tumor suppresser gene p53 . The toxicity and immunogenicity of this vector and vector-mediated p53 gene expression in vivo were studied in immunocompetent C3H and C57BL/6 mice . Expression of the late viral gene product, hexon protein, was observed in C3H and C57BL/6 mice injected with E4 wild-type adenovirus constructs Adv-cmv-beta-Gal (BG), Adv-cmv-hp53 (WT), and empty E1- vector Adv-E4 (EW) 3 to 28 days after injection, but was undetectable in mice treated with E4 modified empty E1- vector Adv-GAL4 (EG) or Adv-cmv-hp53-GAL4 (G4) . Expression of the p53 gene was observed in both WT- and G4-injected C3H and C57BL/6 mouse livers from days 3 to 28 . Ten weeks after injection, p53 gene expression was still detected in G4-treated C57BL/6 mice at similar levels, but was not detectable in WT-treated mice . Vector-induced liver toxicity was evaluated by analyzing serum transaminases (SGOT and SGPT) activities . In all cases, SGOT and SGPT activities were markedly decreased in EG-treated C3H and C57BL/6 mice compared with those in EW-treated mice on days 3, 7 and 14 after injection . In C57BL/6 mice, the total anti-adenoviral CTL activities were two- to three-fold higher in animals treated with EW vector than in those treated with EG vector . These results suggest that inactivation of the E4 promoter efficiently diminished the viral replication and the late viral gene expression, reduced host immune response and consequently reduced toxicity and prolonged the duration of transgene expression in vivo.

Gene Ther, 1999 Mar, 6(3), 385 - 92
Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins; Rade JJ et al.; We constructed a hirudin cDNA cassette, HV-1.1, that encodes mature hirudin variant-1 fused to the signal peptide of human tissue-type plasminogen activator (t-PA) . The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro . Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively . Transduced cells secreted up to 35 +/- 2 ng/10(6) cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks . Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 +/- 0.2 antithrombin units per microgram (ATU/microgram), compared with specific activities of approximately 12 ATU/microgram for both native leech hirudin and recombinant hirudin produced in yeast . Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity . We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone . Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 +/- 0.2 ATU/microgram . Protein sequencing and mass spectroscopic analysis demonstrated proper cleavage of the growth hormone signal peptide . Thus, we achieved high level retrovirus-mediated secretion of biologically active hirudin from endothelial cells in vitro . Use of these vectors may permit sustained local antagonism of thrombin activity in vivo.

Biochim Biophys Acta, 1999 Aug 5, 1428(2-3), 161 - 8
Evidence for the interaction between Translin and GADD34 in mammalian cells; Hasegawa T et al.; To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with the murine GADD34 gene product . We utilized, as bait, the product of the GADD34 cDNA deletions including the PEST region and the gamma(1)34.5 domain . One of the cDNAs cloned encoded murine Translin which is known to bind to the DNA sequence detected in the DNA translocation . The interaction between GADD34 and Translin was also confirmed by an in vitro binding assay and in vivo two-hybrid analysis in NIH 3T3 cells . Although GADD34 expression was significantly elevated with methyl methanesulfonate treatment, we could not detect the induction of Translin mRNA.

Development, 1999 Sep, 126(17), 3937 - 45
The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila; Emmons RB et al.; The Drosophila spineless (ss) gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth . ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor . Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt) . Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons . In this report, we present evidence that Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo) . We show that the ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss(-) mutations . The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers . Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability . During embryogenesis, Tgo is localized to the nucleus at sites of ss expression . This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus . Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage . This suggests that the interaction of Ss and Tgo does not require additional signals, unlike the ligand-dependent interaction of Ahr and Arnt . Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved.

Nat Genet, 1999 Aug, 22(4), 388 - 93
A YAC-based physical map of the mouse genome; Nusbaum C et al.; A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research . Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project . Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse . We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map . The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome . We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X . The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome . The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome . More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Trends Plant Sci, 1999 Aug, 4(8), 315 - 319
Trehalose metabolism in plants; Goddijn OJ et al.; It has long been thought that the biosynthesis of trehalose, a sugar present in all kingdoms, is absent from the vast majority of higher plants . However, recent experiments have indicated that genes from Arabidopsis are able to complement yeast strains deficient in trehalose metabolism . In yeast, trehalose has been suggested as a regulatory component in the control of glycolytic flux and in a variety of stress survival strategies . Thus, the occurrence of complimentary genes in Arabidopsis and yeast might lead to the development of strategies and applications for improvement of crop plants.

Trends Genet, 1999 Aug, 15(8), 294 - 7
The Dictyostelium genome project: an invitation to species hopping; Kay RR et al.; Dictyostelium allows some of the general problems of eukaryotic biology to be addressed by using molecular genetic tools that are more normally associated with yeast . The genome project, now underway, marks an important increase in the attractiveness of Dictyostelium as an experimental organism and will invite increased 'species hopping' by experimenters . We provide a brief guide to the problems that are being addressed in Dictyostelium, to the genome project itself and to the molecular genetic tools available for its exploitation.

Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9252 - 7
Fine-mapping of quantitative trait loci by identity by descent in outbred populations: application to milk production in dairy cattle; Riquet J et al.; We previously mapped a quantitative trait locus (QTL) affecting milk production to bovine chromosome 14 . To refine the map position of this QTL, we have increased the density of the genetic map of BTA14q11-16 by addition of nine microsatellites and three single nucleotide polymorphisms . Fine-mapping of the QTL was accomplished by a two-tiered approach . In the first phase, we identified seven sires heterozygous "Qq" for the QTL by marker-assisted segregation analysis in a Holstein-Friesian pedigree comprising 1,158 individuals . In a second phase, we genotyped the seven selected sires for the newly developed high-density marker map and searched for a shared haplotype flanking an hypothetical, identical-by-descent QTL allele with large substitution effect . The seven chromosomes increasing milk fat percentage were indeed shown to carry a common chromosome segment with an estimated size of 5 cM predicted to contain the studied QTL . The same haplotype was shown to be associated with increased fat percentage in the general population as well, providing additional support in favor of the location of the QTL within the corresponding interval.

Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8895 - 900
Identification and characterization of a human tRNA-specific adenosine deaminase related to the ADAR family of pre-mRNA editing enzymes; Maas S et al.; The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification . We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala) . It represents the functional homologue of the recently identified yeast protein Tad1p {Gerber, A., Grosjean, H., Melcher, T . & Keller, W . (1998) EMBO J . 17, 4780-4789} . The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a Drosophila melanogaster ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins . In vitro, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A(37) in the anticodon loop of tRNA(Ala) from higher eukaryotes and with lower efficiency from lower eukaryotes . It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2 . The anticodon stem-loop of tRNA(Ala) alone is not a functional substrate for hADAT1 . The enzyme is expressed ubiquitously in human tissues and is represented by a single gene . The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA(Ala), which is conserved from yeast to man.

Clin Chem, 1999 Aug, 45(8 Pt 1), 1248 - 54
Structural investigations of a new familial dysalbuminemic hyperthyroxinemia genotype; Petersen CE et al.; BACKGROUND: In a previous study, we found that the amino acid substitution R218H in human serum albumin (HSA) was the cause of familial dysalbuminemic hyperthyroxinemia (FDH) in several Caucasian patients . Subsequently the substitution R218P was shown to be the cause of FDH in several members of a Japanese family . This study attempts to resolve discrepancies in the only other study of R218P HSA and identifies two new Japanese R218P FDH patients unrelated to those described previously . METHODS AND RESULTS: Recombinant R218H, R218P, and wild-type HSA were synthesized in yeast, and the affinities of these HSA species for l- and d-thyroxine were determined using fluorescence spectroscopy . The dissociation constants for the binding of wild-type, R218P, and R218H HSA to l-thyroxine were 1.44 x 10(-6), 2.64 x 10(-7), and 2.49 x 10(-7) mol/L, respectively . The circular dichroism spectra of thyroxine bound to R218H and R218P HSA were markedly different, indicating that the structure of the thyroxine/HSA complex is different for either protein . CONCLUSIONS: The K(d) values for l-thyroxine bound to R218P and R218H HSA determined in this study were similar . The extremely high serum total-thyroxine concentrations reported previously for R218P FDH patients (10-fold higher than those reported for R218H FDH patients) are not consistent with the K(d) values determined in this study . Possible explanations for these discrepancies are discussed.

Mamm Genome, 1999 Aug, 10(8), 824 - 30
A first-generation porcine whole-genome radiation hybrid map; Hawken RJ et al.; A whole-genome radiation hybrid (WG-RH) panel was used to generate a first-generation radiation map of the porcine (Sus scrofa) genome . Over 900 Type I and II markers were used to amplify the INRA-University of Minnesota porcine Radiation Hybrid panel (IMpRH) comprised of 118 hybrid clones . Average marker retention frequency of 29.3% was calculated with 757 scorable markers . The RHMAP program established 128 linkage groups covering each chromosome (n = 19) at a lod >/= 4.8 . Fewer than 10% of the markers (59) could not be placed within any linkage group at a lod score >/=4.8 . Linkage group order for each chromosome was determined by incorporating linkage data from the swine genetic map as well as physical assignments . The current map has an estimated ratio of approximately 70 kb/cR and a maximum theoretical resolution of 145 kb . This initial map forms a template for establishing accurate YAC and BAC contigs and eventual positional cloning of genes associated with complex traits.

EMBO J, 1999 Aug 2, 18(15), 4348 - 58
Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIPalpha but not importin alpha; Jullien D et al.; Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism . RPA consists of three subunits (70, 33 and 14 kDa) . We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA . XRIPalpha homologues are found in human and in Drosophila but not in yeast . XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta . We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA . Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate . RPA import can be restored by addition of recombinant XRIPalpha . Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA . GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro . Finally, RPA import can be reconstituted from the recombinant proteins . We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.

Eur J Immunol, 1999 Jul, 29(7), 2065 - 71
The 5' part of the mouse immunoglobulin kappa locus; Roschenthaler F et al.; The 5' region of the mouse kappa locus comprises 63 Vkappa genes in six contigs of together 1.5 Mb, including one which links the region to the central part of the locus . The structures of the contigs were established by detailed restriction mapping of cosmid clones prepared from libraries of mouse C57BL/6 DNA and of yeast and bacterial artificial chromosomes (YACs, BACs with mouse DNA inserts) . Pulsed-field gel electrophoresis of yeast artificial chromosome digests indicated that the gaps between the contigs were 10 to 60 kb, comprising together about 160 kb . The region of the kappa locus described here contains Vkappa1, Vkappa2, Vkappa9/10, Vkappa11, Vkappa12/13, Vkappa20, Vkappa24, Vkappa32, Vkappa33/34 and Vkappa38C genes as well as the VkappaRF gene and, towards the center of the locus, a number of Vkappa4/5 genes . Near the 5' end of the locus interspersed alpha-tubulin gene-like sequences were found . At its 3' side the region borders on the Vkappa4/5 contigs of the central region of the locus which is described in the accompanying report (Eur . J . Immunol . 1999 . 29: 2057-2064) . Structural details are to be found in the Internet at au/kappa.htm . In a concluding section the main features of the structure of the mouse kappa locus are summarized.

Eur J Immunol, 1999 Jul, 29(7), 2057 - 64
The central part of the mouse immunoglobulin kappa locus; Kirschbaum T et al.; At the present state of analysis the central part of the kappa locus comprises four contigs of together 1.2 Mb and contains 55 Vkappa genes . It is flanked by the 3' part of the locus with 22 Vkappa genes in 0.4 Mb (T . Kirschbaum et al., Eur . J . Immunol . 1998 . 28: 1458-1466) and the 5' part with 63 Vkappa genes in six contigs of together 1.5 Mb (F . Roschenthaler et al., accompanying report) . The 5' and the central regions have one large contig in common . A part of the central region is linked to the 3' region resulting in a 1.1-Mb contig . The structure of the contigs was established mainly by the analysis of overlapping cosmid clones derived from genomic DNA and yeast and bacterial artificial chromosomes (YACs and BACs) and by PCR techniques . Pulsed-field gel electrophoresis of YAC digests indicated that three gaps between the contigs of the central region are 10-40 kb in size, comprising together about 90 kb . Internal duplications in this part of the locus and rearranged YACs were the major problems of the structural work . Structural details are to be found on the Internet at au/kappa.htm . In a concluding section of the report the mouse kappa locus is compared to the human one and some aspects of the evolution of the kappa locus are discussed.

J Biol Chem, 1999 Aug 6, 274(32), 22847 - 54
A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates; Schulke N et al.; Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane . These translocases reversibly cooperate during protein import . We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Delta(1)-pyrroline-5-carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A . When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes . Consequently, the two membranes of mitochondria become progressively "zippered" together, forming long stretches in which they are in close contact (Schulke, N., Sepuri, N . B . V., and Pain, D . (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 7314-7319) . We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth . Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70 . Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.

J Biol Chem, 1999 Aug 6, 274(32), 22618 - 26
Hormone-independent transcriptional activation and coactivator binding by novel orphan nuclear receptor ERR3; Hong H et al.; Orphan nuclear receptors share sequence homology with members of the nuclear receptor superfamily, but ligands are unknown or unnecessary . A novel orphan receptor, estrogen receptor-related protein 3 (ERR3), was identified by yeast two-hybrid screening, using the transcriptional coactivator glucocorticoid receptor interacting protein 1 (GRIP1) as bait . The putative full-length mouse ERR3 contains 458 amino acids and is closely related to two known orphan receptors ERR1 and ERR2 . All the ERR family members share an almost identical DNA-binding domain, which has 68% amino acid identity with that of estrogen receptor . ERR3 bound specifically to an estrogen response element and activated reporter genes controlled by estrogen response elements, both in yeast and in mammalian cells, in the absence of any added ligand . A conserved AF-2 activation domain located in the hormone-binding domain of ERR3 was primarily responsible for transcriptional activation . The ERR3 AF-2 domain bound GRIP1 in a ligand-independent manner both in vitro and in vivo, through the LXXLL motifs of GRIP1, and GRIP1 functioned as a transcriptional coactivator for ERR3 in both yeast and mammalian cells . Expression of ERR3 in adult mouse was restricted; highest expression was observed in heart, kidney, and brain . In the mouse embryo no expression was observed at day 7, and highest expression occurred around the 11-15 day stages . Although ERR3 is much more closely related to ERR2 than to ERR1, the expression pattern for ERR3 was similar to that of ERR1 and distinct from that for ERR2, suggesting a unique role for ERR3 in development.

J Biol Chem, 1999 Aug 6, 274(32), 22586 - 90
Rpb4p is necessary for RNA polymerase II activity at high temperature; Maillet I et al.; Rpb4p and Rpb7p are two subunits of the yeast RNA polymerase II, which form a subcomplex that can dissociate from the enzyme in vitro . Whereas RPB7 is essential, RPB4 is dispensable for cellular viability . However, the rpb4 null mutant is heat-sensitive, and it has been suggested that Rpb4p is an essential component for cellular stress response . To examine this hypothesis, we used two-dimensional gel electrophoresis to analyze the protein expression pattern of the rpb4 null mutant in response to heat shock, oxidative stress, osmotic stress, and in the post-diauxic phase . We show that this mutant is not impaired in stress induced transcriptional activation: the absence of heat shock response of the mutant is due to a general defect in RNA polymerase II activity at high temperature . Under this condition, Rpb4p is necessary to maintain the polymerase activity in vivo . The heat growth defect of the rpb4 null mutant can be partially suppressed by overexpression of RPB7, suggesting that Rpb4p maintains or stabilizes Rpb7p in the RNA polymerase . We also demonstrate that rpb4 null mutant is an appropriate tool to analyze the involvement of transcriptional events in the survival and adaptation to heat shock or other stresses.

FASEB J, 1999 Aug, 13(11), 1385 - 93
The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans; Honda Y et al.; Longevity is regulated by the daf-2 gene network in Caenorhabditis elegans . Mutations in the daf-2 gene, which encodes a member of the insulin receptor family, confer the life extension (Age) phenotype and the constitutive dauer (a growth-arrested larval form specialized for dispersal) formation phenotype . The Age phenotype is mutually potentiated by two life extension mutations in the daf-2 gene and the clk-1 gene, a homologue of yeast CAT5/COQ7 known to regulate ubiquinone biosynthesis . In this study, we demonstrated that the daf-2 mutation also conferred an oxidative stress resistance (Oxr) phenotype, which was also enhanced by the clk-1 mutation . Similar to the Age phenotype, the Oxr phenotype was regulated by the genetic pathway of insulin-like signaling from daf-2 to the daf-16 gene, a homologue of the HNF-3/forkhead transcription factor . These findings led us to examine whether the insulin-like signaling pathway regulates the gene expression of antioxidant defense enzymes . We found that the mRNA level of the sod-3 gene, which encodes Mn-superoxide dismutase (SOD), was much higher in daf-2 mutants than in the wild type . Moreover, the increased sod-3 gene expression phenotype is regulated by the insulin-like signaling pathway . Although the clk-1 mutant itself did not display Oxr and the increased sod-3 expression phenotypes, the clk-1 mutation enhanced them in the daf-2 mutant, suggesting that clk-1 is involved in longevity in two ways: clk-1 composes the original clk-1 longevity program and the daf-2 longevity program . These observations suggest that the daf-2 gene network controls longevity by regulating the Mn-SOD-associated antioxidant defense system . This system appears to play a role in efficient life maintenance at the dauer stage.

FEBS Lett, 1999 Jul 16, 455(1-2), 31 - 5
Identification of a new gene encoding pericentromeric dodeca-satellite binding protein in Drosophila melanogaster; De Felice B et al.; Dodeca-satellite (CCCGTACTCGGT)n is a type of tandemly repeated DNA sequence located in the pericentromeric region of the third chromosome of Drosophila melanogaster and that cross-hybridizes with DNA from other species such as Arabidopsis, mouse and human . This evolutionary conservation suggests that dodeca-satellite might play an important role in the centromeric function . Therefore, the aim of our research was the isolament of genes encoding proteins that might help stabilize these DNA structures, in vivo . To identify D . melanogaster sequence DNAs encoding dodeca-satellite binding proteins, we used the in vivo yeast assay, known as 'one-hybrid system' . Here, we identified a novel gene sequence that encoded pericentromeric dodeca-satellite binding protein and described its sequence characteristics.

Z Gastroenterol, 1999 Jun, 37(6), 519 - 23
Association of lymphocytic colitis with linear IgA dermatosis; Stuber E et al.; The case of a 66-year-old female patient is presented, who suffered from chronic watery diarrhea . In addition, she developed linear IgA dermatosis after oral treatment of a presumed yeast infection with nystatin . To evaluate the reason for her diarrhea, colonoscopy was performed . The macroscopic aspect of the colon mucosa was described as normal with no specific alterations for chronic inflammatory bowel disease or for bacterial infections . In contrast, the histologic examination revealed the typical characteristics of lymphocytic colitis . This disease is thought to be caused by immunological reactions against as yet unknown luminal antigens . After treatment with steroids and dapsone the diarrhea as well as the skin disease disappeared . To our knowledge, the present report describes for the first time the association of linear IgA dermatosis with lymphocytic colitis after oral treatment with nystatin . A possible causative link between these two disease entities is discussed.

Antonie Van Leeuwenhoek, 1999 Apr, 75(3), 261 - 6
An acetylsalicylic acid-sensitive aggregation phenomenon in Dipodascopsis uninucleata; Kock JL et al.; Aggregation of ascospores has been discovered in the yeast Dipodascopsis uninucleata . When this yeast is cultivated to reach the sexual reproductive stage, small ascospores are individually released from the tip of a sac-like ascus which then aggregate in orderly clusters . Acetylsalicylic acid (ASA) inhibited ascospore release and subsequent ordered aggregation process . We suggest that novel ASA-sensitive oxidised fatty acids (3R-hydroxy-oxylipins) and small hooks located on the surface of these ascospores, are involved.

Dev Comp Immunol, 1999 Jun-Jul, 23(4-5), 421 - 7
Opsonic complement system of the solitary ascidian, Halocynthia roretzi; Nonaka M et al.; To elucidate the molecular architecture and function of the possibly primitive complement system of the solitary ascidian . Halochynthia roretzi, cDNA clones for the third component (C3) and mannose-binding lectin (MBL)-associated serine protease (MASP) were isolated from the hepatopancreas cDNA library . The deduced primary structure of ascidian C3 (AsC3) shows overall similarity to mammalian C3 including a typical thioester site . Two distinct ascidian MASPs, termed AsMASPa and AsMASPb, have the same domain structure as mammalian Clr/ Cls/MASP-1/MASP-2 . Both of them show a closer similarity to mammalian MASP-1 than to mammalian Clr/Cls/ MASP-2 . Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and an antibody against AsC3 inhibits this opsonic activity . These results indicate that the lectin-dependent, opsonic complement system was present prior to the emergence of the vertebrates and well ahead of the establishment of adaptive immunity.

Genet Res, 1999 Jun, 73(3), 189 - 203
A gene responsible for a cuticular hydrocarbon polymorphism in Drosophila melanogaster; Coyne JA et al.; Drosophila melanogaster is polymorphic for the major cuticular hydrocarbon of females . In most populations this hydrocarbon is 7,11-heptacosadiene, but females from Africa and the Caribbean usually possess low levels of 7,11-heptacosadiene and high quantities of its position isomer 5,9-heptacosadiene . Genetic analysis shows that the difference between these two morphs is due to variation at a single segregating factor located on the right arm of chromosome 3 near map position 51.5 and cytological position 87C-D . This is precisely the position of a desaturase gene previously sequenced using primers derived from yeast and mouse, and localized by in situ hybridization to the polytene chromosomes of D . melanogaster . Alleles of this desaturase gene may therefore be responsible for producing the two hydrocarbon morphs . Mating tests following the transfer of these isomers between females of the two morphs show that, in contrast to previous studies, the hydrocarbon profiles have no detectable effect on mating behaviour or sexual isolation.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1157 - 63
Caldivirga maquilingensis gen . nov., sp . nov., a new genus of rod-shaped crenarchaeote isolated from a hot spring in the Philippines; Itoh T et al.; Two novel hyperthermophilic, rod-shaped crenarchaeotes were isolated from an acidic hot spring in the Philippines . Cells were mostly straight or slightly curved rods 0.4-0.7 micron in width . Bent cells, branched cells, and cells bearing globular bodies were commonly observed . The isolates were heterotrophs and grew anaerobically and microaerobically . The addition of archaeal cell extract or a vitamin mixture to the medium significantly stimulated growth . The isolates grew over a temperature range of 60-92 degrees C, and optimally around 85 degrees C and grew over a pH range of 2.3-6.4, and optimally at pH 3.7-4.2 . The isolates utilized glycogen, gelatin, beef extract, peptone, tryptone and yeast extract as carbon sources . They required sulfur, thiosulfate or sulfate as electron acceptors . The lipids mainly consisted of various cyclized glycerol-bisdiphytanyl-glycerol tetraethers . The G+C content of the genomic DNAs was 43 mol% . The 16S rDNA contained two small introns . The comparison of the 16S rDNA exon sequences revealed that they represented an independent lineage in the family Thermoproteaceae . The two strains were included in a single species because of high levels of DNA-DNA relatedness . From these results, Caldivirga maquilingensis gen . nov., sp . nov . is proposed in the family Thermoproteaceae to accommodate these isolates . The type strain of C . maquilingensis is strain IC-167T (= JCM 10307T = MCC-UPLB 1200T = ANMR 0178T).

Comp Biochem Physiol B Biochem Mol Biol, 1999 May, 123(1), 115 - 23
Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri; Ballarin L et al.; A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography . This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose . On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions . This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis . The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition . The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells . The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

J Nat Prod, 1999 Jul, 62(7), 963 - 8
Synthesis of furanonaphthoquinones with hydroxyamino side chains; Wu C et al.; Several furanonaphthoquinones have shown useful activity in a yeast assay for DNA-damaging agents and cytotoxicity in mammalian cell culture assays . These results, together with the planar aromatic character of the furanonaphthoquinones, suggested that they might be acting as DNA intercalators . In an attempt to improve this activity, various analogues containing a hydroxyamino side chain have been synthesized . The analogues were prepared by standard methods, but some unexpected reactions were observed nonetheless . Thus, 8-formyl-5-methoxy-4,9-dihydronaphtho{2,3-b}furan-4,9-dione (24) showed an unusual reactivity toward reductive amination, with the reaction proceeding further to give one of two different cyclized products, depending on the amination reagent used . Bioassay results indicated that only simple furanonaphthoquines showed activity in a yeast assay for DNA-damaging agents; compounds with a substituted hydroxyamino side chain were uniformly inactive in this assay . Most of the compounds with a substituted hydroxyamino side chain on the furan ring did, however, show cytotoxicity, although none of them was any more active than the simple aldehyde 2-formyl-4, 9-dihydronaphtho{2,3-b}furan-4,9-dione (14) . This evidence tends to suggest that the furanonaphthoquinones do not serve primarily as DNA intercalators, because if this were the case, they would have been expected to show an increased activity on conversion to their hydroxyamino side chain derivatives.

Izv Akad Nauk Ser Biol, 1999 Mar-Apr, (2), 133 - 44
{Homeobox genes: their structure and expression during development and regeneration}; Znoiko IIu et al.; Molecular mechanisms controlling the most important biological functions are highly conservative . This phenomenon can be seen in homeobox genes, which encode the regulatory proteins controlling basic developmental processes . The homeobox was first described about ten years ago in Drosophila melanogaster as a highly conservative gene region with characteristic structure . Later, homologous consequences were observed in many eukaryotic genes found in organisms ranging from yeast to humans . Biochemical, biophysical, and genetic studies have proved irrefutably that the homeobox controls a DNA-binding domain that allows proteins containing this domain to exert regulatory functions . The decisive role played by homeobox genes during development has stimulated great interest among researchers in this superfamily of genes . At present, more than 300 homeobox genes have been identified in animals of various taxa and their number is constantly growing . This review summarizes the published data about the structure and functions of homeobox genes and their role in development and regeneration.

Biochem Pharmacol, 1999 Jul 15, 58(2), 201 - 7
Sphingosine-1-phosphate: extracellular mediator or intracellular second messenger?
Hla T, Lee MJ, Ancellin N, Liu CH, Thangada S, Thompson BD, Kluk M.
Sphingosine-1-phosphate (SPP), a polar sphingolipid metabolite, has received much attention recently as an extracellular mediator and an intracellular second messenger . It regulates a wide range of biological responses such as cell growth, death, differentiation, and migration . Recent identification of plasma membrane receptors and the cloning of SPP metabolizing enzymes have increased our understanding of the biology of SPP synthesis and action . However, controversy exists regarding the mode of action of this molecule . EDG-1 and related G-protein-coupled receptors were identified recently as plasma membrane receptors for SPP . In light of this recent discovery, many of the functions of SPP previously thought to be due to intracellular second messenger action should be reevaluated . In addition, signaling properties and functions of the three known receptors for SPP need to be fully delineated . The structures and the evolutionary conservation of SPP metabolizing enzymes from yeast to mammals support the hypothesis that SPP also plays a role as an intracellular second messenger . However, definitive assignment of the intracellular role of SPP awaits purification/molecular cloning of elusive intracellular receptors . Better knowledge of the molecular basis of SPP action is needed to assess the physiological and pathophysiological significance of this bioactive lipid mediator.

Mycopathologia, 1998-99, 144(1), 15 - 20
In vitro activity of antimycotic agents determined by E-test method against vaginal Candida species; Candido RC et al.; Vaginal candidiasis continues to be a common cause of vaginal discharge, pruritus and other local complaints in women worldwide . Although numerous antimycotic agents are available for the treatment of yeast vaginitis there is little comparative data on the in vitro activity of these drugs . The objectives of this study were to isolate and identify the Candida species in the vagina and anus of patients treated in a gynaecology clinic, as well as determine the susceptibility to azolic compounds measured by the E-test method . Vaginal and rectal swabs were collected from 80 adult non-pregnant patients, seen at a gynaecological clinic, aged 18-59 years, with sexual activity, with and without vaginitis . The swabs were processed by methods routinely used for the detection of pathogenic yeasts . The susceptibility of the isolates to fluconazole, ketoconazole and itraconazole, was measured by the agar diffusion method (E-test), using RPM1 1,640 medium with 2% glucose and phosphate buffer . Candida species (33) strains were isolated from 17 patients at similar proportions from both anatomical sites, and 12 patients harboured 24 strains of C . albicans in the vaginal and rectal tracts . Twenty one percent of the strains of C . albicans were resistant to ketoconazole, 54% were resistant to itraconazole and 0% were resistant to fluconazole . The sensitivity of strains isolated from the two sites were similar, indicating that these are strains of the same phenotype.

Mycopathologia, 1998-99, 144(1), 9 - 14
Host organism defense by a peptide-polysaccharide extracted from the fungus Sporothrix schenckii; Carlos IZ et al.; A peptide-polysaccharide, a peptide-rhamnomannan, was isolated from the pathogenic yeast form of the fungus Sporothrix schenckii . This substance, which may play a role in fungal virulence, was tested in an animal model of systemic disease, and depression of the immune response was observed in the animals between the 4th and 6th week of infection . Concomitantly, this compound showed mitogenic activity when challenged with normal lymphocytes and was also found to be involved in the inflammatory response . These results provide further information for the understanding of fungal implantation in tissues and of the pathogenicity of this systemic mycosis.

J Biol Chem, 1999 Jul 30, 274(31), 22095 - 101
Cloning and characterization of human polyamine-modulated factor-1, a transcriptional cofactor that regulates the transcription of the spermidine/spermine N(1)-acetyltransferase gene; Wang Y et al.; The increased transcription and ultimate superinduction of the spermidine/spermine N(1)-acetyltransferase (SSAT) gene has been associated with the antineoplastic activity of several new antitumor polyamine analogues . In sensitive tumor cell types, the transcriptional induction appears to be regulated by the constitutive association of the transcription factor Nrf-2 with the recently discovered polyamine-responsive element . Using the yeast two-hybrid system, a new transcriptional cofactor, polyamine-modulated factor-1 (PMF-1), has been identified as a partner protein of Nrf-2 that, in combination with Nrf-2, regulates the polyamine analogue-induced transcription of SSAT . The human PMF-1 gene, located on chromosome 1 near the 1q12/1q21 border, yields an mRNA transcript of approximately 1.2 kilobases that codes for a 165-amino acid protein with a predicted molecular mass of approximately 20 kDa . The PMF-1 mRNA appears to increase in response to analogue exposure only in analogue-responsive cells . In addition to the transcriptional regulation of SSAT, PMF-1 or similar factors should be considered in the regulation of other polyamine-dependent genes.

Genes Dev, 1999 Jul 15, 13(14), 1774 - 9
Transcription elongation factor hSPT5 stimulates mRNA capping; Wen Y et al.; RNA polymerase II nascent transcripts are capped during pausing before elongation . Here we report that hSPT5, the human homolog of yeast elongation factor SPT5, interacts directly with the capping enzyme . hSPT5 stimulated capping enzyme guanylylation and mRNA capping by severalfold . Although RNA 5'-triphosphatase activity was unaffected, binding to this domain in the full-length enzyme is likely involved in the stimulation, as hSPT5 did not increase the activity of the guanylyltransferase fragment . Consistent with capping enzyme binding, TFIIH-phosphorylated CTD stimulated guanylylation, and this increase was not additive with hSPT5.

J Comput Biol, 1999 Summer, 6(2), 187 - 207
An algorithmic approach to multiple complete digest mapping; Fasulo D et al.; Multiple Complete Digest (MCD) mapping is a method of determining the locations of restriction sites along a target DNA molecule . The resulting restriction map has many potential applications in DNA sequencing and genetics . In this work, we present a heuristic algorithm for fragment identification, a key step in the process of constructing an MCD map . Given measurements of the restriction fragment sizes from one or more complete digestions of each clone in a clone library covering the molecule to be mapped, the algorithm identifies groups of restriction fragments on different clones that correspond to the same region of the target DNA . Once these groups are correctly determined the desired map can be constructed by solving a system of simple linear inequalities . We demonstrate the effectiveness of our algorithm on real data provided by the Genome Center at the University of Washington.

Chem Biol Interact, 1999 May 14, 119-120, 53 - 60
Association of tetramers of human butyrylcholinesterase is mediated by conserved aromatic residues of the carboxy terminus; Altamirano CV et al.; Human butyrylcholinesterase (BChE) is composed predominantly of tetramers . Our laboratory has shown that up to 40 carboxy terminal residues of each subunit contribute to the stabilization of tetramers (R.M . Blong, E . Bedows, O . Lockridge, The tetramerization domain of butyrylcholinesterase is at the carboxy-terminus, Biochem . J . 327 (1997) 747-757) . To better define the residues which participate in tetramer stabilization, the in vivo interaction of the BChE C-terminus 46 residue peptide was quantitated for wild type and mutant BChE using the yeast two-hybrid system . The wild type C-terminal peptides interacted with one another in this system . The K-variant (A539T) and C571A peptides showed interaction similar to that of the wild type . However, only 11.7% of the interaction seen with the wild type peptide was observed with the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) had been altered to alanines (aromatics off mutant) . When these seven mutations were incorporated into the complete BChE molecule and expressed in 293T cells, only monomers and dimers were observed . The addition of poly-L-proline to the medium of 293T cells expressing wild type BChE resulted in the increase of the tetrameric form, similar to that observed by Bon et al . (S . Bon, F . Coussen, J . Massoulie, Quaternary associations of acetylcholinesterase II . The polyproline attachment domain of the collagen tail, J . Biol . Chem . 272 (1997) 3016-3021) for acetylcholinesterase expressed in COS cells . However, no increase in tetramers was observed with poly-L-proline addition to the medium of 293T cells expressing the aromatics off BChE mutant . These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues, Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564, and that the poly-L-proline induced increase in tetrameric BChE is mediated through these seven aromatic residues.

Mol Cells, 1999 Jun 30, 9(3), 281 - 5
The cytoplasmic domain of HIV-1 gp41 interacts with the carboxyl-terminal region of alpha-catenin; Kim EM et al.; To know the cellular protein interactions with the viral protein can give an insight into the molecular mechanisms of the virus life cycle . As the function of the cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) gp41 is not known clearly, we searched for a cellular protein that interacts with the cytoplasmic domain of the HIV-1 gp41 using the yeast two-hybrid assay system . Screening of HeLa cell cDNA library yielded alpha-catenin cDNA . The cytoplasmic domain of the HIV-1 gp41 and the simian immunodeficiency virus (SIV) gp41 were able to interact with the carboxyl-terminal region of alpha-catenin specifically . Mapping of the interaction sites revealed that the interaction between the domain containing the second helix structure from the carboxyl-terminus of HIV-1 gp41 and the carboxyl-terminal region of alpha-catenin was stronger than other domains of gp41.

Jpn Heart J, 1999 Mar, 40(2), 199 - 208
A novel A-kinase anchoring protein in the heart interacts with G alpha 13; Suzuki M et al.; A cDNA of a tentative A-kinase anchoring protein, presumably coupled with heterotrimeric GTP binding protein alpha 13 subunit (G alpha 13), was cloned from a human heart cDNA library . It was approximately 650 bases and its mRNA was expressed in the heart . Homology search of DNA sequences revealed that it was a novel cDNA with 84% homology with the partial sequence of rabbit cDNA of AKAP 120 without a stop codon . 3'-Rapid Amplification of cDNA Ends (3'-RACE) and yeast functional assay were performed to determine the 3'-end of the cDNA and ribosomal frameshifting was suggested as a translational mechanism . Here we report that a protein encoded by the cDNA may be involved in intracellular signal transduction via the G alpha 13 and PKA in hearts.

J Chromatogr A, 1999 Jun 18, 846(1-2), 143 - 56
Characterization of the isoforms of PIXY321, a granulocyte-macrophage-colony stimulating factor-interleukin-3 fusion protein, separated by preparative isoelectric focusing on immobilized pH gradients; Balland A et al.; We present here the purification and the characterization of the isoforms of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast . The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients . Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields . Subsequent analysis of purified isoforms of PIXY321 by peptide mapping and mass spectrometry linked the microheterogeneity of the original molecule to three parameters, the presence of deamidated residues, charged glycans and the pattern of O-linked glycosylation along the peptide sequence . This last parameter emphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms.

Mikrobiologiia, 1999 Mar-Apr, 68(2), 160 - 3
{Effect of conditions od Trichosporon pullulans culturing on the synthesis of immunomodulating glycoprotein}; Eroshin VK et al.; An extracellular glycoprotein (GP) exhibiting immunomodulating activity produced by the yeast Trichosporon pullulans grown in a defined ethanol-containing medium differed substantially in its composition from that of the yeast cell walls: therefore, it cannot be considered a structural component of the cell walls . In batch culture, the greatest GP production (40 mg/l) occurred in the exponential phase of the yeast growth . Under continuous cultivation, in both chemostat and pH-auxostat regimes, the specific rate of GP synthesis (qGP) increased with the increasing specific growth rate (mu) and reached 1.55 mg/(g h) at mumax . Under limitation of the yeast growth by zinc qGP was three times lower than under nitrogen or iron limitation . The rate of GP production depended inversely on the oxygen concentration.

J Biol Chem, 1999 Jul 30, 274(31), 21507 - 10
Modulation of rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein; Jordan JD et al.; We used the yeast two-hybrid system to identify proteins that interact directly with Galpha(o) . Mutant-activated Galpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons . We found that Galpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP . This study focuses on Rap1GAP, which selectively interacts with Galpha(o) and Galpha(i) but not with Galpha(s) or Galpha(q) . Rap1GAP interacts more avidly with the unactivated Galpha(o) as compared with the mutant (Q205L)-activated Galpha(o) . When expressed in HEK-293 cells, unactivated Galpha(o) co-immunoprecipitates with the Rap1GAP . Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin . When unactivated Galpha(o) was expressed, the amount of activated Rap1 was greatly increased . This effect was not observed with the Q205L-Galpha(o) . Expression of unactivated Galpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner . These results identify a novel function of Galpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2 . Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions.

J Food Prot, 1999 Apr, 62(4), 414 - 7
Inhibition of aflatoxin-producing fungi by Welsh onion extracts; Fan JJ et al.; Welsh onion ethanol extracts were tested for their inhibitory activity against the growth and aflatoxin production of Aspergillus flavus and A . parasiticus . The survival of spores of A . flavus and A . parasiticus depended on both the extract concentration and the exposure time of the spores to the Welsh onion extracts . The mycelial growth of two tested fungi cultured on yeast extract-sucrose broth was completely inhibited in the presence of the Welsh onion ethanol extract at a concentration of 10 mg/ml during 30 days of incubation at 25 degrees C . The extracts added to the cultures also inhibited aflatoxin production at a concentration of 10 mg/ml or permitted only a small amount of aflatoxin production with extract concentration of 5 mg/ml after 2 weeks of incubation . Welsh onion ethanol extracts showed more pronounced inhibitory effects against the two tested aflatoxin-producing fungi than did the same added levels of the preservatives sorbate and propionate at pH values near 6.5.

J Steroid Biochem Mol Biol, 1999 Apr-Jun, 69(1-6), 45 - 50
Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors; Takimoto GS et al.; Pharmacological antagonists of steroid receptor action had been thought to exert their effects by a passive mechanism driven principally by the ability of the antagonist to compete with agonist for the ligand binding site . However, recent analyses of antagonist-occupied receptor function suggest a more complex picture . Antagonists can be subdivided into two groups, type I, or pure antagonists, and type II, or mixed antagonists that can have variable transcriptional activity based upon differential dimerization and DNA binding properties . This led us to propose that receptor antagonism may not simply be a passive competition for the ligand binding site, but may, in some cases, involve active recruitment of corepressor or coactivator proteins to produce a mixed transcriptional phenotype . We used a yeast two-hybrid screen to identify proteins that interact specifically with antagonist-occupied receptors . Two proteins have been characterized: L7/SPA, a ribosome-associated protein that is localized in both the cytoplasm and nucleus, but with no known extranucleolar nuclear function; and hN-CoR, the human homolog of the mouse thyroid receptor corepressor mN-CoR . In in vivo transcription assays we show that L7/SPA enhances the partial agonist activity of type II mixed antagonists, and that N-CoR and the related corepressor, SMRT, suppresses it . The coregulators do not affect agonists or pure antagonists . Moreover, the net agonist activity seen with mixed antagonists is a function of the ratio of coactivator to corepressor . Based upon these results, we proposed that in breast tumors the inappropriate agonist activity seen with therapeutic antagonists such as tamoxifen is responsible for the hormone-resistant state . To confirm this, we are quantitating coactivator/corepressor ratios in breast tumor cells lines and clinical breast cancers . Results should provide new insights into the mechanisms underlying the progression of breast cancer to hormone resistance, and may suggest strategies for delaying or reversing this process.

Lab Invest, 1999 Jul, 79(7), 837 - 47
Intracellular glutathione redox status modulates MCP-1 expression in pulmonary granulomatous vasculitis; Desai A et al.; A wide spectrum of human lung diseases is characterized by the presence of granulomas . Although understanding of the pathways leading to their development remains incomplete, data from in vitro studies suggest that neutrophils, monocytes, and their secreted products (eg, hydrogen peroxide, H2O2) influence the pathogenesis of pulmonary granulomatous disease through the regulation of local chemokine and cytokine production . Using a well-characterized rat model of glucan-induced pulmonary granulomatous vasculitis, we sought to determine the role of intracellular glutathione (GSH) redox status in the expression of monocyte chemoattractant protein-1 (MCP-1) . Previous studies have revealed that vascular wall MCP-1 expression is obligatory for granuloma development and that both neutrophils and hydrogen peroxide are required for MCP-1 induction . Because in vitro expression of MCP-1 is in part mediated by the redox-sensitive transcription factors nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1), we studied their activation as a function of varying intracellular GSH redox status in the pathogenesis of glucan-induced pulmonary granulomatosis . Infusion of particulate yeast cell wall glucan into rats resulted in a rapid decrease in intracellular GSH concentrations which was accompanied by the activation of NF-kappaB and AP-1 . The pattern of AP-1 and NF-kappaB activation in turn correlated temporally with the expression of MCP-1 . Administration of L-buthionine-S, R-sulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase, resulted in a significant reduction in intracellular GSH pools . GSH depletion resulted in a more than 100% increase in pulmonary MCP-1 concentrations and increased cytosolic to nuclear translocation of NF-kappaB while having no effect on AP-1 levels . These observations suggest that in the pathogenesis of pulmonary granulomatous disease, intracellular glutathione redox status modulates the expression of MCP-1 through redox-sensitive transcription factors.

Plant J, 1999 Jun, 18(5), 529 - 39
Structural and functional analysis of the six regulatory particle triple-A ATPase subunits from the Arabidopsis 26S proteasome; Fu H et al.; The 26S proteasome is a multi-subunit ATP-dependent protease responsible for degrading most short-lived intracellular proteins targeted for breakdown by ubiquitin conjugation . The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylindrical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP-dependence and the specificity for ubiquitinated proteins . Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, designated RPT1-6 for regulatory particle triple-A ATPase, that form a distinct family within the AAA superfamily . Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation . Here, we describe the RPT gene family from Arabidopsis thaliana . From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts . Five of the six RPT sub- units are encoded by two genes; the exception being RPT3 which is encoded by a single gene . mRNA for each of the six proteins is present in all tissue types examined . Five of the subunits (RPT1 and 3-6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent . Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non-redundant functions.

Science, 1999 Jul 23, 285(5427), 553 - 6
Light-dependent sequestration of TIMELESS by CRYPTOCHROME; Ceriani MF et al.; Most organisms have circadian clocks consisting of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness . In this study, CRYPTOCHROME (CRY), a protein involved in circadian photoperception in Drosophila, is shown to block the function of PERIOD/TIMELESS (PER/TIM) heterodimeric complexes in a light-dependent fashion . TIM degradation does not occur under these conditions; thus, TIM degradation is uncoupled from abrogation of its function by light . CRY and TIM are part of the same complex and directly interact in yeast in a light-dependent fashion . PER/TIM and CRY influence the subcellular distribution of these protein complexes, which reside primarily in the nucleus after the perception of a light signal . Thus, CRY acts as a circadian photoreceptor by directly interacting with core components of the circadian clock.

Biochem J, 1999 Aug 1, 341 ( Pt 3), 601 - 11
Analysis of DNase-I-hypersensitive sites at the 3' end of the cystic fibrosis transmembrane conductance regulator gene (CFTR); Nuthall HN et al.; The cystic fibrosis transmembrane conductance regulator gene (CFTR) exhibits a complex pattern of expression that shows temporal and spatial regulation, although the control mechanisms are not fully known . We have mapped DNase-I-hypersensitive sites (DHSs) flanking the CFTR gene with the aim of identifying potential regulatory elements . We previously characterized DHSs at -79.5 and -20.9 kb with respect to the CFTR translational start site and a regulatory element in the first intron of the gene at 185+10 kb . We have now mapped five DHSs lying 3' to the CFTR gene at 4574+5.4, +6.8, +7.0, +7.4 and +15.6 kb that show some degree of tissue specificity . The DHSs are seen in chromatin extracted from human primary epithelial cells and cell lines; the presence of the +15.6 kb site is tissue-specific in transgenic mice carrying a human CFTR yeast artificial chromosome . Further analysis of the 4574+15.6 kb DHS implicates the involvement of CCAAT-enhancer-binding protein (C/EBP), cAMP-response-element-binding protein (CREB)/activating transcription factor (ATF) and activator protein 1 (AP-1) family transcription factors at this regulatory element.

Gene, 1999 Jul 22, 235(1-2), 93 - 101
Identification of a novel gene encoding a p53-associated protein; Zhou R et al.; p53 exerts important physiological functions in cell-cycle control, gene regulation, cell differentiation, apoptosis and tumor suppression by interacting with many cellular proteins . Using the yeast two-hybrid system, we screened a HeLa cDNA library and identified a novel gene encoding a p53-binding protein (p53BP3) . The full-length cDNA of p53BP3 was isolated from a HeLalambdagt10 cDNA library . This predicted protein was composed of 815 amino acids . Sequence analysis indicated that p53BP3 contained two bipartite nuclear localization signals and was confirmed to be a nuclear protein . FISH mapping results showed that this novel gene was located at human chromosome 12, region p11.2-p12.1 . Northern blot analysis suggested that p53BP3 was broadly expressed in human tissues . A further study showed that p53BP3 had a homologue in mouse.

Gene, 1999 Jul 22, 235(1-2), 43 - 50
Transcript map of the human chromosome Xq11-Xq21 region: localization of 33 novel genes and one pseudogene; Villard L et al.; The human Xq11-Xq21.3 region has been implicated in several inherited disorders including dystonia-parkinsonism (DYT3), sideroblastic anemia and several specific and non-specific forms of mental retardation (MR) syndromes . As part of a positional cloning effort to identify MR genes, we have generated a YAC-based transcript map . We first constructed a YAC/STS framework by extending previously published contigs . This framework map consists of a minimal set of 119 clones, covering approximately 20 Megabases (Mb) and allowing the precise ordering of 71 STSs between DXS136 and DXS472 . This YAC contig was then used to define the positions of genes and expressed sequence tags (ESTs) assigned to the Xcen-Xq21.3 region . In addition to the genes previously localized to this part of the X chromosome, 18 transcription units corresponding to additional known genes or gene family members, one pseudogene and 15 novel transcripts were mapped . This transcriptional map incorporates 51 transcription units and provides a useful resource of candidate genes for some of the disorders assigned to this region of the X chromosome.

J Neurosci, 1999 Aug 1, 19(15), 6519 - 27
Cloning and characterization of neuropilin-1-interacting protein: a PSD-95/Dlg/ZO-1 domain-containing protein that interacts with the cytoplasmic domain of neuropilin-1; Cai H et al.; Neuropilin-1 (Npn-1), a receptor for semaphorin III, mediates the guidance of growth cones on extending neurites . The molecular mechanism of Npn-1 signaling remains unclear . We have used a yeast two-hybrid system to isolate a protein that interacts with the cytoplasmic domain of Npn-1 . This Npn-1-interacting protein (NIP) contains a central PSD-95/Dlg/ZO-1 (PDZ) domain and a C-terminal acyl carrier protein domain . The physiological interaction of Npn-1 and NIP is supported by co-immunoprecipitation of these two proteins in extracts from a heterologous expression system and from a native tissue . The C-terminal three amino acids of Npn-1 (S-E-A-COOH), which is conserved from Xenopus to human, is responsible for interaction with the PDZ domain-containing C-terminal two-thirds of NIP . NIP as well as Npn-1 are broadly expressed in mice as assayed by Northern and Western analysis . Immunohistochemistry and in situ hybridization experiments revealed that NIP expression overlaps with that of Npn-1 . NIP has been independently cloned as RGS-GAIP-interacting protein (GIPC), where it was identified by virtue of its interaction with the C terminus of RGS-GAIP and suggested to participate in clathrin-coated vesicular trafficking . We suggest that NIP and GIPC may participate in regulation of Npn-1-mediated signaling as a molecular adapter that couples Npn-1 to membrane trafficking machinery in the dynamic axon growth cone.

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